Origins of Life: The Primal Self-Organization

Origins of Life: The Primal Self-Organization . Richard Egel Dirk-Henner Lankenau Armen Y. Mulkidjanian l l Edito...

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Origins of Life: The Primal Self-Organization

.

Richard Egel Dirk-Henner Lankenau Armen Y. Mulkidjanian l

l

Editors

Origins of Life: The Primal Self-Organization

Editors Richard Egel University of Copenhagen Biocenter Department of Biology Ole Maaløes Vej 5 2200 Copenhagen N Denmark [email protected]

Dr. Dirk-Henner Lankenau Hinterer Rindweg 21 68526 Ladenburg Germany [email protected]

A.Y. Mulkidjanian School of Physics University of Osnabrueck Osnabrueck 49076 Germany and A.N. Belozersky Institute of Physico-Chemical Biology Moscow State University Moscow 119991 Russia [email protected]

ISBN 978-3-642-21624-4 e-ISBN 978-3-642-21625-1 DOI 10.1007/978-3-642-21625-1 Springer Heidelberg Dordrecht London New York Library of Congress Control Number: 2011935879 # Springer-Verlag Berlin Heidelberg 2011 This work is subject to copyright. All rights are reserved, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilm or in any other way, and storage in data banks. Duplication of this publication or parts thereof is permitted only under the provisions of the German Copyright Law of September 9, 1965, in its current version, and permission for use must always be obtained from Springer. Violations are liable to prosecution under the German Copyright Law. The use of general descriptive names, registered names, trademarks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. Printed on acid-free paper Springer is part of Springer Science+Business Media (www.springer.com)

Preface

One more book on the origin of life. . . What for, if there are so many books on tentative origins of life already? In fact, the planning of this book arose from a visionary notion. – If theoretical physicists can seriously entertain canonical “standard models” not only of star formation but even for big-bang generation and the accelerated expansion of the entire universe, why cannot life scientists and geochemists reach consensus on how cellular and organismal life most likely has emerged and settled on this planet? – The biggest gap in our understanding persistently separates bottom-up inferences (from tentative primordial conditions on early Earth) and anthropic top-down extrapolations (from contemporary life forms to common ancestral lineages and states). Staying in the framework of this general view, the editors have started this book project keeping in mind two specific goals which, at first glance, seem incompatible with one another. First we wanted to expand the space of chemical possibilities, drawing attention to chemically plausible synthetic pathways that may appear bizarre for modern life and, therefore, were not fully exploited in origin-of-life considerations. Second, we tried to identify constraints which could confine the virtually unlimited number of the hypothetical origin-of-life scenarios. Surprisingly even to the editors, the presentation of these two topics side by side has led to a certain synergy – the tough energetic constraints discard some traditionally popular scenarios, such as spontaneous formation of complex life forms in a primordial soup – which is thermodynamically implausible, and, instead, draw attention to uncommon processes, which, however, have a solid physical and chemical background – such as abiotic photosynthesis at the surface of semiconducting minerals or metal-catalyzed formation of abiotic peptides. In the opening prologue chapter, the sources of free energy which may have supported the first life forms are considered; it is argued that the nature of these energy fluxes has decisively shaped life as we know it. Part I is devoted to uncommon synthetic pathways, including the tentative primordial metabolism of reduced phosphorus compounds, such as phosphate and hypophosphite, as well as photosynthetic CO2 assimilation at the surface of primordial minerals, which,

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amazingly, could proceed with a high efficiency. Part II exploits the possibilities of prebiotic formation of peptides and lipids and of their interactions under anoxic conditions on the primordial Earth. Among other facets, this part is deliberately putting focus on the potential impact of stochastic uncoded peptides in kick-starting an ever tightening co-evolution of peptides/proteins and nucleic acids. Part III highlights the transition from the physicochemical world of mass and energy to an additional component crucial for the emergence of life, i.e., physically encoded information, engraved in covalent molecular structures and stable over many cycles of replication. These entities are termed informational replicators. The final epilogue chapter connects multiple ends to a tentatively integrating overview from geochemical to biological inferences, referring to all the individual chapters in due course. Furthermore, it also draws attention to another still pending problem, which is not specifically addressed in any other chapter. This concerns the inherently complex nature of precellular organization before the consolidation of integral genomes. From such primordial complexity, eukaryotic cell organization may have evolved more naturally, than what is more commonly assumed to have commenced from prokaryote-like precursory models. Many of the views expressed in this book cannot be considered mainstream yet. If this book can motivate dedicated researchers to seriously consider the presented alternative mechanisms for future scrutiny, it will have served its purpose well. As the coordinating editors, we cordially thank all the authors for their invaluable efforts in preparing the individual chapters. We certainly have learned a lot ourselves in commenting and revising the initial drafts. Without multiple interactions with all the participants we would not have been able to put forth that integral perspective, and we sincerely acknowledge the multiple feedbacks we have received in the editorial process. In putting out this book, we are deeply indebted to the editorial staff at Springer for making this project possible. We would especially like to acknowledge editor Sabine Schwarz at Springer Life Sciences (Heidelberg), desk editor Ursula Gramm (Springer, Heidelberg) and project manager Monisha Mohandas (SPi Content Solutions – SPi Global, Chennai, India) for their proficient and expeditious involvement in the production process. Copenhagen Ladenburg Osnabru¨ck

Richard Egel Dirk-Henner Lankenau Armen Y. Mulkidjanian

Contents

Prologue 1

Energetics of the First Life . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Armen Y. Mulkidjanian

Part I

Primeval Syntheses

2

A Hypothesis for a Unified Mechanism of Formation and Enantioenrichment of Polyols and Aldaric, Aldonic, Amino, Hydroxy and Sugar Acids in Carbonaceous Chondrites . . . . . . . . . . . . . 37 H. James Cleaves II

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On the Origin of Phosphorylated Biomolecules . . . . . . . . . . . . . . . . . . . . . . . 57 Matthew A. Pasek and Terence P. Kee

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Abiotic Photosynthesis: From Prebiotic Chemistry to Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85 Marcelo I. Guzman

Part II

Facets of an Ancestral Peptide World

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Salt-Induced Peptide Formation in Chemical Evolution: Building Blocks Before RNA – Potential of Peptide Splicing Reactions . . . . . . 109 Daniel Fitz, Thomas Jakschitz, and Bernd M. Rode

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Scenario of the Primary Pump: Emergence and Operation of an Automatic Engine to Generate Primordial Peptides and Beyond Nucleic Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129 Auguste Commeyras

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Contents

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The Relevance of Peptides That Bind FeS Clusters, Phosphate Groups, Cations or Anions for Prebiotic Evolution . . . . . . . . . . . . . . . . . . 155 E. James Milner-White

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Peptide-Dominated Vesicles: Bacterial Internal Membrane Compartments as Model Systems for Prebiotic Evolution . . . . . . . . . . 167 James N. Sturgis

Part III

RNA Worlds: Ancestral and Contemporary

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Nicotinamide Coenzyme Synthesis: A Case of Ribonucleotide Emergence or a Byproduct of the RNA World? . . . . . . . . . . . . . . . . . . . . . 185 Nadia Raffaelli

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On Alternative Biological Scenarios for the Evolutionary Transitions to DNA and Biological Protein Synthesis . . . . . . . . . . . . . . . 209 Anthony M. Poole

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Two RNA Worlds: Toward the Origin of Replication, Genes, Recombination, and Repair . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225 Dirk-Henner Lankenau

Epilogue 12

Integrative Perspectives: In Quest of a Coherent Framework for Origins of Life on Earth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 289 Richard Egel

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 361

Prologue

Chapter 1

Energetics of the First Life Armen Y. Mulkidjanian

Abstract Life can exist only when supported by energy flow(s). Here, the tentative mechanisms of coupling between the natural energy fluxes and the first life forms are discussed. It is argued that the evolutionarily relevant, continuous fluxes of reducing equivalents, which were needed for the syntheses of the first biomolecules, may have been provided by the inorganic photosynthesis and by the redox reactions within hot, iron-containing rocks. The only primordial environments where these fluxes could meet were the continental geothermal systems. The ejections from the hot, continental springs could contain, on the one hand, hydrogen and carbonaceous compounds and, on other hand, transition metals as Zn and Mn, which precipitated around the springs as photosynthetically active ZnS and MnS particles capable of reducing carbon dioxide to diverse organic compounds. At high pressure of the primordial CO2 atmosphere, both the inorganic photosynthesis and the abiotic reduction of carbon dioxide within hot rocks should have proceeded with high yield. Among a plethora of abiotically produced carbonaceous molecules, the natural nucleotides could accumulate as the most photostable structures; their polymerization and folding into double-stranded segments should have been favored by the further increase in the photostability. It is hypothesized that after some aggregates of photo-selected RNA-like polymers could attain the ability for self-replication, the consortia of such replicating entities may have dwelled in honeycomb-like ZnS-enriched mineral compartments which provided shelter and nourishment. The energetics of the first life forms could be driven by their ability to cleave the abiogenically formed organic molecules and by reactions of the phosphate group transfer. The next stage of evolution may be envisaged as a selection for increasingly tighter envelopes of the first organisms; this selection may have

A.Y. Mulkidjanian (*) School of Physics, University of Osnabrueck, Osnabrueck 49076, Germany A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow 119991, Russia e-mail: [email protected] R. Egel et al. (eds.), Origins of Life: The Primal Self-Organization, DOI 10.1007/978-3-642-21625-1_1, # Springer-Verlag Berlin Heidelberg 2011

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eventually yielded ion-tight lipid membranes able to support the sodium-dependent membrane bioenergetics. Lastly, the proton-tight, elaborate membranes independently emerged in Bacteria and Archaea, and enabled the transition to the moderntype proton-dependent bioenergetics. The first primeval step would appear to be indicated by the union of single crystalloidal inorganic molecules to form inorganic colloids, and that these meta-stable colloids acting on inorganic carbon compounds, such as carbon dioxide, in presence of water and sunlight, and taking energy from the sunlight, built up at first simple organic bodies, and now these in turn reacting with one another formed more and more complex organic compounds. In any such transformation external energy is necessary, because the reacting bodies, carbon dioxide and water, are fully oxidised, and must be reduced with . . . uptake of energy in what is called an endothermic reaction. To this reaction, the inorganic colloid plays the part of an activator or catalyst, the solar energy being converted into chemical energy of the organic compound, so serving as a reservoir of the energy necessary for the coming living organic world. B. Moore and T.A. Webster (1913)

1.1

Introduction

Living organisms can exist only when supported by energy (Bauer 1935; Schr€odinger 1945; Glansdorff and Prigogine 1971; Williams and Frausto da Silva 2006; Danchin 2009). Therefore, from the very beginning, the life forms should have exploited the natural energy fluxes. Here, I focus on the tentative mechanisms of coupling between the first life forms and the natural energy fluxes at different stages of the early evolution. Because of the energetic continuity requirement – which follows from the Darwinian evolutionary continuity principle (see Lahav (1999); Wolf and Koonin (2007) and references therein) – the energy flows that deserve attention in evolutionary context are those that remain constant on the evolutionary relevant, geological timescale. This consideration essentially discounts the evolutionary importance of occasional energy inputs from impact bombardment, atmospheric electric discharges, shock waves, volcanic explosions, and so on. It is also unlikely that life could notably depend on the chemical compounds that were produced or delivered during such occasional events. It seems implausible that the first life forms could wait from one occasional event (e.g., volcanic explosion) to another to get energy and nourishment. There are no known organisms that obligatorily depend on such irregular sources of energy and matter. Mauzerall has insightfully noted that the energy requirements of the first living beings had to be compatible with those of modern organisms (Mauzerall 1992). He argued that “the ur-cell would be simpler, but it would also be less efficient”. More rigorously speaking, the intensity of the energy flux(es) that supported the emergence of life should be either comparable with the intensity of modern lifesupporting energy flows or stronger.

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As will be shown below, there are only few energy fluxes in nature that fulfil these criteria and, hence, may have been used by the first organisms. Therefore, the energetic constraints are very useful upon testing different hypotheses on the early evolution of life.

1.2

Reducing Power for the First Syntheses

The geochemical and cosmochemical data indicate that the primordial atmosphere on Earth was dominated by carbon dioxide (Nisbet 1991; Nisbet and Sleep 2001; Kasting and Howard 2006; Zahnle et al. 2007). Therefore, energy was initially needed for reducing CO2 to compounds that could further participate in prebiological syntheses (see Lazcano and Miller (1996); Bada (2004); Miller and Cleaves (2006); Lazcano (2010)) and references therein). More specifically, the reduction of CO2 should have required electrons with high reducing potential. The first step of the reduction of CO2 can be described by an equation: CO2 þ 2Hþ þ 2e ! HCOOH where 2e are the two required electrons. The standard redox potential of this reaction is as low as ~ 0.6 eV (at room temperature, neutral pH, and atmospheric pressure of 1 bar), therefore the task of CO2 reduction is far from being trivial. Currently, the reduction of CO2 by living organisms is supported by the two fluxes of reducing power, at least. The communities at the Earth’s surface depend, via photosynthesis and its products, on the solar light. Upon photosynthesis, the energy of light quanta is used to produce electrons with high reductive potency within sophisticated, membrane-embedded, (bacterio)chlorophyll-carrying proteins, called photochemical reaction centers (see Mulkidjanian et al. (2006) and references therein). The biotopes at the sea floor, besides consuming the organic fall-out from the upper, inhabited photic zone (where photosynthesis takes place), can also exploit the redox potential difference between reduced hydrothermal fluids and oxygenated ocean waters by coupling the downhill transfer of electrons to oxygen with an uphill electron transfer to CO2 (Kelley et al. 2002). This mechanism, however, could not be used by the first life forms, since the redox energy span of >1 eV between the reduced compounds of hydrothermal fluids and the sea waterdissolved oxygen became exploitable only after the ocean waters – some 2–2.5 Ga ago – became saturated by molecular oxygen, a waste product of cyanobacterial photosynthesis (Bekker et al. 2004; Mulkidjanian et al. 2006). Some prokaryotes can use other electron acceptors instead of oxygen, then however, the yield of CO2 reduction is much lower (Thauer et al. 1977, 2008). It is noteworthy that the hydrothermal fluids may already contain organic molecules; they are believed to stem, at least partly, from the so-called serpentinization reactions within the rocks of the oceanic crust (Russell and Arndt 2005).

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These reactions occur when iron-containing rocks interact with water at temperatures of 200–500 C. Under these conditions, part of the Fe2+ ions in the rock get oxidized to Fe3+ yielding magnetite (Fe3O4). The electrons that are released upon this reaction are taken either by protons of water or by the available carbon (usually present as water-dissolved CO2), so that H2 and diverse hydrocarbons are produced according roughly to the following equation (Martin et al. 2008): ðMg; FeÞ2 SiO4 þH2 O þ C ! Mg3 SiO5 ðOHÞ4 þ MgðOHÞ2 þ Fe3 O4 þH2 þ CH4 þ C2 C5 (1.1) Thus it has been argued that the organic compounds, as produced upon the serpentinization reactions, contribute to the energy budget of the marine organisms (see Martin et al. (2008) and references therein). Not surprisingly, the aforementioned sources of reducing power have been suggested also as promoters of the primeval synthetic reactions, which ultimately could lead to the origin of life. Some scholars, starting from Moore – a quote from his paper (Moore and Webster 1913) serves as epigraph to this chapter – have suggested that solar radiation served as the driving force upon the emergence of life (Haldane 1929; Granick 1957; Skulachev 1969; Hartman 1975; Halmann et al. 1980; Mauzerall 1992; Skulachev 1994; Hartman 1998; Mulkidjanian et al. 2003; Mulkidjanian and Galperin 2007; Guzman and Martin 2009). Indeed, the very lack of oxygen in the primordial atmosphere should have favoured light-driven chemical syntheses. Without the ozone shield, the solar light reaching Earth contained a UV component that was by orders of magnitude stronger than it is today (Sagan 1973; Vazquez and Hanslmeier 2006) and could drive diverse chemical reactions, in particular, the carbon fixation. No other known energy source could compete with solar irradiation in terms of strength and access to the whole of the Earth’s surface (Miller and Orgel 1973). At least two UV-driven abiogenic processes of CO2 reduction are known to proceed with efficiency comparable to that of modern photosynthesis. One is the photo-oxidation of Fe2+ ions in solution, which may lead to the reduction of CO2 to formaldehyde with a quantum yield of up to 2–3% (Getoff 1962; Borowska and Mauzerall 1988). Next, several naturally occurring minerals, in particular TiO2 (anatase/rutile), WO3 (wolframite), MnS (alabandite), and ZnS (wurtzite, sphalerite), possess the properties of broad-band semiconductors and can photoreduce CO2 at their surfaces (Inoue et al. 1979; Reiche and Bard 1979; Halmann et al. 1980, 1981; Henglein 1984; Henglein et al. 1984; Kisch and K€unneth 1991; Eggins et al. 1993; Fox and Dulay 1993; Hagfeldt and Gratzel 1995; Inoue et al. 1995; Yoneyama 1997; Schoonen et al. 1998; Xu and Schoonen 2000; Schoonen et al. 2004; Zhang et al. 2004, 2007; Guzman and Martin 2009; see also Chap. 4 for details). The highest quantum yield of 80% has so far been reported for the reduction of CO2 to formate at the surface of colloidal ZnS particles (Henglein 1984; Henglein et al. 1984). The crystals of ZnS, which is commonly known as “phosphor” (from

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“phosphorescence”), can trap the energy of light and store it on a time scale of minutes. In addition, these crystals are the most powerful photocatalysts known in nature. Particles of ZnS and other semiconducting minerals can produce diverse organic compounds from CO2 (Fox and Dulay 1993; Eggins et al. 1998), including the intermediates of the Krebs cycle (Zhang et al. 2007; Guzman and Martin 2009), can catalyze photocondensation of HCN or formamide (Senanayake and Idriss 2006; Liu et al. 2008), and, generally, can drive various transformations of carbon- and nitrogen-containing substrates (see (Henglein 1984; Yanagida et al. 1985; Kisch and Twardzik 1991; Hagfeldt and Gratzel 1995; Kisch and Lindner 2001; Marinkovic and Hoffmann 2001; Ohtani et al. 2003) and Chap. 4). The Fe2+-mediated CO2 reduction and the ZnS/MnS-mediated photosynthesis are not mutually exclusive; the addition of Fe2+ ions was shown to enhance the ZnS-mediated reduction of CO2 (M. Schoonen, personal communication). The possibility of the ZnS/MnS-mediated inorganic photosynthesis is particularly remarkable since the evolutionarily oldest proteins, which could be traced to the Last Universal Common Ancestor (LUCA), showed particular enrichment in the atoms of Zn and, to lesser extent, Mn as cofactors and structural elements (Mulkidjanian and Galperin 2009, 2010b), which implies availability of these metals for the primordial life forms. Where could these metals be found on the primordial Earth? The levels of transition metals in the aqueous systems are determined by the solubility constants of the respective salts and oxides. Specifically, the primordial anoxic ocean, as compared to the modern ocean, should have been enriched in well-soluble Fe2+ ions but depleted of Zn2+ ions (with estimated Zn concentration of <1013 M (Williams and Frausto da Silva 2006; Anbar 2008)). As argued elsewhere (Mulkidjanian et al. 2008b; Mulkidjanian and Galperin 2009, 2010a, b), the LUCA could hardly possess ion-tight membranes and the membrane pumps needed to maintain large gradients of Zn2+ ions between the Zn-rich cell interior and the Zn-depleted primeval anoxic waters. Thus, the high content of Zn and Mn in the “oldest” proteins could be explained by thriving of the first life forms not in the water column of the primordial ocean, but in some habitats that were enriched in these transition metals (Mulkidjanian and Galperin 2009, 2010a, b). On the modern Earth, the enrichment in transition metals, such as Zn and Mn, is routinely reported for the sites of geothermal activity, e.g., at the deep-sea hydrothermal vents. Here, the extremely hot hydrothermal fluids reach metal ions from the crust and bring them to the sea floor (Kelley et al. 2002; Tivey 2007). Because of high pressure at the sea bed, the ejected fluids with temperatures reaching 400 C remain liquid and can carry transition metal ions. Since hydrothermal fluids are rich in H2S, their interaction with cold ocean water leads to the precipitation of metal sulfide particles that form “smoke” over the “chimneys” of hydrothermal vents (Kelley et al. 2002; Tivey 2007). These particles eventually aggregate, settle down, and, ultimately, form sponge-like structures around the vent orifices. The sulfides of Fe and Cu precipitate promptly (Seewald and Seyfried 1990; Metz and Trefry 2000) even inside the orifices of hydrothermal vents (Kormas et al. 2006). The sulfides of Zn and Mn precipitate more slowly (Seewald and Seyfried 1990; Metz and Trefry 2000) and can spread over, forming halos around the iron-sulfur apexes of hydrothermal vents (Tivey 2007).

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The ZnS precipitates that cover the deep-sea vents cannot catalyze photosynthesis. Therefore, in relation to the problem of plausible energy sources for the earliest life forms, it was suggested that, at high CO2 pressure of the primordial atmosphere, very hot springs, similar to the deep-sea vents, could deliver metalliferous fluids within reach of solar light, namely at shallow hydrothermal vents or at geothermal systems on land (Mulkidjanian 2009). Accordingly, it was speculated that the illuminated ZnS/MnS precipitates (hereafter, for simplicity, denoted as ZnS precipitates) may have catalyzed the photosynthesis of different organic compounds from CO2, as well as the formation of first polymers, and may have served as feeding grounds for the first life forms (Mulkidjanian 2009; Mulkidjanian and Galperin 2009; see also Chap. 4). The feasibility of the ZnS precipitation within the reach of solar light is supported by the data from the modern Kamchatka volcanic system where the level of Zn2+ ions in the acidic, thermal waters can reach 0.2 mM (Karpov et al. 2009) yielding Zn-enriched precipitates around the hot springs (Kardanova 2009; Karpov et al. 2009). In relation to the aforementioned abundance of Zn2+ ions in the evolutionarily old proteins (Mulkidjanian and Galperin 2009, 2010b), it is noteworthy that the ZnS-mediated photosynthesis is accompanied by the release of Zn2+ ions (Henglein 1984; Kisch and K€ unneth 1991), yielding steadily Zn-enriched milieu at the ZnS surfaces according to the following equation: ZnS þ CO2 þ 2Hþ ! HCOOH + Zn2þ þ S Hence, the prevalence of Zn in the “oldest” proteins can be tentatively explained by the thriving of the first cells in illuminated, ZnS-enriched habitats where photoreleased Zn2+ ions could be preferably recruited as metal cofactors by the proteins and RNA molecules of the first cells (Mulkidjanian and Galperin 2009, 2010b). Alternatively to the inorganic photosynthesis, some authors have focused on the sources of reductive power at the floor of the primordial ocean. Specifically, W€achtersh€auser has proposed a detailed chemical mechanism where oxidation of FeS to FeS2 was suggested to drive the reduction of CO2 (W€achtersh€auser 1988, 1990, 1992; Huber and W€achtersh€auser 1997; W€achtersh€auser 2006, 2007). Indeed, the free energy of the redox transition of FeS to FeS2, at least under some conditions, seems to be sufficient to drive the reduction of CO2. However, as argued by Schoonen and co-workers (Schoonen et al. 1999), the favorable thermodynamics alone is not sufficient in the case of redox reactions. In addition, the redox potential of the electron donor has to be lower than that of the electron acceptor. To drive CO2 reduction at an appreciable rate, one needs a reducing agent with a redox potential that is lower than the redox potential of the CO2/formate redox pair (0.6 V), whereas the reducing potential of the FeS/FeS2 redox pair is higher than that, at least, under conditions that are compatible with life. The reducing power of the iron-containing minerals, however, depends on temperature, pressure, and the exact chemical composition of the minerals. While below 100 C, the oxidation of Fe2+ cannot drive the reduction of CO2, at

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temperatures of 200–500 C and high pressures, the interaction of iron-containing rocks with water could, as discussed above, lead to the production of hydrogen, hydrocarbons, and organic compounds. Building on this phenomenon of serpentinization, Russell and coworkers have suggested that life could emerge at the deepsea hydrothermal vents being supported by the delivery of organic compounds and hydrogen, as produced within hot rocks (see (Russell 2007; Martin et al. 2008)). Hence, while the model of W€achtershauser implies a direct coupling between the redox reactions of mineral iron and the syntheses of biological molecules, the model of Russell of coworkers suggests that the reductive formation of organic molecules may have taken place within hot rocks, at temperatures which were incompatible with life; the subsequent interactions of these organic molecules and the eventual condensation reactions could then take place at the sea floor or at hydrothermal vents, after the ejection and cooling of hydrothermal fluids. Besides the reduced carbon, biological macromolecules contain large amounts of nitrogen. The atmospheric dinitrogen is chemically inert; therefore, nitrogen could participate in the primeval syntheses as chemically reactive ammonia. Since hydrothermal fluids contain ammonia (Kelley et al. 2002; Karpov et al. 2009), it has been argued that dinitrogen could be reduced to ammonia within hot hydrothermal systems (Brandes et al. 1998; Wander et al. 2008; Wander and Schoonen 2008). Experimentally, it was shown that dinitrogen (at partial pressure 50 bar) was reduced in dilute hydrogen sulfide (H2S) solutions to ammonium at 120 C; the ammonia yield increased in the presence of iron monosulfide (Wander et al. 2008; Wander and Schoonen 2008). Nitrogen could also enter the primeval syntheses as formamide. Since formamide is, in fact, a stand-alone peptide bond, its reactions can yield amino acids (Miller and Cleaves 2006). In addition, the synthesis of nucleotides can be also relatively easy achieved at elevated temperatures either by starting from formamide (Saladino et al. 2009, 2010) or in formamide solutions (Powner et al. 2009). One more key element of life is phosphor. The reactions of phosphate group transfer make the essential part of cellular chemical reactions, and the concentration of phosphate within the cells is on the order of 10 mM. Large part of the ubiquitous, evolutionarily oldest proteins are involved in the phosphate group transfer, mostly as ATPases and GTPases (Koonin 2003; Mulkidjanian and Galperin 2009). Hence, the importance of phosphate and its high level within modern cells indicates the abundance of phosphorus compounds in the habitats of the first life forms (CairnsSmith et al. 1992; Schwartz 2006; Mulkidjanian and Galperin 2007; Danchin 2009). Still, the concentration of PO43 in sea water is low (the phosphates of Ca and Mg are hardly soluble in water). Here we have a conundrum: On the one hand, the concentration of free phosphate in natural aqueous systems could never be high enough to satisfy the cellular needs. On other hand, the first life forms could not accumulate sufficient amounts of phosphate since they had neither tight membranes nor phosphate-scavenging pumps. This conundrum has first been noted by Gulick (1955); in the following decades, several tentative solutions were put forward (see Schwartz (2006) for a review). Specifically, several authors have speculated that more reduced phosphorus-containing compounds such as hypophosphite

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(phosphinate, PO23) and/or phosphite (phosphonate, PO33), which have better solubility in water, could have been abundant under primordial reduced conditions (Gulick 1955; Glindemann et al. 1999; Buckel 2001; Bryant and Kee 2006; Schwartz 2006; Pasek 2008; see also Chap. 3). It is important that (hypo)phosphite ions can exergonically interact with diverse organic molecules in exergonic reactions yielding their phosphorylated derivates (Hagan 1996; Schwartz 2006; Bryant et al. 2010). Obviously, de-phosphorylation of such derivates could provide living organisms with energy. The idea of (hypo)phosphite-utilizing biochemistry is highly attractive; its plausibility, however, depends on finding a continuous primordial source of inorganic (hypo)phosphite. One suggestion is that the reduced oxidation state phosphorus originated from extraterrestrial material that fell from space and persisted in the mildly reducing atmosphere long enough to support the emergence of life (Pasek 2008). An alternative source of (hypo)phosphite is suggested by the recent finding of compatible amounts of phosphate and phosphite in the pristine geothermal pool at Hot Creek Gorge near Mammoth Lakes, California, which is fed by hot, bicarbonate-rich geothermal waters (Pech et al. 2009). The finding of phosphite in nature, on the one hand, explains why diverse prokaryotes possess systems of hypophosphite and phosphite oxidation (see (White and Metcalf 2007) for a review). On other hand, the presence of (hypo)phosphite in the geothermal waters of modern Earth strongly supports the possibility of its abundance in the geothermal fluids of the more reduced primordial Earth. Where may the phosphite of the Hot Creek Gorge have come from? The serpentinization-type reactions are not limited to the sea bed, but proceed within any ultramafic rock in the presence of water and at ~ 300 C (see (Sleep et al. 2004)), for a review of the continental reactions of hydrothermal alteration. There are many reports of hydrocarbon production by terrestrial rocks; in the case of the rocks of the Canadian Shield, at least, the abiogenic origin of these hydrocarbons was demonstrated (Sherwood Lollar et al. 2002). The standard redox potentials of CO2 reduction to formate and of phosphate reduction to phosphite have similar standard redox potentials of about 0.6 V. It is tempting to speculate that the hydrothermal rock alteration may account for the reduction of insoluble mineral phosphate to soluble (hypo)phosphite in geothermal fluids (Pech et al. 2009). Analogously, the high ammonia content in the hot springs of Kamchatka (that reach 130 mg/L in the mud pot solutions (Bortnikova et al. 2009)) might be due to the abiogenic reduction of dinitrogen to ammonia within the hot volcanic rocks. Hence, continuous fluxes of reducing equivalents, which were needed for the first syntheses, may have been provided by: (1) The inorganic photosynthesis; and (2) The redox reactions within hot iron-containing rocks. The only primordial environments where these fluxes could meet were the continental geothermal systems. On the one hand, the hot springs should have ejected transition metals that precipitated as photosynthetically active ZnS and MnS particles. On the other hand, the same hydrothermal fluids could be enriched in hydrogen and carbonaceous compounds. At high pressure of the primordial CO2 atmosphere, both the inorganic photosynthesis and the abiotic reduction of CO2 within hot rocks should have proceeded with higher yield than nowadays. The content of (hypo)phosphite and ammonia in the

1 Energetics of the First Life

11

H2S-enriched hydrothermal fluids should have been also higher on the anoxic primeval Earth, not to mention sulfur, which is also essential for life. It is noteworthy that the formamide molecules, which could serve as building blocks for amino acids and nucleotides, can build from formate and ammonia after the elimination of water; hence, formamide could concentrate in continental locations upon evaporation, thanks to its high boiling temperature (210 C). In sum, only at continental geothermal springs, the formation of organic nitrogen-, phosphor-, and sulfur-containing compounds could be supported by two different fluxes of reducing equivalents resulting from the abiotic photosynthesis and the hydrothermal alteration of the iron-containing rocks. In addition, only continental environments could be characterized by wet/dry cycles, favorable for condensation reactions (Bernal 1951; Miller and Orgel 1973; Lahav and Chang 1976; Lahav 1999; Bada 2004; Miller and Cleaves 2006), see also Chap. 12.

1.3

Energetics of Natural Selection

The countdown of life, as argued by many authors (Eigen 1971; Orgel 1986; Lazcano and Miller 1996; Bada 2004; Orgel 2004; Wolf and Koonin 2007; Orgel 2008; Koonin 2009; Lazcano 2010), started to run with the emergence of the first replicating systems. Since the modern replicating systems are based on nucleic acids, the earliest steps of evolution are usually considered in the framework of the “RNA World” scenario, according to which RNA-like molecules capable of both self-reproduction and simple metabolism were the first inhabitants of Earth (see (Belozersky 1959; Woese 1967; Crick 1968; Orgel 1968; Eigen 1971; Gilbert 1986; Benner et al. 1989; Ellington 1993; Spirin 2005; Koonin et al. 2006; Ellington et al. 2009; Lincoln and Joyce 2009; Powner et al. 2009) and Chaps. 9–11). This scenario is supported by the discovery of RNA enzymes (ribozymes) with different catalytic activities (see (Curtis and Bartel 2005; Chen et al. 2007; Joyce 2007; Scott 2007; Lincoln and Joyce 2009) and references therein), by the ability of RNA colonies to grow and propagate on gels or other solid media (Chetverin and Chetverina 2007), by the documented interactions of RNA molecules with metabolites (Shu and Guo 2003; Roth and Breaker 2009), in particular in the case of riboswitches (Gelfand et al. 1999; Cochrane and Strobel 2008), and by the demonstration of a RNA enzyme complex capable of self-replicating (Lincoln and Joyce 2009). The RNA world model implies a step-by-step emergence of increasingly complex RNA-like molecules. Some scholars have considered such a scenario to be implausible. The problem of increasing complexity, however, is not limited to the transition from simple organic molecules to the first replicating biopolymers. The evolutionary development from the simplest prokaryotes to mammals is accompanied by a comparable increase in complexity. This complexity increase, however, is not considered as improbable, but is taken for granted; it is routinely explained by the Darwin’s principle of natural selection (Darwin 1859; Smith and Szathma´ry 1995; Danchin 2009).

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The role of selection in the abiotic generation of increasingly complex macromolecules has been addressed by several authors who suggested that the higher stability of complex macromolecules, as compared to the simpler ones, could favour the relative accumulation of the former. It was speculated that the lower susceptibility of more complex polymer molecules to the lysis or hydrolysis might drive accumulation of increasingly complex polymers (Quastler 1964; de Duve 1987). This speculation might, with certain reservations, be applicable to proteins (see below), but not to the RNA-like oligomers. In the RNA oligomers, the binding of new monomers can proceed only at the two terminal positions, while hydrolysis can implicate any bond. Hence, the probability of hydrolysis should increase with the length and prevent the formation of long polymers (D. Cherepanov, personal communication, (see also (Mulkidjanian et al. 2003; Mulkidjanian 2009)). Therefore, it is unlikely that the first RNA-like polymers were selected as particularly resistant to hydrolysis. Why then have they been selected? In biology, it is often possible to identify the major selective factor(s) just by inspecting particular organisms. For example, the long ears, large eyes, and strong legs of hares indicate that the survival of their ancestors depended on their ability to catch sight of hunting predators and to run away as fast as possible. Elsewhere (Mulkidjanian et al. 2003), we applied the same naı¨ve rationale to polynucleotides and attempted to reconstruct the circumstances of their emergence from their specific traits. The exclusive feature of natural nucleobases is their unique photostability (Cadet and Vigny 1990). Since this trait is not related to the storage of genetic information, several authors (Skulachev 1969; Sagan 1973; Cadet and Vigny 1990) have noted that this property could have been of some use when the UV flux at the surface of primordial Earth – owing to the absence of the ozone layer – was much stronger than it is now. Nucleobases apparently can absorb excess energy quanta from sugar-phosphate moieties and protect them from photo-dissociation (Goossen and Kloosterboer 1978). This feature explains why the UV damage to the backbones even of modern RNA and DNA molecules is 103–104 times less frequent than the destruction of nucleobases proper (Cadet and Vigny 1990). Under the assumption that the unique photostability of nucleobases could hardly be incidental, we have argued that natural nucleotides should have been selected within the reach of solar light and that nucleobases may have protected the first RNA-like polymers from photodissociation (Mulkidjanian et al. 2003). More recently, the reason for the particularly high photostability of nucleotides and nucleobases has been clarified (Sobolewski and Domcke 1999; Marian 2005; Perun et al. 2005a, b; Merchan et al. 2006; Sobolewski and Domcke 2006; Frutos et al. 2007; Serrano-Andres and Merchan 2009). Two types of photochemical reaction paths, which lead to an extremely fast transition from the excited state into the ground state, have been identified, namely: (1) The torsion of certain C-N bonds of rings and (2) The de-protonation of azine or amino groups (Perun et al. 2005a; Sobolewski and Domcke 2006). It has been argued that the high photostability is not affected by the alkylation of nucleobases; such alkylated, mostly methylated, derivates (minor bases) are found in the structures of tRNA

1 Energetics of the First Life

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and rRNA (Nagaswamy et al. 2000). Together, major and minor nucleobases could represent the initial pool of primeval photostable compounds from which the major nucleobases were gradually selected by evolution. Important experimental evidence of the particular photostability of natural nucleotides has recently been provided by Sutherland and co-workers, who have obtained activated pyrimidine ribonucleotides from cyanamide, cyanoacetylene, glycolaldehyde, glyceraldehyde, and inorganic phosphate in a reaction that bypassed free ribose. The synthesis yielded activated ribonucleotide b-ribocytidine-20 ,30 cyclic phosphate (natural cytidine) as a major product as well as several co-products (Powner et al. 2009). Prolonged irradiation of this mixture by 254 nm light caused the destruction of various co-products and the partial conversion of ribonucleotide b-ribocytidine-20 ,30 -cyclic phosphate into ribonucleotide b-ribouridine-20 ,30 -cyclic phosphate (natural uridine). The authors concluded that there must be some (photo) protective mechanism functioning with natural nucleotides but not with other pyrimidine nucleosides and nucleotides (Powner et al. 2009). Admittedly, the least physically plausible step in the hypothetical RNA World scenario is the polymerization of the abiogenically produced nucleotides into oligomers, followed by their joining into complex systems capable of self-replication, with each oligomer involved being composed of 50–100 nucleotides, or more (Eigen 1971; Bada 2004; Lincoln and Joyce 2009). In modern organisms, the enzymes that synthesize RNA molecules are driven by hydrolysis of nucleoside triphosphates. In the RNA world, where these NTP-driven enzymes were absent, the polymerization could be catalyzed by mineral surfaces, as demonstrated in several systems, (see (Ferris 2006) and references therein). The polymerization may also have been promoted by the UV quanta, specifically in the presence of photoactive ZnS crystals (Senanayake and Idriss 2006; Liu et al. 2008). In addition, the aforementioned thermodynamically favourable oxidation of phosphite to phosphate could potentially provide free energy for polymerization reactions. And still, it is unclear whether these factors alone could have been sufficient for maintaining a notable steady-state population of RNA-like polymers – needed as a starting material for further evolutionary transformations. The results of our earlier Monte-Carlo simulations of primordial photochemistry (Mulkidjanian et al. 2003) suggest one more, rather paradoxical, way to channel external energy into the syntheses of increasingly complex compounds. The aim of the simulations was to quantify the significance of the UV-protection mechanism for the evolution of primordial RNA-like polymers by modelling the polymerization of sugar-phosphate monomers in the presence of nucleobases under varying conditions (Mulkidjanian et al. 2003; Mulkidjanian 2009). The simulation has shown that the ability of nucleobases to protect the adjoining sugar-phosphate units from UV-dissociation could alone result in a dramatic increase both in the length of formed polymers and in the fraction of nucleobase-carrying sugar-phosphate units, as compared to the simulations where no UV-protection was “switched on” (Mulkidjanian et al. 2003). This finding is not trivial since in both types of simulations, with the UV protection switched on and switched off respectively, the utilization of radiation

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energy for any kind of synthesis was not “permitted”. The fraction of complex polymers grew only owing to the breakage of less photostable molecules by the UV quanta. The broken pieces steadily re-arranged anew into more photostable and, accordingly, more complex polymers. Therefore – and this is the key point – the enrichment in more complex RNA-like polymers was driven by the photo-dissociation of their less stable counterparts, in the absence of any direct coupling between the energy flow and the synthetic reactions. From the experimental viewpoint, the results of this modelling might be related to the aforementioned enrichment of photostable natural nucleotides on account of non-natural nucleotides after prolonged UV illumination (Powner et al. 2009). From the theoretical viewpoint, the described mechanism of “indirect” energy utilization might be related to the so-called Landauer principle (Landauer 1961; Bennett and Landauer 1985; Bennett 2003). According to this principle, the creation of information requires energy in any case, but the energy can be utilized in two different ways: (1) For creating the information itself, and/or (2) For erasing the “garbage” from the system. More recently, Danchin has applied this principle to biological systems (Danchin 2009). Danchin describes a selection process that has to “actively discriminate between entities that are in some degree functional and those that cannot function. This is because the process needs to avoid destroying the elements that carry increased information and this is where energy comes in. Energy has to be consumed to make innovations stand out in a discriminant process; energy is used to prevent degradation of functional entities, permitting destruction of the nonfunctional ones” (quoted from (Danchin 2009)). This approach seems to be applicable to the primordial RNA-like polymers in illuminated environments, provided that we consider the more photostable constructs as “functional” ones and the solar UV radiation as the selecting and erasing energy input.1 Earlier, we have hypothesized that the excitonic interaction between the stacked nucleotides as well as their Watson-Crick pairing may have been favoured by higher photostability of the resulting structures in a UV-irradiated environment (Mulkidjanian et al. 2003; Mulkidjanian and Galperin 2007). This suggestion is built on the empirical evidence of the higher UV stability of double-stranded RNA samples as compared to single-stranded molecules (Bishop et al. 1967; Jagger

1 Incidentally, the interplay between supporting and erasing energy fluxes, which was disclosed during the aforementioned simulations (Mulkidjanian et al. 2003; Mulkidjanian 2009), should also persist on a higher, organismic level. Indeed, let us take a hare as an example. Via consumed plants, the hare is supported by solar energy. How can the “erasing” flux of solar energy affect the hare? The hare, of course, has a chance to dye of sunstroke. The probability of such indiscriminative elimination is, however, negligibly small. Much larger is the probability of encoutering a fox that (i) exploits a different flow of solar energy than the hare and (ii) can catch the hare, but only if the latter is not smart enough. Hence, one energy flow sustains the population of hares, while the other energy flux, which supports the foxes, cares for the fitness of hares. It is tempting to speculate that the interplay between supporting and erasing energy fluxes might represent the physical essence of biological evolution (see Lotka (1922); Darlington (1972); Danchin (2009) for related discussions).

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1976; Koonin et al. 1980). Recently, the physical background of the higher photostability of paired nucleotides has been clarified. For the nucleotides that form a Watson–Crick pair, the lifetime of the excited state has been estimated to be as low as a few femtoseconds (Abo-Riziq et al. 2005), i.e., ca. one hundred times shorter than that of single bases (Cadet and Vigny 1990; Cohen et al. 2004; CrespoHernandez et al. 2004, 2005; Satzger et al. 2006). This extremely short lifetime has been attributed to excited-state deactivation via electron-driven proton shuttling between the bases (Sobolewski et al. 2005; Perun et al. 2006; Frutos et al. 2007). It is noteworthy that other possible (non Watson–Crick) conformers of paired nucleobases have not shown these unique photochemical properties (Abo-Riziq et al. 2005). It has been suggested “that the biologically relevant Watson-Crick structures of GC and AT are distinguished by uniquely efficient excited-state deactivation mechanisms which maximize their photostability” (quoted from (Sobolewski and Domcke 2006)). Hence, the flux of solar energy may have supported the formation of RNA-like polymers in two complementary ways, namely (1) by driving fortuitous photopolymerization events, and (2) by supporting the relative enrichment of the photostable RNA-like polymers via the systematic photo-dissociation of more labile molecules. The minerals of the aforementioned ZnS-enriched precipitates around continental hot springs, besides catalyzing abiogenic photosynthesis of useful metabolites and serving as templates for the synthesis of longer biopolymers from simpler building blocks, could protect the first biopolymers from photo-dissociation by absorbing the excess radiation (Biondi et al. 2007; Mulkidjanian 2009). In addition, Zn2+ ions are known for their exclusive ability to catalyze the formation of the naturally occurring 30 –50 linkages upon abiogenic polymerization of nucleotides (Bridson and Orgel 1980; van Roode and Orgel 1980). The selection of first proteins may have differed from the selection of nucleotides. Proteins are synthesized by the RNA molecules of ribosomes (Steitz and Moore 2003). Recently, the ancient active center, supposedly capable of catalyzing peptide bond formation, have been identified within the large ribosomal subunit (see (Bokov and Steinberg 2009; Davidovich et al. 2009) and the chapter by Lankenau). Therefore, polypeptides could begin evolving only after the emergence of the first pro-ribosomes2. The exclusive property, which is common for proteins and distinguishes them from other random polymers, is the large energy gap between the energy of the most stable, native protein structure and the energy spectrum of other possible structures, e.g., misfolded or denatured states (Finkelstein and Ptitsyn 2002). In other words, proteins, unlike random polymers, can endure large thermal quanta without changing their conformation. Because the large part of their peptide bonds is tightly packed within the hydrophobic protein

2 These considerations do not rule out the possibility of abiotic peptide formation (see Chaps. 5–8 and 12). Abiogenically formed peptides may have paved the way to the biogenic protein synthesis by serving as nourishment for the first RNA-based organisms and by protecting them from different hazards (Mulkidjanian 2009).

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core, the folded proteins are more resistant to hydrolysis, cf. (de Duve 1987). Hydrolysis is driven by thermal energy, via the Brownian motion of water molecules. Hence, while the energy of light may have served as a selection force for the first abiogenically synthesized (poly)nucleotides, the proteins, after being produced by pro-ribosomes, may have been selected by thermal energy; this selection process could favour the accumulation of folded proteins on account of simpler, but unfolded polypeptides. It is tempting to speculate that one of the initial functions of proteins could be the protection of RNA-like polymers from overheating and denaturation (Mulkidjanian 2009). In summary, the unique photochemical properties of nucleotides and their natural polymers suggest the involvement of UV light as a selecting factor during the initial stages of evolution and point to illuminated habitats as the tentative cradles of life.

1.4

Compartmentalization and the Emergence of Membrane Bioenergetics

In modern prokaryotes, the energetics is intimately coupled with compartmentalization since the main energy-converting machinery is embedded in the cell membrane. This machinery consists of diverse enzyme complexes that can pump protons and/or sodium ions out of the cell yielding an electrochemical ion gradient across the membrane. This gradient can be used by the rotary membrane ATP synthases for synthesis of ATP (see (Cramer and Knaff 1990; Mulkidjanian et al. 2009) for reviews). In the vast majority of cases, including the animal mitochondria and the plant chloroplasts, the ATP synthases are driven by proton gradients; only in some prokaryotic organisms, the ATP synthases translocate sodium ions. Not surprisingly, several authors suggested that the very first organisms could utilize pH gradient for synthetic reactions, (see e.g., (Russell 2007; Lane et al. 2010)). This beguiling scheme, however, is not supported by phylogenomic analysis of the membrane rotary ATPases (Mulkidjanian et al. 2007, 2008a, 2008b; Mulkidjanian et al. 2009; Dibrova et al. 2010). This analysis is instrumental since the rotary ATPase was operative already in the LUCA (Gogarten et al. 1989; Mulkidjanian et al. 2007, 2008b) and is the only ubiquitous enzyme that is involved in membrane bioenergetics. The membrane ATP synthases are complex rotary machines that couple ATP synthesis and hydrolysis with the translocation of protons or sodium ions across the membrane, (see (Mulkidjanian et al. 2009) for a review). The direction of rotation depends on whether enzyme synthesizes ATP by using the ion gradient or, the other way around, translocates ions by hydrolyzing ATP. These ATP synthases/ATPases are present in all organisms and exist in distinct archaeal (A/V-type) and bacterial (F/N-type) versions (Hilario and Gogarten 1998; Dibrova et al. 2010). Both archaeal and bacterial ATPases can be proton- or sodium-translocators, respectively. Comparative analyses of the crystal structures of the ion-binding membrane

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subunits of Na+-translocating archaeal and bacterial ATPases (Meier et al. 2005; Murata et al. 2005) have shown that nearly identical sets of amino acid residues were involved in Na+ binding; these sets almost perfectly superimpose in space (Mulkidjanian et al. 2008b). Since a convergent emergence of the same set of Na+ ligands in several lineages looked extremely unlikely, the similarity of the Na+binding sites in the two prokaryotic domains led to the suggestion that the last common ancestor of the extant rotary ATPases possessed a Na+-binding site (Mulkidjanian et al. 2008b). The ion specificity of the rotary ATPases is decisive for the nature of the bioenergetic cycle in any organism. Although membrane ion gradients can be generated by a plethora of primary sodium or proton pumps, the rotary ATPases are unique in their ability to use these gradients for producing ATP (Cramer and Knaff 1990). Therefore, evolutionary primacy of the sodium-binding ATPase indicates the evolutionary primacy of the sodium-based bioenergetics. This evolutionary primacy could be explained by the higher resistance of lipid membranes to sodium ions as compared to protons (Deamer 1987; Nagle 1987; Konings 2006). As argued in more detail elsewhere (Mulkidjanian et al. 2008b, 2009), creating a nonleaky membrane that can hold a large proton potential, needed to drive the synthesis of ATP, is a harder task than making a sodium-tight membrane. The representatives of the three domains of life employ distinct solutions to make their membranes tighter to protons, (see (Haines 2001; Konings 2006; Mulkidjanian et al. 2008b) for details). This fact supports the suggestion on the independent transition from the sodium to proton bioenergetics in different lineages. This transition was accompanied by losses of different Na+-ligands in different lineages and happened after the separation of Bacteria from Archaea (Mulkidjanian et al. 2008b, 2009). Hence, the phylogenomic analysis (Mulkidjanian et al. 2007; Mulkidjanian and Galperin 2010a, b; Mulkidjanian et al. 2009; Dibrova et al. 2010) rules out the possibility that the first cells could rely on proton-dependent bioenergetics. The membrane proteins show a non-random distribution of hydrophobic and hydrophilic amino acid residues; hence, they should have emerged after the watersoluble proteins in which this distribution is quasi-random (Finkelstein and Ptitsyn 2002). Arguably, the emergence of modern, ion-tight membranes should have taken place relatively late, after the separation of Bacteria from Archaea (Mulkidjanian et al. 2008b, 2009), since the membranes of Archaea and Bacteria are fundamentally different (Pereto et al. 2004; Koonin and Martin 2005; Koga and Morii 2007). These and some other considerations have prompted the “lipids late” evolutionary scenarios (Koonin and Martin 2005; Koonin 2006; Mulkidjanian et al. 2009; Mulkidjanian and Galperin 2010a) where the advent and evolution of simple, virus-like, RNA/protein life forms preceded the emergence of lipid membranes3.

3 This suggestion does not contradict the possibility of abiotic formation of amphiphilic molecules, see the chapters by Egel and Sturgis in this volume. Such abiotically formed molecules, by serving as food for the first organisms, may have paved the way to the biogenic syntheses of lipids via reversion of the respective catabolic chains (Mulkidjanian 2009).

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The suggested late emergence of lipid membranes then posits the question of the compartmentalization of the first life forms. The separation of replicating entities, which nowadays is achieved by lipid membranes, is a condition of the biological evolution and should have taken place from its very beginning. The separation of the first life forms into discrete units could, however, proceed even without lipids. Russell and co-workers have hypothesized that the early stages of evolution may have taken place inside the “FeS bubbles” – the pores of the ironsulfide precipitates at the aforementioned deep-sea hydrothermal vents (Russell and Hall 1997; Martin and Russell 2003; Russell and Hall 2006). This “inorganic” solution of the compartmentalization problem could be applied also to the precipitates at the sites of continental geothermal activities (Mulkidjanian 2009; Mulkidjanian and Galperin 2009). Compartmentalized structures should have formed around terrestrial hot springs as well; they form around hot springs and fumaroles even in the modern volcanic systems (Kardanova 2009; Morgan et al. 2009). Such habitats could accommodate the first life forms; the affinity of the evolutionary old proteins to the Zn2+ ions (Mulkidjanian and Galperin 2010b, 2009) indicate that the first replicators may have been shaped within mineral compartments that were Zn-enriched. The replicating entities in each compartment could share a common pool of metabolites and genes, so that each interacting consortium would comprise a distinct evolutionary unit. Such a scheme, with an extensive (gene) exchange between the members of one consortium but not between different, mechanistically separated consortia solves a major conundrum between the notion of extensive gene mixing that is considered a major feature of early evolution (Woese 1998) and the requirement of separately evolving units as subjects of Darwinian selection (Koonin and Martin 2005; Mulkidjanian et al. 2009). The advantage of the sub-aerial ZnS-enriched porous precipitates, as compared to the FeS bubbles at the sea floor (Russell et al. 1988; Martin and Russell 2003; Russell 2007), is that the Zn ions are redox inert while the redox active Fe2+ ions and FeS clusters can generate harmful hydroxyl radicals and are therefore detrimental for the “naked” RNA molecules (Meares et al. 2003; Cohn et al. 2004; Luther and Rickard 2005; Cohn et al. 2006). In addition, the suggested scenarios of the emergence of life at the deep-sea hydrothermal vents (Russell et al. 1988; Martin and Russell 2003; Russell 2007; Martin et al. 2008) do not account for the exceptional photostability of natural nucleotides and their polymers. Generally, the central theme in the evolution of compartmentalization should have been the increasing tightness of cell envelopes. Szathma´ry and co-workers have recently developed and modeled a set of evolutionary scenarios that exemplified the crucial importance of the interaction and exchange between the primeval replicating entities for the stability of their populations (Szathma´ry 2006; Szathma´ry 2007; K€ onnyu˝ et al. 2008; Branciamore et al. 2009). According to Szathmary and co-workers, an increase in the complexity of pro-cells should have been accompanied by their progressive sequestering from the environment, so that the gradual build up of enzymatic pathways inside the pro-cells would be

1 Energetics of the First Life

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accompanied by decrease in membrane permeability, ultimately paving the way to the membrane bioenergetics that required ion-tight membranes. In sum, the intimate coupling between the membrane bioenergetics and the cellular compartmentalization, which is observed in modern prokaryotes, should have been absent during the first evolutionary steps. The first compartments inhabited by consortia of replicating entities may have been inorganic, they may have been built of ZnS-enriched sediments around continental hot springs. The membrane bioenergetics should have appeared much later, only after the emergence of ion-tight lipid membranes, with the sodium-dependent energetics appearing first and the proton-dependent energetics emerging last.

1.5

Evolution of the Early Energy-Converting Mechanisms

Figure 1.1 suggests how the early energy-converting mechanisms may have evolved. The scheme places the emergence of the first replicating entities within the ZnS-enriched, photosynthesizing precipitates around continental hot springs. As in the case of modern continental volcanic systems (Karpov and Naboko 1990; Kardanova 2009; Karpov et al. 2009), the Zn2+ ions of metalliferous fluids, being carried by geothermal waters, should ultimately precipitate as ZnS particles. Since ZnS is the slowest precipitating transition metal sulfide (Seewald and Seyfried 1990; Metz and Trefry 2000), the ZnS-enriched precipitates should fall out further off the geothermal springs and form interwoven “haloes” around them (Mulkidjanian and Galperin 2009). While carried by geothermal fluids, the precipitating ZnS particles should have mixed with the clay particles, the products of rock-weathering; in this case, compartmentalized clay-ZnS sediments may have formed. ZnS crystals, as powerful photocatalysts, may have photosynthesized diverse organic compounds from CO2 under the solar UV light. In addition, the hydrothemal fluids should have brought organic molecules synthesized within the hot rocks (Sleep et al. 2004). The organic compounds could become phosphorylated in the presence of hydrothermally derived (hypo)phosphite, yielding diverse phospho-derivates that could condensate into larger molecules, in particular, during occasional dry periods. Among a plethora of different carbonaceous molecules (see Chap. 2), the natural nucleotides could accumulate as the most photostable structures, their polymerization and folding into double-stranded segments should be favored by further increases in the photostability (Mulkidjanian et al. 2003; Sobolewski and Domcke 2006; Mulkidjanian 2009; Powner et al. 2009; SerranoAndres and Merchan 2009). Occasionally, some aggregates of photoselected RNA-like polymers may have shown the ability of self-replication. The multiplication of such replicators should have led to the depletion of the abiotically synthesized nucleotides. The competition for “building blocks” may have favoured the emergence of catalytic activities enabling the synthesis of nucleotides from the available simpler organic molecules by ribozymes. Ultimately, the broadening of the catalytic repertoire of ribozymes

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E, V surface electron trap Conduction band H 2S

S

2-

Primary electron acceptor

-2 +

Secondary electron acceptors

CO2+2H

-

NAD(P)+

HCOOH 0

e

H2S

e

-

NAD(P)H

2+

S+2H

+

S2

Visible light

+

S+2H Valenceband

UV light

2

10 nm

Photochemical reaction center

ZnS nanoparticle

H+-tight membranes

Diverse archaea

Sodium protobacteria Diverse bacteria

Sodium proto-archaea Na+-tight membranes

Emergence of the membrane bioenegetics Na+

ATP

LUCA?

ADP+P Na+

ATP

ADP+P

ancestors of the rotary ATPases Porous membranes

Fig. 1.1 The proposed scenario for the evolution of the early energy-converting mechanisms. The scheme suggests the emergence of the first replicating entities within ZnS-enriched precipitates around continental hot springs. Note that the ZnS particles (grey dots) precipitate farther than other transition metal sulfides (The picture is modified from Mulkidjanian and Galperin (2010a) and is based on data from Metz and Trefry (2000); Kelley et al. (2002); Russell (2006)). The evolution of membranes is shown as a transition from primitive, porous membranes that were leaky both to Na+ and H+ (dotted lines), via membranes that were Na+-tight but H+-leaky (dashed lines) to the modern-type membranes that are impermeable to both H+ and Na+ (solid lines). As the common

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could result in the synthesis of polymers other than ribozymes, proteins in the first line. As argued elsewhere (Mulkidjanian and Galperin 2007; Danchin 2009; Mulkidjanian 2009; Mulkidjanian and Galperin 2009), the enzymatic repertoire of the evolutionary oldest proteins indicates that the energetics of the first organisms was based on the reactions of phosphate group transfer. As argued by Watson and Milner-White (Watson and Milner-White 2002), the phosphatebinding “nests” are formed by the protein backbone and therefore could be present already in the very first polypeptides (see also Chap. 7). In the described system, ATP could be recycled not only chemically, via substrate level phosphorylation (i.e., by phosphate group transfer from the abiogenically produced phosphorylated compounds), but also photochemically, via the light-driven phosphorylation of ATP at mineral surfaces (see (Kritsky et al. 2007)). The honeycomb-like, ZnSenriched mineral compartments could provide, besides shelter and nourishment, an ample supply of reducing equivalents for biosyntheses. The ZnS precipitates would have attenuated the UV component of solar light, thus providing shade for the inhabitants of the deeper-lying compartments. Hence, a stratified system could be established with the illuminated external layers being involved in the “harvesting” of reduced organic compounds and the deeper, less productive, but more protected layers providing shelter for the first replicating entities in their pores (see Fig. 1.1). As argued elsewhere (Mulkidjanian 2009; Mulkidjanian and Galperin 2009), the ZnS-enriched precipitates should have formed large topologically connected fields around the hot springs. The displacement of life forms within these fields by water flows could ensure the exchange the spreading of the genetic material. The precipitation, proper, should have led to a continuous formation of new, empty compartments at the surface. The life forms should have thrived to propagate into these new compartments; otherwise, they could become buried and isolated from the sources of organic molecules. The porosity of the ZnS-enriched precipitates (Takai et al. 2001; Petersen et al. 2005) would have enabled some metabolite transport between the surface layers. Both the gradient of light and the interlayer metabolite exchange are typical of

ä Fig. 1.1 (continued) ancestor of the rotary membrane ATPases possessed a Na+-binding site (Mulkidjanian et al. 2008a, 2009), the LUCA, most likely, could possess sodium energetic (see Mulkidjanian et al. (2007, 2008b, 2009) for details). The emergence of the membrane energetic is shown as a reversion of the Na+-translocating ATPase in a sodium-rich sea water. The emergence of elaborated proton-tight membranes is shown to be followed by the rise of the (bacterio) chlorophyll-based photosynthesis among bacteria; the phototropic bacteria are shown in green (see main text, and Mulkidjanian et al. (2006); Mulkidjanian and Galperin (2009) for details). Insert: A comparison of energy diagrams for a photosynthesizing ZnS nanoparticle (left panel, see also Chap. 4 where the mechanism of semiconductor-mediated photosynthesis is discussed) and a Bacterial photochemical reaction center (right panel, a primitive, sulfide-oxidizing reaction center complex of green sulfur Bacteria is shown schematically as an example, see Frigaard and Bryant (2004); Jagannathan and Golbeck (2008) for reviews on this type of reaction centers) (The diagram is taken from Mulkidjanian and Galperin (2009))

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modern stratified, phototrophic communities (e.g., stromatolites (Nisbet 1991; Nisbet and Sleep 2001; Westall et al. 2006)). The next stage of evolution is envisaged as selection for increasingly tighter cellular envelopes, perhaps initially protein-made. Such envelopes may have prevented the uncontrolled escape of polymers, but still enabled the sharing of small molecules (Mulkidjanian et al. 2009). At this stage, the energetics consisted in the interplay between the delivery of abiogenically produced organic molecules (metabolites) at the sites of geothermal activity and the increasingly complex heterotrophy of the first organisms (see also (Lazcano and Miller 1999)). This heterotrophy may have been based on coupling the exergonic breakdown of (phosphorylated) metabolites with endergonic (bio)synthetic reactions. The same interplay of autotrophy (phototrophy and chemotrophy), on the one hand, and heterotrophy, on other hand, drives the majority of terrestrial communities today. The emergence of the first lipid membranes may have been coupled with the spreading of the life forms into the Fe-enriched habitats. Generally, the environments of the primordial Earth should have been enriched in well-soluble ions of Fe2+, the dominating transition metal ion of the primordial aqueous systems (with an estimated content of 106105 M (Williams and Frausto da Silva 2006; Anbar 2008)). Since lipids can prevent the damaging action of iron-containing minerals on RNA (Cohn et al. 2004), the need to protect polynucleotides from the Fe2+ ions may have driven the transition towards lipid-encased life forms. The isolation by lipid membranes should have prompted the emergence of enzymes that could extract reducing equivalents, needed for synthetic reactions, from inorganic electron donors such as H2 and H2S, and for the transportation of these reducing equivalents into the cells. First such enzymes, most likely, could use the FeS clusters, which are least structurally demanding (Watson and Milner-White 2002; Russell et al. 2007), as redox cofactors. Still, unless the lipid membranes became electrically tight, no coupling between the redox reactions and the synthesis of ATP via the membrane potential was possible. The very difference between the ionic compositions of cellular cytoplasm and of the seawater (Williams and Frausto da Silva 2006) implies that the cellular organisms could colonize the seawater only after attaining ion-tight membranes. Indeed, according to the principle of chemistry conservation (see e.g. (Macallum 1926; Mulkidjanian and Galperin 2007)), primordial cells would strive to keep their internal chemistry similar to the chemical compositions of the brine in which the first life forms had emerged. Since the cytoplasm of all cells contains more potassium than sodium, and the translation systems specifically require K+ ions for functioning (Bayley and Kushner 1964; Conway 1964; Spirin et al. 1988), the first life forms were likely to emerge in K+ -rich environments (Macallum 1926; Natochin 2007; Mulkidjanian 2009; Mulkidjanian and Galperin 2010a). Hence, besides striving to maintain a high internal Zn level, the first cells should also have been challenged by the high sodium content in the ocean. Therefore, the propagation of cells in sea water should have specifically required ion-tight membranes and ion pumps capable of ejecting Na+ ions out of the cell. As argued elsewhere (Mulkidjanian et al. 2009; Dibrova et al. 2010), the common ancestor of the rotary

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ATPases may have acted as an ATP-driven, outward Na+-pump. This enzyme complex, owing to its rotating scaffold, would be potentially able to translocate Na+ ions in both directions. At high ocean salinity, a reversal of the rotation would result in Na+-driven synthesis of ATP by this primordial rotary machine (see Fig. 1.1). Already in the Archean eon, the concentration of Na+ in the ocean water was approximately 1 M (DeRonde et al. 1997; Foriel et al. 2004; Pinti 2005), i.e., it was high enough for the rotary machine to switch from ATP hydrolysis to the ATP synthesis mode. This event marked the birth of membrane bioenergetics; together with the ancient outward Na+ pumps, the ancestral rotary ATP synthases would complete the first, sodium-dependent bioenergetic cycle in a cell membrane. Only after this stage, further membrane-embedded energy-converting systems, such as methanogenesis or anoxic respiration (Thauer et al. 1977; Thauer et al. 2008), could start to emerge. The final evolutionary step in the present scenario is envisaged as a transition(s) to the proton-tight, elaborate membranes that provided better protection to the cells. These membranes, in addition, were more lucrative from the point of view of energetics, since the proton transfer events could be chemically coupled to redox reactions, especially those of water and diverse quinones, thus enabling the advent of efficient redox-and light-driven generators of membrane potential (Mulkidjanian et al. 2008b, 2009; Mulkidjanian and Galperin 2010a). Since the membranes are fundamentally different in Archaea and Bacteria (Pereto et al. 2004; Koonin and Martin 2005; Koga and Morii 2007), it is tempting to speculate that the separation of the two domains might have been coupled with spreading of the two branches of the LUCA’s descendants, perhaps, differing in their habitats and metabolic strategies, away from the ZnS-enriched habitats (Mulkidjanian and Galperin 2009). The decreased dependence on the ZnS-mediated photosynthesis may also have paved the way to the emergence of the (bacterio)chlorophyll-based photosynthesis. Figure 1.1 (insert) illustrates the similarity between physical mechanisms of the (bacterio)-chlorophyll-based and ZnS-based photosyntheses, which both include light-induced charge separation followed by energy loss needed for stabilization of reduced states (see also (Halmann et al. 1980; Mulkidjanian 2009; Mulkidjanian and Galperin 2009) and Chap. 4). This figure also shows that the same reaction of sulfide oxidation could be used to re-fill the photo-generated electron vacancies (holes). Hence, the emergence of biogenic photosynthesis might represent a clearcut case of a functional takeover – with the primeval photochemical reaction centers and primordial Calvin cycle together accomplishing the function that the ZnS-enriched precipitates could perform alone, namely the utilization of solar energy for fixation of CO2. After the ancestors of cyanobacteria attained the ability to use the solar light for extracting electrons from water (Mulkidjanian et al. 2006), oxygen, the byproduct of this reaction, shifted the redox balance of the biosphere. This development led to the emergence of increasingly complex organisms, striving to decrease the oxidative damage to their cells (Broda 1975; Shnol 1981; Skulachev 1998; Williams and Frausto da Silva 2006). The consideration of these relatively recent evolutionary events is outside the scope of this volume.

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Outlook

Here, I have considered the natural energy fluxes that could be involved in sustaining the first life forms. Apparently, solar light should have played the major role. Rather counter-intuitively, the gradients of thermal energy could also be supportive. They may have been involved in the formation of hydrogen and carbonaceous compounds within the hot, iron-containing rocks, as well as in the selection of the folded, thermostable proteins. The evolution of biological energy conversion may have started from the selection of RNA-like polymers that could promptly get rid of the UV quanta or, in other words, “process” these quanta without undergoing photodamage. The ability to discard the energy of light is less structurally demanding than the ability to store and exploit energy; so, this ability may have been inherent already in simple organic molecules. The energy requirements of the first replicators could have been covered by the breakdown of “nutrients” that were produced in abiogenic reactions. Only the emergence of biogenic dielectrics, namely proteins and lipid membranes, enabled the storage of the energy of light in the form of charge-separated states, physically analogous to those involved in the abiotic, ZnS-mediated photosynthesis. As the accessible assets of reducing power ran out with the oxygenation of the atmosphere, living organisms had to increase their complexity, if they wanted to access and exploit new energy sources. The common energetic denominator of living organisms – throughout the whole evolutionary time span – may have been not the ability to exploit high-energy quanta, which could not be inherent in the simplest life forms, but the ability to “deal” with such quanta – in a general sense. From this viewpoint, one can follow a continuous evolutionary thread from the emergence of life to the modern struggle of mankind for energy. Acknowledgements Valuable discussions with Drs. A.V. Bogachev, A.Y. Bychkov, D.A. Cherepanov, M. Eigen, M.Y. Galperin, E.V. Koonin, K.S. Makarova, M.J. Russell, V.P. Skulachev, R. Thauer, N.E. Voskoboynikova, and R.J.P. Williams are greatly appreciated. While editing this volume I have learned a lot from my co-editors, Drs. R. Egel and D. Lankenau, as well as from all the authors of this book. This study was supported by the Deutsche Forschungsgemeinschaft and the Russian Government.

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Part I

Primeval Syntheses

Chapter 2

A Hypothesis for a Unified Mechanism of Formation and Enantioenrichment of Polyols and Aldaric, Aldonic, Amino, Hydroxy and Sugar Acids in Carbonaceous Chondrites H. James Cleaves II

Abstract Carbonaceous chondrites are a type of meteorite with a high organic content, which formed early in the history of the solar system. In addition to large amounts of insoluble high molecular weight organic polymer, they contain a variety of small organic molecules such as polycyclic aromatic hydrocarbons, polyols and aldaric, aldonic, amino, hydroxy and sugar acids, among others. Intriguingly, some of the amino acids and hydroxy acids have been found to display a chiral bias. The types of compounds present, their isotopic distributions and relative abundances may provide clues to the mechanisms by which they were synthesized in meteorite parent bodies. A unified set of mechanisms which may explain some of these properties is presented here. It involves simple addition reactions of cosmically abundant 1-carbon compounds such as HCHO and HCN undergoing wellestablished aqueous chemistry. Although aqueous processes could amplify and pass a chiral bias from one type of compound to another, circularly polarized light or another pre-accretion mechanism may have been required to initiate the chiral bias observed. Most of the small organic molecules present in these meteorites (90 to > 99% by number) to date remain unidentified; these mechanisms may provide a framework for identifying them.

Abbreviations a-AIB CCs DAMN DHA

a-aminoisobutyric acid carbonaceous chondrites diaminomaleonitrile dihydroxyacetone

H.J. Cleaves II (*) Geophysical Laboratory, The Carnegie Institution of Washington, 5251 Broad Branch Rd. NW, Washington, DC 20015, USA e-mail: [email protected] R. Egel et al. (eds.), Origins of Life: The Primal Self-Organization, DOI 10.1007/978-3-642-21625-1_2, # Springer-Verlag Berlin Heidelberg 2011

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a-HMA a-HMG a-HMS HMT MEEs

2.1

a-hydroxymethylalanine a-hydroxymethylglyceric acid a-hydroxymethyl serine hexamethylenetetramine methylene ether esters

Introduction

Carbonaceous chondrites (CCs) contain an enormous variety of organic compounds, many of biological interest (Botta and Bada 2002; Pizzarello et al. 2006; Sephton 2002). It is likely that significant amounts of extraterrestrial organic material were delivered to the early Earth before the origin of life (Chyba and Sagan 1992). CCs may be good analogs for the prebiotic inventory, and this may be more likely to be true if the Earth’s early atmosphere was not reducing (Kasting 1993), as atmospheric organic synthesis appears to be less robust in neutral or oxidizing atmospheres (Miller and Schlesinger 1984). However, they may be even better analogs if the early atmosphere was reducing, given the correspondence between the products of Miller-Urey type experiments (Miller 1953) using reduced gas mixtures and the organics identified to date in CCs (Ring et al. 1972; Wolman et al. 1972). CCs remain the most abundant natural examples of abiotic organic chemistry we have available for laboratory study.

2.1.1

Organics in CCs

One CC, the Murchison meteorite, has been especially well studied, partly due to its high carbon content, and partly due to the large amount of sample collected and curated. A recent investigation of the Murchison meteorite using high resolution mass spectrometry (Schmitt-Kopplin et al. 2010) found tens of thousands of organic compounds with distinct molecular formulas, and estimated based on typical organic structural isomerism that the true number of compounds could be on the order of several millions. However, to date only some 500 distinct compounds have been conclusively identified (Table 2.1). Clearly, the vast majority of compounds present in these bodies remain to be identified. CCs are highly variable in their organic content (Table 2.2) both from meteorite and even spatially within individual meteorites, a fact that suggests they have complicated histories. Soluble organic matter is typically 1–30% of the total organic material in CCs, the remainder (70–99%) being insoluble organic material (Pizzarello et al. 2006). Coincidentally, this is similar to the fraction of the organic products of Miller-Urey type electric discharge experiments rendered as “tar” (Miller 1953). There is also a striking correspondence between the amino acid contents of both types of sample

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Table 2.1 Partial list of the organic compounds identified in the Murchison meteorite (adapted from Pizzarello et al. 2006) Class Concentration (ppm) Number of compounds identified Aliphatic hydrocarbons >35 140 Aromatic hydrocarbons 15–28 87 Polar hydrocarbons <120 10 Carboxylic acids >300 48 Amino acids 60 74 Hydroxy acids 15 7 Dicarboxylic acids >30 17 Dicarboximides >50 2 Pyridine carboxylic acids >7 7 Sulfonic acids 67 4 Phosphonic acids 2 4 N-heterocycles 7 31 Amines 13 20 Polyols 30 19

Table 2.2 Comparison of the organic contents of three representative CC types (adapted from Alexander et al. 2007) Type Name Organic content % Location found CM Murchison 0.83 Australia CR GRA95229 0.68 Antarctica CI Orgueil 2.00 France Carbonaceous chondrites are grouped according to their similarity to a characteristic type specimen. CI (Ivuna type) CCs have experienced a high degree of aqueous alteration and are highly oxidized. They are possibly the most “primitive” of the CCs. Most CM (Mighei type) CCs have also experienced extensive aqueous alteration. CR (Renazzo type) CCs are rich in metallic Fe-Ni, and many, but not all, have experienced extensive aqueous alteration. For a more extensive discussion of CC classification and mineralogy, see Lauretta and McSween (2006)

with respect to type and abundance, which is perhaps less coincidental if the products of both were formed by similar chemical mechanisms (Peltzer et al. 1984; Ring et al. 1972; Wolman et al. 1972). CCs are remnants of the formation of the early solar system, which occurred ~4.5 billion years ago. These meteorites likely formed when various types of dust and small grains present in the early solar system accreted to form primitive asteroids. Over this long time interval, they have been processed, for example, by thermal, aqueous and radioactive alteration (Cody et al. 2008), which has modified their original organic contents. If the delivery of CC type organics was important for the origin of life, the original (hypothetical but deductable) inventory is perhaps of more interest than the present one. Laboratory models good approaches to this problem. A variety of sources likely contributed to the synthesis of the organics in CCs. Based on mineralogical evidence (for example, the presence of hydrous minerals such as phyllosilicates; see for example Lauretta and McSween (2006)), the parent

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bodies of CCs appear to have undergone a period of relatively low temperature aqueous processing, somewhere below 100 C and at a pH between 7 and 12 (Anders 1991; Cody et al. 2008; Ehrenfreund and Cami 2010; Lauretta and McSween 2006; Pizzarello 2004, 2007), which may have lasted for as long as several hundred or several thousand years (Browning et al. 1996). This may have been the period in which precursor molecules collected from the solar nebula, such as NH3, HCN and HCHO, reacted to form the more complex species observed in CCs, for example by forming amino acids via the Strecker synthesis (Cronin et al. 1995).

2.1.2

Chiral Biases in CC Organics

It is becoming increasingly accepted that some of these organic compounds, in particular non-protein amino acids (Glavin and Dworkin 2009; Pizzarello et al. 2008) and a-hydroxy acids (Pizzarello et al. 2010), are enantiomerically enriched. Even more intriguingly, in the case of some of the non-biological amino acids, such as isovaline, and a-hydroxy acids, this enrichment is often, but not always, in the preferred (or L-) biological orientation (Glavin and Dworkin 2009; Pizzarello et al. 2010). Some reported enantiomeric enrichments of organic compounds reported in CCs are shown in Table 2.3. The origin of biological homochirality has attracted much speculation (Bonner 1995; Meierhenrich 2008; Meierhenrich and Thiemann 2004; Popa 1997), and remains difficult to explain. Since many of the compounds in CCs which display enantiomeric enrichments are not common biochemicals and have isotopic enrichments atypical of terrestrial biological organic compounds (Pizzarello et al. 2006), the observed chiral bias is likely indigenous. Since the bias is in the same direction as that observed in biology, it has been speculated that extraterrestrial processes may have contributed to or determined biology’s choice of molecular handedness (Bonner 1999; Glavin and Dworkin 2009; Weber and Pizzarello 2006). Furthermore, since some of these compounds racemize extremely slowly (Pollock et al. 1975), it is possible that these chiral biases were created during their formation Table 2.3 Some reported organic molecules from CCs which display enantioenrichment Compound Meteorite Enantiomeric enrichment (%) Reference Cronin and Pizzarello (1997) a-Methylnorvaline Murchison L (2.8) Isovaline Murchison L (15.2 0.2) Pizzarello et al. (2003) L (15.2 4.0) Glavin and Dworkin (2009) Isovaline Orgueil Isovaline Murchison L (18.5 2.6) Glavin and Dworkin (2009) Isovaline LEW90500 L (3.3 1.8) Glavin and Dworkin (2009) Pizzarello et al. (2008) Isoleucine GRA95229 L (14 0.8) Lactic acid Murchison L (6.1) Pizzarello et al. (2010) Pizzarello et al. (2010) Lactic acid GRA95229 L (2.9) Pizzarello et al. (2010) Lactic acid LAP02342 L (5.2)

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during low temperature aqueous processing (Glavin and Dworkin 2009). It has also been suggested that the bias was introduced prior to accretion, for example, by circularly polarized light in the solar nebula (Pizzarello et al. 2008), and perhaps amplified during aqueous alteration (Glavin and Dworkin 2009). It is also possible that the bias was introduced after low temperature aqueous alteration.

2.1.3

Mechanism of Formation of CC Organics

The mechanism of formation under postulated parent body conditions of the polyols and amino and hydroxy acids detected in CCs is an experimentally tractable question, and synthesis and isotopic and enantiomeric enrichments may be mechanistically intertwined. This discussion will focus on hydroxy acids and polyols, but the general scheme is also applicable to amino acids. It has been suggested that the formose reaction (Breslow 1959; Butlerow 1861) was the mechanism by which the sugar acids and polyols observed in CCs were formed (Cooper et al. 2001). Higher polyols which contain chiral centers, however, are not especially abundant in CCs, and their enantiomeric enrichment, if any, has not been reported. Hydroxy acids in CCs have been investigated more extensively (Cronin et al. 1995; Cronin et al. 1993; Peltzer et al. 1984; Pizzarello et al. 2010), and generally display the same enantioenrichment as the amino acids (Pizzarello et al. 2010). Some of the hydroxy acids detected in three CCs in a recent report are shown in Fig. 2.1. Some of the detected polyols and sugar, hydroxy and amino acids and their concentrations in the Murchison meteorite as a function of the number of carbon atoms in the molecule are shown in Fig. 2.2. The yield of compounds within a class generally drops off as a function of the number of carbon atoms in the molecule, suggesting synthesis from smaller, likely 1-carbon, compounds. The especially abundant small polyols (ethylene glycol and glycerol) would require reduction of a parent sugar if the formose reaction were the ultimate source of these compounds, while the sugar acids would require oxidation of a parent sugar. The excess of glycerol over glyceric acid, and of ethylene glycol over glycolic acid, argues against, but does not exclude an oxidative mechanism. The rather low abundance of dihydroxyacetone (DHA) and the absence of glyceraldehyde, which would be expected to be in equilibrium with DHA, and glycolaldehyde also argue against a formose mechanism for these molecules’ synthesis. Modification of formose sugars may thus not be the most parsimonious explanation for the observed distribution of products. D/L isomerism in a-amino and a-hydroxy acids is defined by convention relative to the configuration of the triose glyceraldehyde. The stereochemistry of some related D- and L-amino and hydroxy acids is shown in Fig. 2.3. As mentioned above, it has been suggested based on the decreasing abundance of various small molecules with increasing carbon number in CCs that they were formed from 1-carbon precursors. This is also suggested by 13C isotopic enrichment

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Fig. 2.1 Hydroxy acids detected in CCs by Pizzarello et al. (2010). The cognate amino acids are shown in parentheses, and the measured direction of enantiomeric enrichment is shown in parentheses with an asterisk. In general the enantioenrichment is in the L direction and rather small (see Table 2.3)

patterns (Cronin et al. 1995; Kerridge 1994; Yuen et al. 1984). Figure 2.4 shows some isotopic trends for hydroxy acids and polyols measured in the Murchison and GRA95229 CCs. There is a general decreasing isotopic enrichment trend for linear isomers (solid lines in Fig. 2.4), that is glycolic > lactic > a-hydroxybutyric > a-hydroxypentanoic (data for a-hydroxypentanoic were not reported from GRA95229), which suggests a common mechanism of formation. There is an almost identical slope of the deuterium enrichment of the a,a-dialkyl isomers from lactic > a-methyllactic acid > a-hydroxy-a-methylbutyric acids (dashed lines), and an almost parallel correspondence of the isotopic trend for a-hydroxybutyric to a-hydroxy-a-methylbutyric acids, despite a generally consistent offset in isotopic values (dotted lines). The isotopic measurement for the bulk neutral polyols measured in Murchison by Cooper et al. (2001), a fraction of

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Fig. 2.2 Polyols and related compounds detected in the Murchison meteorite as estimated from the data presented in Cooper et al. (2001) (blue diamonds). Also shown are a-hydroxy acids reported in Pizzarello et al. (2010) (open squares) and a-amino acids as reported in Ehrenfreund et al. (2001) (red circles). aAIB a-amino isobutyric acid, aABA DL-a-aminobutyric acid

Fig. 2.3 The relationship between D/L and R/S stereoisomerism in serine, alanine, glyceric and lactic acids

unknown average molecular weight, suggests it is composed of an average C5-C6 size range, or a difference in deuterium exchange rates and equilibria. The offset but essentially identical pattern of D-enrichment shown by Murchison and GRA952229 compounds makes a very strong argument for similar mechanisms of formation from precursors of the same type but differing initial isotopic composition, or processing via the same mechanisms in water with a different D-enrichment. It has been argued that meteorite organics are largely derived from formaldehyde (HCHO), a reactive 1-carbon compound (Cody et al. 2009). However, another likely precursor, which does not exclude HCHO from playing a role, is HCN.

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Fig. 2.4 The carbon (left) and deuterium (right) isotopic distribution of the hydroxy acids measured in the Murchison (blue data points) and GRA95229 (red data points) CCs by Pizzarello et al. (2010). Note the correspondence of the general patterns of trends in both isotope systems, that is a general decrease in enrichment in higher linear isomers, and an increase in enrichment in branched isomers, which then again decreases with increasing number of carbon atoms. The 13C isotopic measurement of the bulk neutral polyol fraction from Murchison by Cooper et al. (2001) is an average but is consistent with a common synthetic precursor for both types of compound. In the deuterium plot (right), the isotopic composition of hydroxy acids as a function of the ratio of sp2 to sp3 hybridized carbon is shown (data from Pizzarello et al. 2010). This would be significant if HCN is the source of the carboxyl groups, and HCHO the source of the other carbon atoms in each molecule, and the starting HCN and HCHO pools have different initial isotopic compositions. A plot of the deuterium abundance vs. carbon number is essentially topologically identical. Note that the numbering of the structures shown in the left panel is used again on the right

HCHO has been detected in significant concentrations in CCs (Breger et al. 1972), and HCHO and HCN are both abundant interstellar molecules (Ehrenfreund and Cami 2010), components of comets (Anders 1991; Chyba et al. 1990; Oro et al. 1992), and likely components of the material that was incorporated into CCs when they formed (Anders 1991). They are also among the very few 1-carbon compounds that are reactive enough to form carbon-carbon bonds under the conditions of pressure and temperature thought to have prevailed during parent body aqueous alteration.

2.2

A Possible Unified Mechanism for the Formation of CC Organics

The point of this discussion is to explore whether experimentally testable mechanisms can reproduce the observed trends in molecular diversity, chirality and isotopic enrichment in CC organic compounds. Using these various constraints (parent body conditions, types of synthetic precursors), a mechanism for the formation of the structural diversity seen in CC organic compounds is proposed which may also provide a mechanism for chiral amplification.

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The starting point for this could be glycolic acid, which is readily derived from a cyanohydrin synthesis (Peltzer et al. 1984; Schlesinger and Miller 1973) (Fig. 2.5). Glycolic acid is a major product of the UV irradiation of pre-cometary ice analogs (Nuevo et al. 2010) and electric discharge reactions (Miller 1957) and is also quite abundant in CCs (Pizzarello et al. 2010) (see Fig. 2.2), demonstrating the likely ease and plausibility of this mechanism. Glycolonitrile and its amide and acid may undergo HCHO addition in a manner reminiscent of the malonic ester synthesis, due to the acidity of the protons bonded to the a-carbon atom (C2 in Fig. 2.5) and which has been demonstrated for glycine and its nitrile (Akabori et al. 1956; Ivanov and Ivanov 1983; Subbaraman et al. 1975) (Fig. 2.6). This would produce a carbanion which can attack HCHO to form racemic glyceronitrile, which hydrolyzes to give racemic glyceric acid, which is abundant in CCs (Cooper et al. 2001) and UV irradiation experiments mimicking precometary ices (Nuevo et al. 2010). Minerals are known to catalyze the decarboxylation of various a-hydroxy acids (Hoa et al. 2009), which with glyceric acid would yield ethylene glycol, which is again abundant in the Murchison meteorite (Cooper et al. 2001). A second addition of HCHO to glyceric acid derivatives (the parent nitrile and amide) via a similar mechanism would yield a-hydroxymethylglyceric acid (a-HMG) derivatives (Fig. 2.7), which have not been reported in CCs. a-HMG is a rarely reported molecule in any context, suggesting that it has either been deemed uninteresting, or is unstable. It is suggested here that it is unstable and

Fig. 2.5 Formation of glycolonitrile from HCHO and HCN. Note the C1 atom is sp2 hybridized while the C2 atom is sp3 hybridized, owing to the different oxidation states of the precursors (HCN and HCHO, respectively). The 13C enrichment of the starting HCN and HCHO pools could be quite different, with HCN more enriched

Fig. 2.6 Proposed addition of HCHO to glycolic acid derivatives to give glyceric acid derivatives, and the formation of ethylene glycol by decarboxylation of glyceric acid

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Fig. 2.7 Proposed conversion of glyceric acid to a-hydroxymethylglyceric acid and finally glycerol

Fig. 2.8 Proposed mechanism for the synthesis of lactic acid and alanine from glyceronitrile. The potential significance of this mechanism is it does not depend on the availability of acetaldehyde, a 2-carbon compound, as a precursor for lactate or alanine via the Strecker-cyanohydrin mechanism

especially given to decarboxylation, which would yield glycerol as shown in Fig. 2.7, and which is more abundant than the most abundant amino acids in the Murchison meteorite (Cooper et al. 2001) (see Fig. 2.2), and which is also a product of the UV irradiation of pre-cometary ice analogs (Nuevo et al. 2010). Glyceric acid readily decomposes under basic conditions to give lactic acid (Albert and Upson 1935; Gaud 1894), which is very stable under basic conditions (Abelson 1957) and unusually abundant in CCs (Pizzarello et al. 2010) (see Fig. 2.2). The cognate reaction with serine, which is among the most unstable of the biological amino acids (Abelson 1957) and rarely identified in CCs (Pizzarello et al. 2008), would give alanine (Bada et al. 1982) (Fig. 2.8). Unfortunately, in both cases it is unclear what the hydride donor would be, but the chemistry appears to be robust. The half life for the conversion of serine to alanine has been estimated at approximately 1 month at 100 C near neutral pH (Bada et al. 1982).

2.2.1

Sugars in CCs and the “Miller Paradox”

It has been suggested that the polyhydroxy acids and polyols detected in Murchison may be derived from the oxidation of formose sugars (Cooper et al. 2001). The synthesis of these compounds and hydroxy acids observed in CCs from sugars may

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however be problematic for three well-understood chemical reasons: the formation of hexamethylene tetramine (HMT), the formation of glycolonitrile and the occurrence of the Maillard reaction. While HCHO condenses to give sugars under basic conditions and in the presence of minerals (Breslow 1959; Butlerow 1861; Reid and Orgel 1967; Ricardo et al. 2004; Schwartz and Degraaf 1993), criteria that appear to have been met on the parent bodies of CCs (Lauretta and McSween 2006), it is also known that HCHO reacts rapidly and almost quantitatively with ammonia (NH3) to give hexamethylene tetramine (HMT) (Walker 1964). Recent measurements of NH3 in CCs (Pizzarello and Holmes 2009) suggest that it was surprisingly abundant, and observations of cometary comae suggest that NH3 is often as abundant as HCHO in the materials that likely formed coevally with CCs (Festou et al. 2004). HCHO would thus have to have been present in significant excess over NH3 for formose chemistry to occur in CC parent bodies. Although HCN is generally less abundant in cometary comae than HCHO or NH3 (Festou et al. 2004), it also reacts quantitatively with HCHO under a wide range of pH and concentration conditions to give glycolonitrile (Schlesinger and Miller 1973), as noted above. In the context of the RNA World model for the origin of life, which supposes that RNA was spontaneously generated in primitive environments (Gesteland et al. 1999), this has been dubbed the “Miller paradox” (Arrhenius et al. 1994), as it makes the synthesis of the nitrogenous bases (such as adenine, guanine and uracil, which are products of concentrated HCN solutions (Levy et al. 1999; Oro and Kimball 1961)) and sugars, specifically ribose, in the same location problematic. Purines among other nitrogenous bases have been detected in CCs (van der Velden and Schwartz 1977) and appear to be indigenous based on isotopic measurements (Martins et al. 2008, 2009). The third barrier to the formation of sugars in CCs is the Maillard or browning reaction, the reaction of amines with reducing sugars (sugars containing ketone or aldehyde groups) (Waller et al. 1983). This reaction occurs readily from almost any amine, including ammonia and amino acids, and any sugar, including glycolaldehyde, and generates a similar though variable set of nitrogen and oxygen heterocycles (Waller et al. 1983). While a variety of nitrogen heterocycles have been detected in CCs (Stoks and Schwartz 1981), they do not appear to be the ones expected from Maillard chemistry, and Maillard chemistry would likely rapidly consume nascent sugars before they could elongate via the formose reaction given the large amounts of ammonia and amines suggested to have been initially present in these meteorites (Peltzer et al. 1984; Pizzarello and Holmes 2009). Indeed, the only true sugar detected to date is DHA which is remarkable given its notorious instability (Riddle and Lorenz 1973). Given the large amounts of glycerol detected, it is possible that DHA is generated by oxidation during sample workup and analysis. The suggestion then that the aldonic (sugar acids bearing one terminal carboxyl group) and aldaric (sugar acids bearing two terminal carboxyl groups) acids detected in Murchison are derived from oxidized sugars (Cooper et al. 2001) is thus problematic. There are two possible resolutions to this apparent paradox: first,

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Fig. 2.9 Yield of insoluble HCN polymer from the room temperature reaction of 1 M NH4CN and various amounts of HCHO for 6 months (mole fraction HCHO ¼ (HCHO)/ (HCHO + NH3 + HCN)). While the yield of polymer drops dramatically with increasing mole fraction of HCHO, polymer formation is apparently not completely inhibited

HCN and HCHO oligomerization can in fact occur simultaneously, through various reversible and irreversible reactions, or second, the aldaric and aldonic acids detected are derived from other mechanisms. To test the first possibility, mixed reactions of 1 M NH4CN with variable amounts of HCHO (0–2 M) were prepared. HCN self-polymerization in water generates an insoluble polymer which is known to be a source of purines (Borquez et al. 2005); however, as mentioned above, HCHO effectively removes HCN from the reaction pool via the formation of glycolonitrile: while small amounts of HCHO actually accelerate HCN oligomerization when HCN is in excess (Schwartz and Goverde 1982), when HCHO is in excess HCN polymerization should not occur. In these simple experiments, after 6 months of reaction at room temperature reactions were simply centrifuged, and the insoluble fraction recovered and assayed for its mass and elemental composition. The mass of insoluble polymer recovered as a function of the concentration of HCHO is shown in Fig. 2.9. Interestingly, the insoluble polymer recovered in all cases has a close to identical elemental composition suggesting it is the same insoluble polymer formed from pure aqueous HCN. This suggests that HCN polymer can still be formed in the presence of an excess of HCHO over HCN, perhaps through reversible formation of glycolonitrile, among other intermediates, and removal of the polymer from the reaction scheme due to its insolubility. This suggests that HCHO is not an impediment to the formation of compounds which are precursors to purines from HCN. Whether the formation of sugars can still occur in the presence of an excess of NH4CN remains to be determined. However, the synthesis of ethylene glycol, glycerol, lactate and glycerate starting from glycolic acid discussed above may provide another explanation.

2.3

Possible Origin of Chiral and Structural Biases

Hydroxy acids, such as glyceric acid, react with HCHO to form cyclic methylene ether-esters (MEEs), and formals in the case of molecules containing multiple alcohol functionalities (Walker 1964) (Fig. 2.10).

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Fig. 2.10 Formation of glyceric acid MEEs (top two products) and glyceric acid formal (bottom) from HCHO and D-glyceric acid. Starting from L-glyceric acid, the opposite configuration would be obtained, the important point being that the stereochemistry may be retained in the product

Fig. 2.11 Reaction of D-glyceric acid MEEs with HCHO to give 4-carbon aldonic acids. DErythronic acid synthesis with retention of configuration should be favored if steric effects direct HCHO addition. L-Glyceric acid would preferentially make L-erythronic acid

HCHO addition to deprotonated glyceric MEEs could yield the 4-carbon aldonic acids, erythronic and threonic acids, as well as branched chain products (Fig. 2.11). Importantly, here the stereochemistry of the starting material could begin to influence the stereochemistry of the product due to steric effects, with the configuration of the a-hydroxyl group favoring addition of HCHO from the opposite side of the ring. The 4-carbon aldonic acids readily form five-membered lactones in solution. In addition to deprotonation of the a-carbons of these, which would lead to the synthesis of a-hydroxymethyl derivatives as discussed earlier, the d-carbons of these may be more easily deprotonated to form carbanions which can undergo further analogous HCHO addition to give the 5-carbon aldonic acids, ribulonic, xylonic, lyxonic and arabinonic acids, again with retention of configuration around the a-carbon, and preferential stereochemical addition at the d-carbon (Fig. 2.12). 5-Carbon aldonic acids also readily cyclize to give five-membered lactones which similarly could add HCHO to give 6-carbon aldonic lactones. Hydrolytic ring-opening and decarboxylation of the 4- to 6-carbon branched-chain aldonic acids would, analogously to the production of glycerol from a-HMG, give straight chain alditols, which are observed in CCs (Cooper et al. 2001) (Fig. 2.13).

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Fig. 2.12 Proposed mechanism for the formation of 5-carbon aldonic lactones from 4-carbon aldonic lactone, L-threonic lactone. L-threonic acid should thus produce L-5-carbon aldonic acids, and D-threonic should yield D-aldonic acids. The Greek letter ordering system for the ring carbons is shown

Fig. 2.13 Proposed mechanism for the formation of 5-carbon alditols from 5-carbon aldonic acid lactones, again with the retention of configuration around many of the chiral centers

Fig. 2.14 Proposed synthesis of tartaric acid from HCN. Compound specific isotopic measurements could determine if this compound is derived from HCN

a-HMG is the hydroxy analog of a-hydroxymethyl alanine (a-HMA), which could be a precursor of a-AIB, which is abundant in CCs (Ehrenfreund et al. 2001), provided that dehydration/reduction of this molecule were feasible. At first glance it does not appear to be so, given the proposed mechanism described in Fig. 2.7, which requires that the a-carbon participate in the formation of a C¼C bond which is not possible for this tetra-substituted carbon atom. a-HMA has not been identified in CCs; however, a-HMA and a-hydroxymethylserine (a-HMS) have been detected in the mixed NH4CN/HCHO reactions described above. It is not presently known if the failure to detect a-HMS, a-MHA and other a-hydroxymethylamino acids in CCs is because they are not present or because they have not been searched for. Chiral biases could be introduced into amino acids by analogous mechanisms as those described here, for example by substituting amino-formals for MEEs. The aldaric (a–O dicarboxylic) sugar acids are harder to account for by this scheme, but they may be rationalizable based on HCN chemistry (Eschenmoser 2007), which was suggested earlier may occur even in the presence of excess HCHO. For example, tartaric acid could be derived from the HCN tetramer diaminomaleonitrile (DAMN), as HCN yields 2,4-diaminosuccinic acid (Ferris et al. 1974), which would by deamination yield tartaric acid (Fig. 2.14).

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51

Discussion

Much of this is no more than paper chemistry, which is infinitely easier than experimentation or analytical chemistry. However, it does describe avenues for experimentation and measurement. CCs are troublesome subjects of experimentation as they may come from a variety of parent bodies (Ehrenfreund et al. 2001) and the materials studied to date have likely experienced varying degrees of parent body alteration and terrestrial contamination. Nevertheless, there may be some overarching chemical unity to these materials. Some simple measurements could confirm or deny these suggestions. First, does HCHO add to glyceric acid? Second, does it do so in a regioselective manner? Third, does HCHO add to the lactones of 4-carbon and 5-carbon aldonic acids, does it do so in the manner suggested, and to what degree does it make branched isomers? Fourth, do these easily decarboxylate to give polyols? Fifth, can a-hydroxymethyl-a-alkyl amino acids dehydrate to yield a-methyl- a-alkyl amino acids (for example, aHMA ! aAIB)? In proposing these pathways, the published literature has been used as a reference, and many compounds exist in CCs that have not been searched for. By understanding the mechanism of formation of small molecules, more complex ones can be identified which could be searched for to verify or falsify these proposed mechanisms. Studies that focus on compounds for which off-the-shelf standards are available may bias our sense of mastery over the molecular composition of CC organics. The previously noted high-resolution mass spectrometry studies of the Murchison meteorite (Schmitt-Kopplin et al. 2010) and a recent study of Miller’s early spark discharge experiments (Johnson et al. 2008), for example, show that numerous species remain to be discovered, as our technologies and search strategies change over time. If compounds found in modern metabolism turn out to be easily produced abiotically, this supports the notion that they were available for the origin of life. Researchers have thus tended to look for compounds which coincide with contemporary theories (sugars for an RNA World model, bases for the same, etc.); but, given that some 90–99.9995% of the small molecules in the soluble fraction of CC organics remain unidentified, it may be premature to suggest that we understand how the compounds identified were formed or what the prebiotic inventory was. Furthermore, it must be borne in mind that modern biochemistry is the result of billions of years of evolution, and thus is likely highly modified from its initial stages. We may never be able to fully deconvolute the thermal and radioactive processes that formed CCs organics, but we can make better analogs of these materials, and having better analogs we can conduct searches for new molecules which will give new insights into these systems. Organic chemistry is, unlike artifactual evidence, an experimental science. A better understanding of the reactivity of small organic molecules is likely to be informative. These phenomena are currently under investigation.

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Unfortunately, the scheme presented here explains little of the ultimate source of the chiral bias observed in CCs, which must lie in some presolar or early solar system process which favored, for example, L over D glyceric acid or L over D serine. It seems likely that this bias occurred at this point based on the observation of chiral biases, which are in the same direction, in more than one CC which likely had different parent bodies. Circularly polarized light thus remains one of the few plausible chiral initiators (Bonner and Rubenstein 1987). Acknowledgments The author would like to thank Dr. M. Fogel of the Carnegie Institution of Washington for elemental analyses and Dr. Jason Dworkin of the NASA Goddard Space Flight Research Center for amino acid analysis assistance.

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Stoks PG, Schwartz AW (1981) Nitrogen-heterocyclic compounds in meteorites – significance and mechanisms of formation. Geochim Cosmochim Acta 45:563–569 Subbaraman AS, Kazi ZA, Choughuley ASU, Chadha MS (1975) Methyleneaminoacetonitrile – possible role in chemical evolution-II. Orig Life Evol Biosph 6:537–539 van der Velden W, Schwartz AW (1977) Search for purines and pyrimidines in the Murchison meteorite. Geochim Cosmochim Acta 41:961–968 Walker JF (1964) Formaldehyde, 3dth edn. Reinhold, New York Waller GR, Feather MS, American Chemical Society, Division of Agricultural and Food Chemistry, American Chemical Society, Division of Carbohydrate Chemistry (1983) The Maillard reaction in foods and nutrition. American Chemical Society, Washington, DC. Weber AL, Pizzarello S (2006) The peptide-catalyzed stereospecific synthesis of tetroses: a possible model for prebiotic molecular evolution. Proc Natl Acad Sci USA 103:12713–12717 Wolman Y, Haverland WJ, Miller SL (1972) Nonprotein amino acids from spark discharges and their comparison with the Murchison meteorite amino acids. Proc Natl Acad Sci USA 69:809–811 Yuen G, Blair N, Des Marais DJ, Chang S (1984) Carbon isotope composition of low molecular weight hydrocarbons and monocarboxylic acids from Murchison meteorite. Nature 307:252–254

Chapter 3

On the Origin of Phosphorylated Biomolecules Matthew A. Pasek and Terence P. Kee

Abstract Phosphorus is a key element in biology, serving in cellular replication, metabolism, and structure. The versatility of phosphorus in biology is due to several unique chemical characteristics that rely on its electronic structure and geochemical abundance. The formation of phosphorylated biomolecules and their activated precursors have hence been a major focus of prebiotic syntheses for the past 50 years. This chapter highlights the basic chemical and physical features that make phosphorus chemicals so valuable within contemporary biochemistry, the putative prebiotic routes to phosphorylated biomolecules, and a growing role for reduced oxidation state phosphorus compounds, including those derived from meteorites, in the development of life on the Earth. We distinguish three primary forms of biological phosphates that form an energetic hierarchy: (i) stable phosphorylated biomolecules that are unreactive and in which the P provides a structural or binding handle; (ii) energetic condensed phosphates including ATP which store metabolic energy; and (iii) reactive phosphorylated biomolecules which are generated during metabolism and transfer phosphates and energy to condensed phosphates for energy storage. We suggest here that: (1) precursors to modern biologic phosphates likely included reduced oxidation state phosphorus compounds; (2) ATP as the main metabolic energy transfer agent likely arose well after the origin of life, and was likely co-opted from its role as a RNA building block into its metabolic role.

M.A. Pasek (*) Department of Geology, University of South Florida, 4202 E Fowler Ave, SCA 528, Tampa 33620, FL, USA e-mail: [email protected] T.P. Kee School of Chemistry, University of Leeds, LS2 9JT, Leeds, UK e-mail: [email protected] R. Egel et al. (eds.), Origins of Life: The Primal Self-Organization, DOI 10.1007/978-3-642-21625-1_3, # Springer-Verlag Berlin Heidelberg 2011

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3.1

M.A. Pasek and T.P. Kee

Introduction

The scientific investigation of the origins of life has advanced significantly over the past 60 years. This advancement has been led in no small part by the investigation of potential prebiotic chemistries arising on the early Earth. The discovery of formation pathways of amino acids (Miller 1953), of sugars (Breslow 1959), and of nucleobases (Oro 1960) highlighted the possibility of a total synthesis of biological compounds starting from simpler starting products. Included among these successes were several demonstrations of nucleoside phosphorylation, the first showing the formation of adenosine, AMP, ADP, and ATP from adenine, ribose, and ethyl metaphosphate (Ponnamperuma et al. 1963). The next 10 years saw a slew of potentially prebiotic phosphorylation successes, many of which formed nucleotides or nucleotide triphosphates using simple precursors such as sodium phosphates or condensed phosphates, nucleosides, and heat (Steinman et al. 1964; Ponnamperuma and Mack 1965; Ponnamperuma and Chang 1971). However, geologists critiqued the use of phosphates – often employed in high concentrations and using highly soluble salts – as being unrealistic in the primitive environment where high aqueous concentrations would have been mediated against by the presence of metal ions such as magnesium, calcium, and iron. Subsequently, several researchers repeated these experiments using an assortment of phosphate minerals, with pertinent results coming from phosphorylation by struvite, MgNH4PO4.6H2O (Handschuh and Orgel 1973). Recent work has continued analyzing the phosphorylating capability of phosphate minerals (e.g., Costanzo et al. 2007), of reduced oxidation state phosphorus compounds (Pasek and Lauretta 2005; Bryant and Kee 2006), and has continued to investigate phosphorylation in the broader context of the origin of replication and metabolism (Powner et al. 2009; Hagan 2010). These studies are especially relevant with evidence for an RNA world preceding the evolution of the modern DNA-RNA-protein biochemistry (Orgel 2004; Lincoln and Joyce 2009). Even before the advent of the putative RNA world, phosphorus likely participated in several critical prebiotic or early biotic functions (de Duve 1987; Eschenmoser 2007; Sharov 2009). This chapter will detail the reasons why phosphorus may have been prebiotically important in the origin of life, the specific chemical properties of phosphorus (P) and orthophosphate in particular that make it so advantageous for use in contemporary biology, provide an overview of phosphorylation experiments using a variety of P-sources, some with greater degrees of prebiotic provenance than others, and detail a possible role for meteorites in the emergence of phosphorylated biomolecules.

3.2

The Ubiquity of Phosphorylated Compounds

With carbon, nitrogen, oxygen, hydrogen, and sulfur, phosphorus is one of the key elements of biology. Phosphorus participates in nearly all biochemical functions including replication, metabolism, cell structure, regulation, and, less frequently,

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catalysis. Phosphorus comprises about 1% of the dry weight of cells (Lange and Heijnen 2001), and is scavenged from the environment principally in the form of orthophosphates and their derivatives (Benitez-Nelson 2000). Marine ecosystems are well regulated with ratios of C:N:P defined by the Redfield ratio (Redfield 1958). Many other ecosystems are P-limited and will only grow to the point at which all available P is consumed (Benitez-Nelson 2000). Indeed, the addition of P to an ecosystem frequently results in algal blooms as organisms reproduce to use up the extraneous P (Smayda 1997). Replication and metabolism are the two biochemical processes that are highly reliant on P on an atomic basis. Phosphorus is 3% of the atomic composition of RNA (10% by weight), and is about 1% of the atomic composition of the metabolome (Srinivasan and Morowitz 2009). Over 44% of all metabolic molecules are phosphorylated (Srinivasan and Morowitz 2009). The high importance of P to these two critical functions suggests a fundamental utility in P and phosphates to contemporary biochemistry, and hence, a critical need for P at the origin of life. Phosphorylated biomolecules can be broadly divided into three major categories: (i) reactive organophosphorus compounds, (ii) stable organophosphorus compounds, and (iii) condensed phosphates. Reactive phosphorylated biomolecules are those in which a phosphate group is bound to a molecule (typically carbon) with sp2 hybridization. Most reactive phosphorylated biomolecules are active participants in cellular (often energy) metabolism, and include so-called “high energy” biomolecules such as phosphoenolpyruvate, acetylphosphate, phosphocreatine (bound through a nitrogen atom), and 1,3-diphosphoglycerate (Fig. 3.1a). In contrast, stable phosphorylated biomolecules are those in which a phosphate group is bound to a molecule (again, typically carbon) with sp3 hybridization (Fig. 3.1b). Stable phosphorylated molecules participate in molecules which need to persist in a cell, and include glycerol phosphate in cell membranes, sugar phosphates in DNA and RNA, and 2,3-bisphosphoglycerate, an active component of respiration. The phosphate in many stable phosphorylated biomolecules acts primarily as a handle in biochemical reactions, tethering the substrate to a highaffinity binding site. As an example, the highly versatile cofactor pyridoxal phosphate is tethered by its phosphate to a Gly-Thr binding site (Mittenhuber 2001; Schnell et al. 2007). These biomolecules may be quite easy to generate via prebiotic reactions (Milner-White and Russell 2005). Phosphate in stable phosphorylated biomolecules also serves a key function structurally, as Mg-phosphate bridges keep RNA folds rigid (e.g., Hsiao and Williams 2009; Petrov et al. 2011). We include with stable phosphorylated biomolecules the phosphonates, which are unique in biology as they possess a P–C bond. Phosphonates can persist for extremely long times in the environment as the P–C bond does not hydrolyze as readily as the P–O or P–N bonds, a feature connected to the lower polarity of the former bond compared to the latter (Freedman and Doak 1957; Murai and Tomizawa 1976), and these compounds have been found to play a major role in the P biogeochemical cycle (Dyhrman et al. 2009; Goldhammer et al. 2010); Pasek (2008) argued for an ancient origin of phosphonates on the basis of these features.

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Fig. 3.1 Organophosphorus compounds discussed in text

The final group of phosphorylated biomolecules in cells is condensed phosphates (Fig. 3.1c). Condensed phosphates are acid anhydrides of orthophosphoric acid, and typically serve in metabolic roles, or in phosphate or energy storage roles in cells. Examples include ATP and ADP, which are the energy carriers in modern metabolism, pyrophosphate, an energy carrier in more ancient metabolisms (Baltscheffsky et al. 1999), and polyphosphates. Pyrophosphate proton-pumps (PPase enzymes) generate pyrophosphate from orthophosphate instead of ATP from ADP (Serrano et al. 2007), though this is not the main function of this enzyme. Polyphosphate acts as phosphate storage (Makino et al. 1989), can transfer phosphate to ADP to make ATP (Ahn and Kornberg 1990), participates in membrane channels (Reusch and Sadoff 1988), and catalyzes several biochemical reactions (Sauer et al. 1969). Polyphosphates have also been proposed to be ancient and to have preceded ATP in early metabolism (Waehneldt and Fox 1967). Both pyrophosphate and polyphosphate are likely precursors to ATP, due to the complementary structural

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features, inherent simplicity, and persistence in modern organisms today. However, significant issues remain over pyrophosphate and polyphosphate as prebiotic phosphorylating agents; so, what may have preceded them en route to ATP is less clear. In general, the energy of dephosphorylation of these biomolecules proceeds as stable phosphorylated biomolecules < condensed phosphates < reactive phosphorylated biomolecules (Nelson and Cox 2005). Due to these energy differences, reactive phosphorylated biomolecules will tend to be metabolic intermediates in the generation of condensed phosphates (i.e., in glycolysis). In turn, condensed phosphates are used in the phosphorylation of organic compounds to form stable phosphorylated biomolecules, and in the anabolic construction of other molecules (Fig. 3.1). These three classes of P compounds follow a metabolic hierarchy; the high energy P compounds are formed from catabolism of organic compounds, which in turn phosphorylate the condensed P compounds, which in turn phosphorylate substrates to afford the stable P compounds. In general, metabolic processes proceed by the transfer of chemical energy either as a phosphoanhydride bond, or as an acetyl bond (Srinivasan and Morowitz 2009). However, energy is stored almost exclusively as phosphoanhydride bonds, typically ATP or polyphosphates. Indeed, acetic anhydride is kinetically unstable in water and hence cannot act as an energy-storing molecule (Nelson and Cox 2005).

3.3

Why Phosphorus?

What specific properties of P make it so critical to biochemical functions? How is P different from other compounds? These questions were first addressed by Todd (1959), expanded upon by Westheimer (1987), and were re-addressed recently by Bowler et al. (2010). We expand on their arguments by adding to their list properties that distinguish P from other compounds in a prebiotic context.

3.3.1

Solubility

Phosphate is soluble in water in the absence of divalent cations. Water is the most abundant solvent on the surface of the Earth and is the substance in which nearly all metabolic reactions must take place; hence, all metabolic reactants must be watersoluble. Many organic compounds are phosphorylated upon entry into the cell membrane to increase solubility by adding a charged functional group. As an example, adenine and adenosine are much less soluble in water (saturated concentrations of 0.009 M and 0.02 M, respectively) than the phosphorylated equivalent of these molecules (0.082 M, Rytting et al. 2005). This is primarily due to the ionic character of the phosphate group in organophosphates.

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M.A. Pasek and T.P. Kee

Ionization

Organophosphates are always negatively charged in biochemical systems as a result of their pKa profiles (pKa1 ~2; pKa2 ~7; pKa3 ~13). Ionization is necessary for biochemistry as ionic compounds are less able to traverse an amphiphilic membrane and are more readily retained by the cell than an unphosphorylated derivative. Negative ionization also serves a second purpose by stabilizing the system toward hydrolysis. Hydrolysis occurs as the nucleophilic, or positive-charge seeking, oxygen in water bonds to an electrophilic, or negative-charge seeking center, which splits the molecule into two parts. The charge on phosphate in organophosphates is localized around the phosphorus atom, and localized negative charges deter the negative oxygen in water from hydrolyzing the organophosphate bond through Coulombic repulsion. Phosphate functional groups have the most negative charge of any functional group employed in biology, minimizing hydrolysis. The acid-base characteristics of orthophosphate are related to the benefits of being ionized. Orthophosphate loses its first proton at a pH of ~2 but retains a proton up to a pH of ~13, which is superior to many organic acids. By retaining a proton, orthophosphate is active in dehydration reactions, encouraging phosphorylation.

3.3.3

Energetics

Organophosphates are ideal metabolic molecules due to the distinct electronic configuration of the phosphate group of organophosphates. Phosphorus biomolecules are typically thermodynamically unstable and will degrade to release energy. Much of this energy comes from the resonance stabilization of the two distinct molecules when an energetic P biomolecule is hydrolyzed. Metabolic reactions involving P biomolecules release intermediate amounts of energy (Table 3.1). The energy of hydrolysis of ATP is about 30 kJmol1 whereas acetic anhydride is about 91 kJ/mol (Nelson and Cox 2005). Acetic anhydride hydrolysis releases three times the energy of ATP, but stores energy poorly, principally due to the rapidity of hydrolysis in water. As a side note, some P biomolecules undergo highly energy releasing (exothermic) reactions, like phosphoenol pyruvate. Others engage in more closely thermoneutral transformations, like the sugar phosphates. With an energy intermediate between these two biomolecules, ATP is particularly well-suited to its role as the energy currency of life. The metabolic breakdown of organic molecules takes place over several chemical steps. If the metabolic currency of life was a high-energy compound like phosphoenol pyruvate, then very few of these breakdown steps would provide the energy necessary to regenerate phosphoenol pyruvate via the phosphorylation of pyruvate, a process requiring

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Table 3.1 Standard free energy of assorted compounds Name Class DG0 0 (kJ/mol) Phosphoenol pyruvate Reactive P compound 61.9 1,3-bisphosphoglycerate Reactive P compound 49.3 Phosphocreatine Reactive P compound 43.0 ADP Condensed phosphate 32.8 ATP Condensed phosphate 30.5 ATP– > AMP + Pyrophosphatea Condensed phosphate 45.6 AMP Stable P compound 14.2 Pyrophosphate Condensed phosphate 19.2 Glucose 1-phosphate Stable P compound 20.9 Fructose 1-phosphate Stable P compound 15.9 Glucose 6-phosphate Stable P compound 13.8 Glycerol 1-phosphate Stable P compound 9.2 Acetyl CoAb Thioester 31.4 Acid anhydride 91.1 Acetic anhydridec Source: Data from Nelson and Cox (2005) and references therein with the exception of those species marked in bold, all reactions consist of loss of a PO3 group. a This reaction is the loss of a pyrophosphate group from ATP. b Acetyl-CoA has a S–C linkage which is hydrolyzed to form SH and HO–C. c The hydrolysis of acetic anhydride forms two acetate groups

61.9 kJmol1 (Nelson and Cox 2005). Conversely, if the metabolic currency was a low energy compound like a sugar phosphate, the amount of energy extracted from each step would be low and catabolism would be a wasteful process. ATP is the happy intermediate between these two extremes, maximizing the energy extracted from each step of metabolism. In effect, ATP is capable of acting as a rechargeable, molecular battery. Whilst ATP is not stored in great quantities in the human body, it is rapidly turned-over catalytically with up to 6 moles of ATP being used per hour; this equates to an energy consumption of ca. 19 AA batteries each hour. This preference for ATP is likely an evolutionary, post-origin-of-life adaptation due to the complexity and specificity of ATP, and alternative chemicals with the potential to act as rechargeable molecular batteries (e.g., polyphosphates may have preceded ATP as the energy carrier for early life (Waehneldt and Fox 1967)).

3.3.4

Kinetics

Despite the thermodynamic instability of many P biomolecules, organophosphates are long-lived, or kinetically stable. The kinetic stability of P biomolecules is due to the hydrolysis-resistant negatively charged P group which inhibits nucleophilic attack from water oxygen at the phosphorus center. Phosphorus biomolecules are resonance-stabilized which allows for the sharing of electrons, increasing stability (as illustrated for triphosphate in Fig. 3.1c). Many P biomolecules are also stabilized by the presence of divalent cations, like Mg2+. Magnesium bonds to

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orthophosphate and polyphosphate groups and reduces the space available for water to attack the phosphate groups. Indeed, ATP is typically bound with Mg2+ as a salt.

3.3.5

Reactivity

Many metabolic reactions use phosphate groups to synthesize novel compounds. Reactions using phosphate groups are typically either substitution reactions, in which phosphate is lost and replaced with another compound, or elimination reactions, in which phosphate is lost and removes nearby functional groups in the process (Westheimer 1987). Phosphate is frequently the leaving group for many metabolic reactions and hence phosphate is used in biosynthetic pathways to change the chemical characteristics of molecules.

3.3.6

Site Specificity

Phosphate groups are easily recognized by enzymes since they bond strongly to divalent cations such as Mg2+. Enzymes that act on phosphates incorporate Mg2+ into their structure and use the Mg/phosphate linkage as a spatial point of reference from which a chemical reaction occurs (e.g., phosphohydrolases and phosphotransferases). Similarly, since phosphate has an affinity to Mg, Mgphosphate complexes are critical to maintaining RNA structure and folding (Hsiao and Williams 2009).

3.3.7

Bridges

The bridging capability of P is especially important to DNA and RNA. Orthophosphate is capable of forming two C-O-P linkages while still retaining a negative charge. Repeating these bridges forms a large polymer which genetically compiles the information necessary for life’s functions and physically tends to separate from other polymers. This ensures that the genetic material is kept separated from other cellular material.

3.3.8

Achirality

The orthophosphate functional group is achiral even when bound to one or two organic groups as the remaining two oxygen atoms engage in resonance which equates their electronic structure (cf: Fig. 3.1c). Achiral functional groups minimize

3 On the Origin of Phosphorylated Biomolecules

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the information necessary for the construction or destruction of an organic molecule. With RNA and DNA, it is unimportant how the orthophosphate groups are connected, which is advantageous given the specificity of the ribose and nucleobase. A lower information requirement for metabolic reactions is always advantageous as it minimizes the variation needed for enzymes and decreases the side products from biochemical reactions. In this context, though it is apposite to point out that ribonucleotides have greater potential reactivity profile than their 20 -deoxy cousins especially toward phosphate binding. This may have some functional benefit in terms of emerging metabolism but may lead to some dilution of informational integrity.

3.3.9

Uniform Oxidation State

P is nearly always P5+ on the surface of the Earth, with the minor exception of the P-C organic compounds (which remain pentavalent but with a formally lower oxidation state at P of +3) and a few unusual sources (Pasek and Block 2009; Pech et al. 2009). Other elements that might substitute for P such as arsenic (As) and vanadium (V) change oxidation state depending on atmospheric composition, ocean depth, fluid composition, and temperature. Phosphorus is uniformly +5 below temperatures of about 1000 K and in the presence of water, when the system is in thermodynamic equilibrium. This is true even under mildly reducing conditions like those hypothesized on the early Earth (and used by Miller and Urey in 1953 to form organics from the atmosphere).

3.3.10 Terrestrial Distribution The only readily available compound with the chemical characteristics described above is orthophosphate. As previously stated, P is frequently the limiting nutrient for life and presumably a similar situation would have existed during the emergence of the first life forms within the terrestrial prebiotic system. However, orthophosphate in all its forms is the most abundant species with the features described above; yet, it is not without difficulty for early organisms to have garnered and used this element. Due to the centrality of P in biochemistry, many modern microorganisms have evolved to extract P from their surroundings, but frequently at the cost of considerable metabolic energy due to the over-expression of enzymes for this process. Since P is a ubiquitous minor element in most rocks, life is not limited to specific localities and is capable of diversifying. Other elements, for instance, vanadium and arsenic (Wolfe-Simon et al. 2009), may be similar or even superior to P for a specific feature, but the sum of the beneficial characteristics of P far outweighs those of its competitors.

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Phosphorylations Using Phosphate

Phosphorus has several chemical properties that make it critical to contemporary biochemistry. Indeed, P can be considered a “Deeply Rooted” element – it lies at the core of many biochemical functions – and hence it seems likely that the origin or the very early evolution of life made use of phosphorylated biomolecules, presumably via energetically acceptable, cyclic processes. If true, then this opens the question, “How was P incorporated into prebiologic compounds?” Historically, attempts to form phosphorylated biomolecules have proceeded via a dehydration reaction between an orthophosphate and an organic molecule containing an –XHn function (e.g., hydroxyl OH or amino NH2 moieties): ROH þ HOPO3 2 ! ROPO3 2 þ H2 O

(3.1)

This simple dehydration reaction was one of the first explored. A number of nucleosides and sugars can be phosphorylated in this manner, albeit at low to moderate yields, using this method (Table 3.2). In general, the temperature of these reactions is of the order of 80-160 C, and the reactions must be heated to dryness. While most of these reactions have focused on the phosphorylation of sugars or nucleosides, recently Maheen et al. (2010) have shown the clay-catalyzed phosphorylation of glycerol occurs by thermal dehydration, highlighting a route to the formation of phospholipids, and possibly other stable phosphorylated biomolecules. Some general rules arise from study of these reactions. Acidic solutions generate more organophosphates, as there are more protons attached to phosphate at pH < 7 which are subsequently available for condensation. Also, in general, higher temperatures of reactions will generate more products as more water is driven off, unless the heating time is too long, at which point the products start to degrade. Longer timescales will tend to make more products, provided that the product is not removed by competing processes. Compounds that have been phosphorylated by this method are stable organophosphorus compounds (examples include many of the key intermediaries in carbohydrate metabolism such as glycolysis and the pentose phosphate pathways), and not reactive organophosphorus compounds. Finally, there appears to be relatively little functional group selectivity under such forcing conditions. Reaction (3.1) is thermodynamically unfavorable in water, so heating of this reaction eliminates water, providing an entropic driving force toward maintaining a non-equilibrium system. An alternative means of forcing reaction (3.1) is to load the reaction with chemical energy in lieu of thermal energy. In this case, two options replace the chemistry presented in reaction (3.1): ROLG þ HOPO3 2 ! ROPO3 2 þ LGOH or

(3.2)

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Table 3.2 Phosphorylation through addition of energy Organic Yield compound P source Conditions Compound produced (%) References St64, H3PO4, Ca, Na, K, UMP 0–16 PM65 NH4 phosphates 160 C, 2 h Uridine Ca, Na, K, NH4 Uridine phosphates 126 C, 2 h UMP 0–9.7 PC71 . Uridine Ca(H2PO4)2 H2O 98 C, 2 days UMP 29.2 PC71 98 C, 14 days UMP 26.8 PC71 Uridine Ca(H2PO4)2 . H2O Urea, 65 C ThP 25 Bi72 Thymidine Na2HPO4 Urea, Uridine Ca3(PO4)2 85 C UMP 30–80 Ha73 (NH4)2C2O4, 90 C ThP 15–30 Sc75 Thymidine Ca3(PO4)2 Trehalose NaH2PO4 56 C Trehalose phosphate 15 TW93 Nucleoside-5Nucleosides, 100 C monophosphates 18 RZ99 urea NaH2PO4 AMP, including 90 C, in formamide cyclic forms 0–3 Co07 Adenosine Phosphate minerals Glycerol H3PO4 100–180 C Glycerol phosphate 1 M10 For Tables 3.2–3.5: Phosphate typically refers to Na2HPO4 in concentrations of ~0.01 MPPi is pyrophosphate, and PPPi is triphosphate, Poly-P is polyphosphate, AMP is adenosine monophosphate, UMP is uridine monophosphate, ThP is thymidine monophosphate, NTP is nucleotide triphosphate, cTMP is cyclotrimetaphosphate . BDL is below detection limitAbbreviations for references used in Tables 3.2–3.5: BO65 Beck and Orgel 1965, Bi72 Bishop et al. 1972, Ch02 Cheng et al. 2002, Co07 Costanzo et al. 2007, dZ04 de Zwart et al. 2004, Fe84 Ferris et al. 1984, Fe93 Ferris and Ertem 1993, Ga68 Gabel 1968, GO00 Gao and Orgel 2000, Ha07 Hagan et al. 2007, Ha69 Halmann et al. 1969, HS70 Halmann and Schmidt 1970, Ha73 Handschuh and Orgel 1973, He90 Hermes-Lima 1990, HV89 Hermes-Lima and Vieyra 1989, Ib71 Ibanez et al. 1971, Ka97 Kanavarioti 1997 KM96 Keefe and Miller 1996, Kr99a Krishnamurthy et al. 1999, Ko97 Kolb et al. 1997, Li05 Lin et al. 2005, LO68 Lohrmann and Orgel 1968, Lo77 Lohrmann 1977, Ma10 Maheen et al. 2010, MP64 Miller and Parris 1964, MS07 Mullen and Sutherland 2007, OO72 Osterberg and Orgel 1972, Oz04 Ozawa et al. 2004, Pi94 Pitsch et al. 1994, PF97 Prabahar and Ferris 1997, PM65 Ponnamperuma and Mack 1965, Le06 Leman et al. (2006), Sa70 Saffhill (1970), PC71 Ponnamperuma and Chang 1971, Ra68 Rabinowitz et al. 1968, RZ99 Reimann and Zubay 1999, Sa92 Sales et al. 1992, Sa81 Saygin 1981, Sa83 Saygin 1983, Sc75 Schwartz et al. 1975, SP68 Schwartz and Ponnamperuma 1968, SO77 Sherwood and Oro 1977, St64 Steinman et al. 1964, St65 Steinman et al. 1965, TW93 Terelli and Wheeler 1993, Tu01 Turian and Rivara-Minten 2001, VW58 Van Wazer 1958, Wa90 Wagner et al. 1990, We81 Weber 1981, We82 Weber 1982, Ya79 Yamagata et al. 1979, Ya81 Yamagata et al. 1981, Ya99 Yamagata 1999, YI97 Yamagata and Inomata 1997; YM82 Yamagata and Mohri 1982

ROH þ LGOPO3 2 ! ROPO3 2 þ LGOH

(3.3)

where LG is a leaving group. These reactions will occur at lower temperatures and are thermochemically biased for the equilibrium to lie to the right hand side. Reaction (3.2) includes several examples, and requires some form of condensing agent, whose job it is to activate the organic substrate. The most popular condensing agents include cyanate and sulfur-containing organics many of which have successfully phosphorylated organic compounds (Table 3.3). However, these

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Table 3.3 Phosphorylation through energetic organic condensing agents Organic compound

P compound

Reaction conditions

Cyanamide, dicyandiamide, glucose

Phosphate

pH 2

Dicyandiamide, glucose Dicyandiamide NCCONH2, Carbodiimide, cyanate, ethylisocyanide, Uridine Cyanamide, cyanogen, ribose Cyanimide

Phosphate ADP + phosphate With clay

Phosphate

Phosphate

Ferricyanide Carbamyl phosphate

Phosphate AMP, ADP

Cyanate

Phosphate Carbamyl phosphate

Acetate Diiminosuccinonitrile, uridine

Glucose-6phosphate Glucose-6phosphate ATP

Electric discharge Under visible light Electric discharge

St65 St65

1–10

LO68

8–20 58

Ha69, HS70 SO77

AMP Carbamyl phosphate ADP, ATP Carbamyl phosphate

0.02

Ya79, Ya81

15 2–17

Sa81 Sa81

6

YM82

Acetyl phosphate

30

Sa83

4

Fe84

22.5

Wa90

47 25

Pi94 Ya99 Ya99

2-Aziridinecarbonitrile

H3PO4:H2O 93:7

Oxiranecarbonitrile Nucleotide Di-P, cyanate Nucleotide Mono-P, cyanate

Phosphate Ca3(PO4)2 Ca3(PO4)2 Phosphate, aminoacids

NDP Aminoacylphosphates

19

Acetyl phosphate

15

Thioacetate

Phosphate

80 C

Uracil, UV light

Reference

1.5–2.4 St64

UMP Glycolaldehyde phosphate Glycolaldehyde phosphate NTP

COS

Phosphate

Yield (%)

1.9 0.5

UMP Ribose-1phosphate dTppdT

Phosphate dTMP

Cyanate, adenosine

Product

Le06 Ha10

compounds frequently require lower pHs than are expected on the surface of the early earth, perhaps consistent with acidic volcanic fumarole environments, and typically have very low yields. As a result, Hulshof and Ponnamperuma (1976) concluded that reaction (3.3) is more likely than reaction (3.2). Reaction (3.3) is of special interest to our study of the roles of P in the emergence of life on earth, as these phosphate compounds are often activated with other phosphates as leaving groups. Reaction (3.3) compounds include the use of condensed phosphates such as pyrophosphate [PPi(V)] and cyclotrimetaphosphate (cTMP). The leaving group in many of these reactions is either phosphate or polyphosphate. Since the chemical energy in reaction (3.3) is preloaded into the phosphate group, side reactions removing the organic compound are minimal.

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In most models of prebiotic systems, the organic compound is usually much rarer than the P compound and hence most prebiotic research on the phosphorylation pathways of organic compounds have used condensed or activated P compounds to drive the reaction forward (Table 3.4). Cyclotrimetaphosphate (cTMP) is an excellent phosphorylating agent, and is capable of phosphorylating organic compounds at concentrations as low as 106 M (Krishnamurthy et al. 1999). It is most active in solutions with NH4+ or amino groups, and the phosphorylation reactions are catalyzed by Mg2+. For these reasons, the formation of cTMP itself has been a goal of prebiotic chemistry. Indeed, many prebiotic reactions have been considered successful if the end product is a condensed P compound: O3 POLG2 þ HOPO3 2 ! O3 POPO3 4 þ LGOH

(3.4)

with O3P–O–PO34- also including larger polyphosphates. Reaction (3.4) has been the focus of several studies (Table 3.5), as the formation of polyphosphates mimics modern metabolism, and several of these reactions are considered “biomimetic.” Polyphosphate production reactions generally require high temperatures, condensing agents, or some other means of activating the reaction. Weber has suggested that inorganic phosphate (Pi) could be activated by thioesters which raises the interesting possibility of Pi sequestration from stable P-chemical intermediates, activation as an acetylphosphate via reaction with a thioester and hence coupling of two key bioenergetic pathways. This idea has potential and some evidence of its efficacy (Weber 1981) but further work on converting the system to a functional cycle would greatly enhance its impact. Despite these successes, many putative phosphorylation attempts are plagued by geochemical problems (Keefe and Miller 1995). Despite the ubiquity of P in biological systems, P is comparatively scarce in aqueous geosystems. The surficial P of the Earth is limited by the geologic environment; in most environments, it exists predominantly as the thermodynamic sink of orthophosphate minerals due to the significant difference in energy between orthophosphate and all other varieties of condensed and reduced P compounds (Fig 3.2). Many orthophosphate based phosphorylation reactions employed elevated phosphate concentrations (10 mM or greater), whereas aqueous geologic concentrations average at about 1 mM. In the presence of divalent cations, especially Ca2+, orthophosphate is highly insoluble and the dissolution of phosphate minerals is buffered by an abundance of Ca2+ in the ocean. These low concentrations may be modified by chelating agents (Schwartz 1972) or, alternatively, by formation of carbonate-apatite (Kakegawa et al. 2002), although neither process is expected to have acted abundantly on the surface of the early earth prior to the development of life. Furthermore, most surficial apatite may be derived from biological deposition (Arrhenius et al. 1997), questioning whether this mineral may have dominated the early earth. In addition, several of these prebiotic pathways use rare or non-existent phosphate minerals (e.g., Na3PO4), or activated phosphates that are hard to justify as

b-hydroxy-n-alkylamines

AMP

Glycolate Glyceraldehyde Oligonucleotides-5 phosphates Nucleobase Adenosine

Uridine Uridine AMP

Adenosine Adenosine Ribose

cTMP

cTMP cTMP cTMP cTMP cTMP

pH 10

100 C

Mg2+ Mg2+ Ni2+

Conditions Room temp, pH ~8 Na4P2O7, Na5P3O10, Nax+2PxO3x+1(Graham’s salt) Nax+2PxO3x+1(Graham’s salt) 100 C, pH ~8 cTMP pH 14 Na2H2P2O7, Na5P3O10, Trimetaphosphate,, Nax+2PxO3x+1 (Graham’s salt) 126 C, 6 h 162 C, 0.1 h Na5P3O10, tripolyphosphate cTMP Mg2+ Hydrotalcite cTMP catalyzed cTMP Mg2+, NH4+

Table 3.4 Phosphorylation through high energy phosphorus compounds Organic P compound

12.9–29.8 7–15.4 90

UMP UMP Adenosine polyphosphates

ADP, ATP b-hydroxy-n-alkylamines phosphates and triphosphates

19–40

1

30 total

37

34 76

0–1.08 1.52 63

AMP AMP Ribose-phosphate

Glycolate phosphate Glyceraldehyde phosphate Oligonucleotides-5 polyphosphates Nucleobase phosphate cyclic-AMP, ATP

Yield (%)

Product

MS07

Oz04

GO00 Tu01 Ch02

Ko97 Kr99

PC71 PC71 Lo77

SP68 SP68 Sa70

Reference

70 M.A. Pasek and T.P. Kee

3 On the Origin of Phosphorylated Biomolecules

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Table 3.5 Synthesis of high energy phosphorus compounds Organic

Cyanate Dicyandiamide Dicyandiamide Carbodiimide Cyanate Cyanate

Dithionite

Urea, deoxythymidine Urea (NH4)2C2O4, Thymidine Diacetyl cysteamine and imidazole Ethanethioic esters

P compound

Conditions

P products

NaH2PO4 NaH2PO4 and KH2PO4 Apatite Phosphate Phosphate Phosphate Apatite Ca3(PO4)2 and Na5P3O10

500–600 C

Trimetaphosphate 100

VW58

200–600 C

Poly-P PPi PPi PPi PPi PPi

100 26 0.2–1.8 <0.1 <0.1 1–2

VW58 MP64 St65 BO65 BO65 BO65 BO65

H3PO4 Na2HPO4 . 7H2O, Na3PO4 . 12H2O, CaHPO4, KH2PO4, Ca3(PO4)2

160 C, 2 h

Trimetaphosphate 1 30–50, PPi, PPPi 1–5

160 C, 2 h

None

Ra68

(NH4)2HPO4

160 C, 2 h

PPi, PPPi

Ca(H2PO4)2 . H2O Phosphate NH4H2PO4, NaH2PO4.H2O Ca, Na, NH4 phosphates Ca(H2PO4)2 . H2O, NaH2PO4 . H2O

160 C, 2 h Under O2

Ra68

PPi, PPPi PPi

BDL 10–30, 5–10 30–50, 10–30 0.24

Ra68 Ga68

160 C, 2 h

PPi, PPPi

32–64

PC71

90 C, 1 week

PPi,PPPi

1–5

PC71

65 C, 2 months

PPi

1

PC71

NH4H2PO4 NH4H2PO4

100 C, 4 days 85–100 C

Trimetaphosphate 23 Poly-P 60–100

OO72 OO72

Ca3(PO4)2

90 C

Poly-P

1–50

Sc75

PPi, PPPi PPi Poly-P PPi PPi PPi

3–8 ~4 50 3.5–4.6 0.057 53

We81 We82 Sa92 HV89 He90 KM96

PPi PPi PPi PPi PPi PPi PPi PPi

26 5 5 4 4 0.25 0.14 0.4–4.5

KM96 KM96 KM96 KM96 KM96 dZ04 dZ04 Ha07

Phosphate NaH2PO4 (0.02 M) W/apatite MgHPO4 250–550 C

Phosphoenolpyruvate NH4HCOO Thiocyanate and H2O2 Maleic anhydride Pantoyl lactone Polymerizing HCN Imidazole and CaCl2 Acetyl phosphate Phosphoenolpyruvate Cyanate

Yield (%) References

H3PO4 Phosphate

37 C

Phosphate Phosphate Phosphate Phosphate Phosphate Fe(II) minerals Fe(II) minerals Phosphate

Ra68

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M.A. Pasek and T.P. Kee

Fig. 3.2 Relationship of oxidation state of a given redox state of P to the Gibbs free energy of formation of the hydride or oxide of the associated P compound. The two energies at oxidation state of zero correspond to pure white P (solid dot) and P in the metal phosphide Fe3P (open dot)

arising from purely geologic phenomena (e.g., ethyl metaphosphate). Furthermore, several of the condensing agents were unlikely to have been abundant on the prebiotic earth Hulshof and Ponnamperuma (1976), and hence it could be argued that condensed phosphates were the likely source of chemical energy for prebiotic phosphorylation. However, a reliance on condensed phosphates for phosphorylations is also difficult to justify. Thus far, only three polyphosphate minerals have been recognized by International Mineralogical Association. These are kanonerovite (MnNa3P3O10.12H2O), canaphite (CaNa2P2O7.4H2O), and wooldridgeite (CaNa2Cu2[P2O7]2.10H2O). Canaphite and kanonerovite are associated with pegmatites, which are course-grained igneous rocks characterized by high concentrations of rare elements and the occurrence of unusual minerals. Woolridgeite has an unknown origin, and has been found as a weathering crust on primary sulfide minerals. Altogether, these polyphosphate minerals likely comprise ~10 kg of material, suggesting few geologic sources of polyphosphate were present on the early earth in mineral form. Yamagata et al. (1991) provided a possible solution to the origin of polyphosphates. Sampling the gases of volcanic fumaroles, Yamagata and others found polyphosphates such as pyrophosphate and linear triphosphates at low (mM) concentrations. The same publication is also frequently used as justification for the use of cTMP as a prebiotically available phosphorylating agent; formed from rapid aqueous quenching of volcanic out-gassed phosphorus oxides such as P4O10. Such

3 On the Origin of Phosphorylated Biomolecules

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quenching reactions do indeed produce cyclic condensed metaphosphates in laboratory studies but it is worth pointing out that Yamagata’s field studies at Mount Usu, whilst locating linear polyphosphates did not provide any evidence for their cyclic analogs (Yamagata et al. 1991). This discovery suggested that early volcanoes may have released the necessary P for the origin of life. Arrhenius et al. (1997) argued that minerals other than apatite may have been abundant on the early earth, and that these minerals may have been more susceptible to condensation. Recent work has continued to explore the origins of phosphorylated biomolecules. Powner et al. (2009) showed that adenosine phosphate forms from glycolaldehyde, glyceraldehyde, cyanoacetylene, cyanamide, and orthophosphate. The orthophosphate acts as a catalyst in this reaction, as well as a buffer. This reaction provides an elegant pathway to critical nucleobases. Costanzo et al. (2007) explored the phosphorylating power of a variety of phosphate minerals, finding that formamide makes a better solvent than water. From a geologic perspective, the best result they obtained was phosphorylation of adenosine by hydroxylapatite, with yields of ~8% using preheated material. Other phosphates used to phosphorylate adenosine were geologically rare. An especially exciting result comes from Hagan (2010). Hagan (2010) showed that under UV light, a mixture of thioacetate and phosphate yields acetyl phosphate. Furthermore, the reaction is catalyzed by uracil, and the acetyl phosphate can react with phosphate to form pyrophosphate and polyphosphate, suggesting this reaction pathway may have acted in early metabolism. Of phosphorylation reactions leading to organophosphates, the most likely production pathway appears to be reaction (3.3). However, the abiotic production of condensed phosphates by the dehydration of phosphates appears to be limited and largely yield pyrophosphate, the solubility of pyrophosphate is especially low and it is a reluctant phosphorylating agent in the absence of a catalyst. An alternative to phosphates are reduced oxidation state phosphorus compounds (hereafter reduced P). Recent results have shown reduced P is a plausible alternative to phosphates on the early earth. Due to a larger chemical reactivity and greater solubility, these compounds may have served in place of phosphates (especially activated phosphates) on the early earth, priming these systems with chemical energy.

3.5

Phosphorylations Using Reduced Oxidation State Phosphorus Compounds

An alternative to phosphates are reduced P compounds, in which the nominal charge on P is less than 5+, the charge on P in orthophosphate. These alternatives include phosphite (also termed H-phosphonate) with P3+, hypophosphite (also termed H-phosphinate) with P1+, hypophosphate with P4+, and phosphine with P3(Fig. 3.3). These compounds were first suggested by Gulick (1955) to have played a role in the development of life due to the increased solubility of phosphite (about 1,000 the solubility of phosphate in the presence of divalent cations) and

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Fig. 3.3 Reduced P compounds discussed in text

hypophosphite (106 more soluble than phosphate in the presence of divalent cations). Gulick (1955) argued that, if these reduced P compounds were abundant on the early earth, then P would not have been limited and would have been much more available to react with primitive organics. This idea was dismissed by Miller and Urey (1959) as Gulick could provide no source of reduced P on the early earth, and because phosphate was presumed to be the dominant carrier of P based on thermodynamic equilibrium models. These ideas were challenged by the discovery of phosphonic acids in the Murchison meteorite (Cooper et al. 1992). The Murchison meteorite, a CM chondrite with abundant organic compounds such as amino acids, sugars, and carboxylic acids, may present the closest analog to the possible organic chemistry on the early earth. What was notable in the Murchison meteorite was not the presence of organophosphates, but organophosphonates. These organophosphonates are organic compounds in which the P is of nominally reduced oxidation state, these compounds are considered phosphite analogs of organophosphates, and where one [P-O] bond has been replaced by a [P-C] bond. The origin of these phosphonates in the Murchison meteorite is unclear; however, they have been attributed to both photolysis of phosphite and organics by UV light (de Graaf et al. 1995) and to gas-phase reactions (Gorrell et al. 2006). Both of these formation routes highlight alternative oxidation states of P that may have been relevant on the early earth. An effective source of reduced P for the prebiotic earth was identified by Pasek and Lauretta (2005) and Bryant and Kee (2006). These studies showed that iron meteorites, which contain the reduced P mineral schreibersite – (Fe,Ni)3P – release phosphite, pyrophosphate (and possibly higher condensed phosphates), hypophosphate, and hypophosphite on reaction with water, in addition to phosphate. The reaction proceeds by oxidation of P by water (Bryant and Kee 2006) to generate phosphite radicals (Pasek et al. 2007) which combine with H and OH radicals to generate the observed products. During this corrosion, electrons also flow from metal to phosphide, driving cathodic reactions at the meteoritic matrix-metal phosphide boundary (Bryant et al. 2009), some of which result in release of gaseous phosphine (PH3) in addition to condensed oxyacids of P (Bryant et al. manuscript in preparation). Condensed phosphates are formed by reaction of reduced P with peroxides in the presence of an iron catalyst (Pasek et al. 2008). The early earth is believed to have experienced a heavy bombardment period 4.5–3.8 billion years ago in which the flux of material striking the surface of the earth would have been 102–105 the present rate. While iron meteorites were the

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focus of the earlier Pasek and Lauretta (2005) and Bryant and Kee (2006) studies, schreibersite and other phosphides have been found in nearly all meteorite types including carbonaceous chondrites such as Murchison. As a result, as much as 1–10% of the total P on the surface of the earth may be exogenous in origin (Pasek and Lauretta 2008). Additionally, lightning may have played a role in the delivery of reduced P, as phosphates in rocks struck by lightning may be reduced to phosphides (Essene and Fisher 1986) or phosphites (Glindemann et al. 1999; Pasek and Block 2009), though the role of lightning is predicted to be much less than the role of extraterrestrial material (Pasek and Block 2009). Reduced P compounds, while unstable on the early earth, may have persisted for 108–109 years, under a mildly reducing atmosphere (Pasek 2008) and indeed, anaerobic corrosion experiments on sectioned iron meteorites, such as Sikhote Alin, are currently under investigation in our laboratories. If a phosphide mineral corrodes in an aqueous solution with simple organic compounds, these organic compounds can become phosphorylated (Pasek et al. 2007). Schreibersite reacts in water with acetate to generate organophosphorus compounds, such as hydroxylmethylphosphonate, acetylphosphonate, and possibly phosphoglycolate. These compounds, while not likely critical to prebiotic chemistry, illustrate a potential utility in reduced P in “one pot” syntheses of organophosphorus compounds. The yields are low but are proportional to the mole fraction of organic compound in solution. The most likely phosphorylation pathway of these organic compounds is a radical reaction with the organic compound, and the compounds most susceptible to phosphorylation were carboxylic acids. Schreibersite corrodes primarily to phosphite under hydrothermal conditions (Pasek and Lauretta 2005), and to hypophosphite under photolytic conditions (Bryant and Kee 2006). These two compounds are both more soluble than phosphate in seawater, and hence may have been more available for prebiotic phosphorylation on the early earth. Additionally, since the oxidation of both compounds is thermodynamically favored, these compounds have an inherent energy that may promote phosphorylation reactions more readily than phosphate. We highlight here a few reactions showing the prebiotic potential of the reduced P species phosphite and hypophosphite. Pasek et al. (2008) showed that hypophosphite and phosphite, when oxidized by H2O2 in the presence of Fe2+/Fe3+, generate pyrophosphate, triphosphate, and trimetaphosphate in 25–35% yield, across a range of starting conditions. This reaction likely proceeds by a radical reaction initiated by Fenton chemistry (e.g., Walling 1975). Compared to prior attempts at forming condensed phosphate, and especially at forming cyclotrimetaphosphate (cTMP), the oxidation of reduced P is by far much more facile. The reaction occurs readily in water and does not occur by dehydration; hence, it is not thermodynamically inhibited in wet systems. The reaction is also robust, occurring at a range of pHs and occurs at concentrations of 103 M and lower. Additionally, H2O2 is among the most likely oxidants present on the early earth (Joo et al. 1999). This reaction offers a way out from the reduced P world, generating especially useful potentially prebiotic products. A possible alternative to H2O2 and Fe2+ was recently identified by Mulkidjanian (2009) – photolysis in the presence of zinc

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sulfide. In such a system, reduced P compounds might have been oxidized at the surface of ZnS, forming a surface of concentrated condensed phosphates. Such a process merits further experimental investigation. Furthermore, the reduced P compounds, phosphite and hypophosphite, are also reactive toward organic compounds. Hypophosphite in particular will readily react with aldehydes via a phospho-aldol addition (Bryant et al. 2010). Hypophosphite reacts with pyruvate to form cyclic P products as shown in Fig. 3.4. Especially noteworthy, we believe, is the reaction of 2 with benzylamine to afford 4, a simple condensation between a carboxylic acid and an amine to afford an amide linkage, this being the fundamental building block of peptides. The resulting product, 4, is an example of a mixed organic-inorganic material which possesses a regular layered structure in the solid state, an example of how easily it is possible to build structural complexity into chemical systems in water. What is perhaps more interesting however, is that such a reaction is chemically very difficult to perform in aqueous solution in the absence of a coupling agent (Montalbetti and Falque 2005). That such a process should take place at all is, we believe, a result of even more subtle chemistry of the precursor compound 2. This compound engages in a pH dependent equilibrium in water with cyclic lactone 3. It is this cyclic lactone which appears to be the key intermediate in the conversion of 2 to 4 (Bryant et al. 2010). These same studies reveal another pathway from reduced P to condensed polyphosphorus species, not a polyphosphate but pyrophosphite, 5, a reduced analog of pyrophosphate (Fig. 3.4). Preliminary investigations suggest pyrophosphate to be active in phosphorylation and dehydrative coupling chemistry, under putative prebiotic environments. These reactions illustrate the potential prebiotic chemistry of reduced P. Several open questions remain: (1) How abundant were reduced P species on the early earth? To this end, it is necessary to identify plausible sources of reduced P,

Fig. 3.4 Some features of the H-phosphinate-pyruvate (HPP) system

3 On the Origin of Phosphorylated Biomolecules

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endogenous and exogenous, and their prevalence. While it is unlikely, reduced P may have been persistent enough on the early earth to have been concentrated in geologic regions, and may still even be found in sedimentary rocks older than the rise of oxygen. (2) What sorts of reactions are available to reduced P? What other reactions might reduced P compounds participate in, given the presence of other minerals like sulfides and clays? (3) What is the true prebiotic potential of reduced P compounds? With the discovery of polyphosphate production during reduced P oxidation, all reduced P can potentially react via the phosphorylation reactions by condensed P outlined previously, but are there any direct routes to other prebiotic compounds? Direct production of the condensed polyphosphorus oxyanion pyrophosphite may be particularly significant as it is both capable of performing transformations that pyrophosphate itself is reluctant to perform, can readily be regenerated from its breakdown constituents and hence function in a cyclic fashion, whilst being seen as a stepping stone to pyrophosphate, to which it can be converted by chemical oxidation.

3.6

Summary and Closing Thoughts

The incorporation of P into biomolecules was likely one of the key steps of prebiotic chemistry that led to the origin of life. The chemical properties of P and phosphate have tied P to its critical role in modern biochemistry and were likely critical in earlier systems as well. It seems logical that prebiotic phosphorylation reactions may have proceeded by chemically simple, dehydration and condensation reactions, as these would be expected to dominate under energetic, non-equilibrium conditions as envisaged within early earth geological environments. However, recent work has suggested reduced P compounds may provide a route to prebiotic organophosphorus compounds, and the oxidation of these compounds has been shown to be quite relevant to prebiotic chemistry, regardless that P redox chemistry does not now play a significant role in contemporary biochemistry. An open question to consider is: What was the first P biomolecule? Did it fill a role in metabolic, replication, or structural processes? We can likely eliminate P in membranes, as although phospholipids have several excellent properties and are stable in a variety of solutions, simple fatty acids behave in a fashion quite similar to phospholipids (Pohorille and Deamer 2009). Indeed, some organisms have replaced their organophosphate (or organophosphonate) lipid membranes altogether with sulfate membranes (Batrakov et al. 1999), suggesting phospholipids were not critical for early life. This leaves us to question whether P first was incorporated into metabolism or into replication. Several replacement molecules for phosphate in RNA have been proposed, including peptide nucleic acids (Nielsen 2007), glyoxylate (Bean et al. 2006), and arsenate (Wolfe-Simon et al. 2009). Comparatively, there are fewer routes around P in metabolic processes. However, with recent showing the relative

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ease of forming nucleotides (Powner et al. 2009), this question may in fact be answered with “both.” The origin of P in stable biomolecules is difficult to determine. If the original role of P was more ancillary to its fundamental properties (e.g., those related to energy transfer), then perhaps the original metabolic function of P was to provide a tethering site for biochemical reactions. Alternatively, perhaps P played a structural role and helped determine the shape and structure of enzymes. Related to this question is: What was the first use of ATP? Was ATP first used to build RNA, or as the metabolic energy currency of life? In modern life, ATP is circulated much more frequently through metabolic processes than in RNA synthesis, but is this necessarily true for the origin of life? Some have speculated that ATP arose first as a metabolic molecule, only to later be used to construct RNA (e.g., Dyson 1999). This seems unlikely. The business end of ATP, the end which stores metabolic energy, is the pyrophosphate [P–O–P] linkage inherent within the triphosphate moiety. The construction of ATP de novo, for the purpose of metabolism, requires ribose and adenine, both of which are essentially superfluous to the main activity of ATP (with the exception of some signal and catalytic recognition sites). Indeed, since both pyrophosphate and polyphosphate can substitute for ATP as a metabolic currency, it seems more likely that ATP was co-opted from its role as an RNA building-block component during the RNA world (White 1976). A final question we would like to address is: What were the likely routes to get P into metabolism? Given the central role of condensed phosphates in metabolism, it seems most likely that the formation of these compounds (or of more reactive relatives such as pyrophosphite which may have evolved chemically into condensed phosphates) was a critical first step. These compounds are capable of phosphorylating other compounds to generate stable organophosphates, which are critical to most of the functions of modern biology. Prebiotic chemistry on the early earth likely generated these compounds through an assortment of reactions (Tables 3.2–3.5), or alternatively, via oxidation of reduced P compounds. Given the relative simplicity of the condensed phosphates in modern metabolism and the comparative ease by which they may have been formed by abiotic processes, condensed phosphates and phosphites were likely present, if not abundant on the early earth. Modern heterotrophic life today generates these compounds during the catabolism of biological products. Early life may have generated these either through condensation reactions, or alternatively through oxidation reactions, like those of phosphite with peroxide. About half of all anabolic reactions are phosphorylation reactions (Srinivasan and Morowitz 2009). The other half comprises primarily acylation reactions. A molecule capable of doing both is acetylphosphate. This compound may have been the key product in early metabolism, and hence prebiotically viable formation pathways of this molecule merit further study. Hagan’s (2010) demonstration of the formation of this molecule suggests its presence on the early earth to have been plausible, and further work may find routes which show how early metabolisms operated.

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Ferris JP, Yanagawa H, Dudgeon PA, Hagan WJ, Mallare TE (1984) The investigation of the HCN derivative diiminosuccinonitrile as a prebiotic condensing agent. The formation of phosphate esters. Orig Life Evol Biosph 15:29–43 Freedman LD, Doak GO (1957) The preparation and properties of phosphonic acids. Chem Rev 57:479–523 Gabel NW (1968) Abiotic formation of phosphoric anhydride bonds in dilute aqueous conditions. Nature 218:354 Gao K, Orgel LE (2000) Polyphosphorylation and non-enzymatic template-directed ligation of oligonucleotides. Orig Life Evol Biosph 30:45–51 Glindemann D, de Graaf RM, Schwartz AW (1999) Chemical reduction of phosphate on the primitive Earth. Orig Life Evol Biosph 29:555–561 Goldhammer T, Bruchert V, Ferdelman TG, Zabel M (2010) Microbial sequestration of phosphorus in anoxic upwelling sediments. Nat Geosci 3:557–561 Gorrell IB, Wang LM, Marks AJ, Bryant DE, Bouillot F, Goddard A, Heard DE, Kee TP (2006) On the origin of the Murchison meteorite phosphonates. Implications for pre-biotic chemistry. Chem Commun 15:1643–1645 Gulick A (1955) Phosphorus as a factor in the origin of life. Am Sci 43:479–489 Hagan WJ (2010) Uracil-catalyzed synthesis of acetyl phosphate: a photochemical driver for protometabolism. Chembiochem 11:383–387 Hagan WJ, Parker A, Steuerwald A, Hathaway M (2007) Phosphate solubility and the cyanatemediated synthesis of pyrophosphate. Orig Life Evol Biosph 37:113–122 Halmann M, Schmidt H-L (1970) Cyanogen-induced synthesis of 18O-labelled Bribofuranose1-phosphate and its acid catalysed hydrolysis. J Chem Soc C 9:1191–1193 Halmann M, Sanchez RA, Orgel LE (1969) Phosphorylation of D-ribose in aqueous solution. J Org Chem 34:3702–3703 Handschuh GJ, Orgel LE (1973) Struvite and prebiotic phosphorylation. Science 179:483–484 Hermes-Lima M (1990) Model for prebiotic pyrophosphate formation: condensation of precipitated orthophosphate at low temperature in the absence of condensing or phosphorylating agents. J Mol Evol 31:353–358 Hermes-Lima M, Vieyra A (1989) Pyrophosphate formation from phospho(enol)pyruvate adsorbed onto precipitated orthosphosphate: a model for prebiotic catalysis of transphosphorylations. Orig Life Evol Biosph 19:143–152 Hsiao C, Williams L (2009) A recurrent magnesium-binding motif provides a framework for the ribosomal peptidyl transferase center. Nucleic Acids Res 37:3134–3142 Hulshof J, Ponnamperuma C (1976) Prebiotic condensation-reactions in an aqueous-medium – review of condensing agents. Orig Life Evol Biosph 7:197–224 Ibanez JD, Kimbal AP, Oro J (1971) Possible prebiotic condensation of mononucleotides by cyanate. Science 173:444–445 Joo H, Lin ZL, Arnold FH (1999) Laboratory evolution of peroxide-mediated cytochrome P450 hydroxylation. Nature 399:670–673 Kakegawa T, Noda M, Nannri H (2002) Geochemical cycles of bio-essential elements on the early Earth and their relationships to the origin of life. Res Geo 52:83–89 Kanavarioti A (1997) Dimerization in highly concentrated solutions of phosphoimidazolide activated mononucleotides. Orig Life Evol Biosph 27:357–376 Keefe AD, Miller SL (1995) Are polyphosphates or phosphate esters pre-biotic reagents? J Mol Evol 41:693–702 Keefe AD, Miller SL (1996) Potentially prebiotic syntheses of condensed phosphates. Orig Life Evol Biosph 26:15–25 Kolb V, Zhang SB, Xu Y, Arrhenius G (1997) Mineral-induced phosphorylation of glycolate ion – a metaphor in chemical evolution. Orig Life Evol Biosph 27:485–503 Krishnamurthy R, Arrhenius G, Eschenmoser A (1999) Formation of glycolaldehyde phosphate from glycolaldehyde in aqueous solution. Orig Life Evol Biosph 29:333–354

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Lange HC, Heijnen JJ (2001) Statistical reconciliation of the elemental and molecular biomass composition of Saccharomyces cerevisiae. Biotechnol Bioeng 75:334–344 Leman LJ, Orgel LE, Ghadiri MR (2006) Amino acid dependent formation of phosphate anhydrides in water mediated by carbonyl sulfide. J Am Chem Soc 128:20–21 Lin C, Fu H, Zhao Y, Cheng C (2005) Synthesis of nucleoside N-phosphoamino acids and peptide formation. Orig Life Evol Biosph 35:11–17 Lincoln TA, Joyce GF (2009) Self-sustained replication of an RNA enzyme. Science 323: 1229–1232 Lohrmann R (1977) Formation of nucleoside-5‘-phosphoramidates under potentially prebiological conditions. J Mol Evol 10:137–154 Lohrmann R, Orgel LE (1968) Prebiotic synthesis: phosphorylation in aqueous solution. Science 171:64–66 Maheen G, Tian G, Wang Y, He C, Shi Z, Yuan H, Feng S (2010) Resolving the enigma of prebiotic C-O-P bond formation: prebiotic hydrothermal synthesis of important biological phosphate esters. Heteroatom Chem 21:161–167 Makino K, Shinagawa H, Amemura M, Kawamoto T, Yamada M, Nakata A (1989) Signal transduction in the phosphate regulon of Escherichia coli involves phosphotransfer between PHOR and PHOB proteins. J Mol Biol 210:551–559 Miller SL (1953) A production of amino acids under possible primitive Earth conditions. Science 117:528–529 Miller SL, Parris M (1964) Synthesis of pyrophosphate under primitive earth conditions. Nature 204:1248–1250 Miller SL, Urey HC (1959) Organic compound synthesis on the primitive earth. Science 130: 245–251 Milner-White EJ, Russell MJ (2005) Sites for phosphates and iron-sulfur thiolates in the first membranes: 3 to 6 residue anion-binding motifs (nests). Orig Life Evol Biosph 35:19–27 Mittenhuber G (2001) Phylogenetic analyses and comparative genomics of vitamin B6 (pyridoxine) and pyridoxal phosphate biosynthesis pathways. J Mol Microbiol Biotechnol 3:1–20 Montalbetti CAGN, Falque V (2005) Amide bond formation and peptide coupling. Tetrahedron 61:10827–10852 Mulkidjanian AY (2009) Origin of life in the Zinc World. 1. Photosynthetic, porous edifices built of hydrothermally precipitated zinc sulfide (ZnS) as cradles of life on Earth. Biol Direct 4:26 Mullen LB, Sutherland JD (2007) Formation of potentially prebiotic amphiphiles by reaction of b-hydroxy-n-alkylamines with cyclotriphosphate. Angew Chem Int Ed Engl 46:4166–4168 Murai T, Tomizawa C (1976) Chemical transformation of S-benzyl O-ethyl phenylphosphonothiolate (Inezin) by ultraviolet-light. J Environ Sci Health B 11:185–197 Nelson DL, Cox MM (2005) Lehninger’s principles of biochemistry, 4th edn. W.H. Freeman and Company, New York, 1100pp Nielsen PE (2007) Peptide nucleic acids and the origin of life. Chem Biodivers 4:1996–2002 Orgel LE (2004) Prebiotic chemistry and the origin of the RNA world. Crit Rev Biochem Mol Biol 39:99–123 Oro J (1960) Synthesis of adenine from ammonium cyanide. Biochem Biophys Res Comm 407–412 Osterberg R, Orgel LE (1972) Polyphosphate and trimetaphosphate formation under potentially prebiotic conditions. J Mol Evol 1:241–248 Ozawa K, Nemoto A, Imai E-I, Honda H, Hatori K, Matsuno K (2004) Phosphorylation of nucleotide molecules in hydrothermal environments. Orig Life Evol Biosph 34:465–471 Pasek MA (2008) Rethinking early Earth phosphorus geochemistry. Proc Natl Acad Sci USA 105:853–858 Pasek MA, Block K (2009) Lightning reduction of phosphate: implications for phosphorus biogeochemistry. Nat Geosci 2:553–556

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Pasek MA, Lauretta DS (2005) Aqueous corrosion of phosphide minerals from iron meteorites: a highly reactive source of prebiotic phosphorus on the surface of the early Earth. Astrobiology 5:515–535 Pasek MA, Lauretta DS (2008) Extraterrestrial flux of potentially prebiotic C, N, and P to the early Earth. Orig Life Evol Biosph 38:5–21 Pasek MA, Dworkin JP, Lauretta DS (2007) A radical pathway for organic phosphorylation during schreibersite corrosion with implications for the origin of life. Geochim Cosmochim Acta 71: 1721–1736 Pasek MA, Kee TP, Bryant DE, Pavlov AA, Lunine JI (2008) Production of potentially prebiotic condensed phosphates by phosphorus redox chemistry. Angew Chem Int Ed Engl 47: 7918–7920 Pech H, Henry A, Khachikian CS, Salmassi TM, Hanrahan G, Foster KL (2009) Detection of geothermal phosphite using high-performance liquid chromatography. Environ Sci Technol 43: 7671–7675 Petrov AS, Bowman JC, Harvey SC, Williams LD (2011) Bidentate RNA–magnesium clamps: On the origin of the special role of magnesium in RNA folding. RNA 17:291–297, 10.1261/rna. 2390311 Pitsch S, Pombovillar E, Eschenmoser A (1994) Chemistry of a-aminonitriles.13. Formation of 2-oxoethyl phosphates (glycolaldehyde phosphates) from racoxiranecarbonitrile and on (formal) constitutional relationships between 2-oxoethyl phosphates and oligo(hexopyranosyl and pentopyranosyl) nucleotide backbones. Helvetica Chimica Acta 77:2251–2285 Pohorille A, Deamer D (2009) Self-assembly and function of primitive cell membranes. Res Microbiol 160:449–456 Ponnamperuma C, Chang S (1971) In: Buvet R, Ponnamperuma C (eds) The role of phosphates in chemical evolution. Chemical evolution and the origin of life. North-Holland, Amsterdam, Netherlands 216–223 Ponnamperuma C, Mack R (1965) Nucleotide synthesis under possible primitive Earth conditions. Science 148:1221–1223 Ponnamperuma C, Mariner R, Sagan C (1963) Synthesis of adenosine triphosphate under possible primitive earth conditions. Nature 199:222–226 Powner M, Gerland B, Sutherland JD (2009) Synthesis of activated pyrimidine ribonucleotides in prebiotically plausible conditions. Nature 459:239–242 Prabahar KJ, Ferris JP (1997) Effect of dinucleoside pyrophosphates on the oligomerization of activated mononucleotides on Na+montmorillonite: reaction of the 50 -phosphoro4-(dimethylamino)pyridinium[4-(CH3)2NPYPA] with A50 ppA. Orig Life Evol Biosph 27: 513–523 Rabinowitz J, Chang S, Ponnamperuma C (1968) Phosphorylation on the primitive Earth. Nature 218:442–443 Redfield AC (1958) The biological control of chemical factors in the environment. Am Sci 46: 205–221 Reimann R, Zubay G (1999) Nucleoside phosphorylation: a feasible step in the prebiotic pathway to RNA. Orig Life Evol Biosph 29:229–247 Reusch RN, Sadoff HL (1988) Putative structure and functions of a poly-beta-hydroxybutyrate calcium polyphosphate channel in bacterial plasma-membranes. Proc Natl Acad Sci USA 85: 4176–4180 Rytting E, Lentz KA, Chen X-Q, Qian F, Venkatesh S (2005) Aqueous and cosolvent solubility data for drug-like organic compounds. AAPS J 7:E78–E105 Saffhill R (1970) Selective phosphorylation of the cis-20 ,30 -diol of unprotected ribonucleosides with trimetaphosphate in aqueous solution. J Org Chem 35:2881–2883 Sales BC, Chakoumakos BC, Boatner LA, Ramey JO (1992) Structural evolution of the amorphous solids produced by heating crystalline MgHPO4.3H2O. J Mater Res 7:2646–2649 Sauer HW, Goodman EM, Babcock KL, Rusch HP (1969) Polyphosphate in life cycle of PhsarumPlycephalum and its relation to RNA synthesis. Biochim Biophys Acta 195:401–409

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Saygin O (1981) Nonenzymatic photophosphorylation with visible light. A possible mode of prebiotic ATP formation. Naturwissenschaften 68:617–619 Saygin O (1983) Nonenzymatic photophosphorylation of acetate by carbamyl phosphate. A model reaction for prebiotic activation of carboxyl groups. Orig Life Evol Biosph 13:43–48 Schnell R, Oehlmann W, Singh M, Schneider G (2007) Structural insights into catalysis and inhibition of O-acetylserine sulfhydrylase from Mycobacterium tuberculosis: crystal structures of the enzyme-a-aminoacrylate intermediate and an enzyme–inhibitor complex. J Biol Chem 282:23473–23481 Schwartz AW (1972) The role of phosphates in chemical evolution In: Rohlfing DL, Oparin AI (eds) Sources of phosphorus on the primitive earth. Molecular evolution prebiological and biological. Plenum Press, New York, pp 216–223 Schwartz AW, Ponnamperuma C (1968) Phosphorylation on the primitive earth. Phosphorylation of adenosine with linear polyphosphate salts in aqueous solution. Nature 218:443 Schwartz AW, Van der Veen M, Bisseling T, Chittenden GJF (1975) Prebiotic nucleotide synthesis – demonstration of a geologically plausible pathway. Orig Life Evol Biosph 6: 163–168 Serrano A, Perez-Castineira JR, Baltscheffsky M, Baltscheffsky H (2007) H+-PPases: yesterday, today and tomorrow. IUBMB Life 59:76–83 Sharov AA (2009) Coenzyme autocatalytic network on the surface of oil microspheres as a model for the origin of life. Int J Mol Sci 10:1838–1852 Sherwood E, Oro J (1977) Cyanamide mediated syntheses under plausible primitive earth conditions II. The polymerization of deoxythymidine 50 -triphosphate. J Mol Evol 10:183–192 Smayda TJ (1997) Harmful algal blooms: their ecophysiology and general relevance to phytoplankton blooms in the sea. Limnol Oceanogr 42:1137–1153 Srinivasan V, Morowitz HJ (2009) Analysis of the intermediary metabolism of a reductive chemoautotroph. Biol Bull 217:222–232 Steinman G, Lemmon RM, Calvin M (1964) Cyanamide: a possible key compound in chemical evolution. Proc Natl Acad Sci USA 52:27–30 Steinman G, Kenyon DH, Calvin M (1965) Dehydration condensation in aqueous solution. Nature 206:707–708 Terelli E, Wheeler SF (1993) Formation of esters, especially phosphate esters, under “dry” conditions and “mild” pH. Chem Industry 1993:164–165 Todd A (1959) Some aspects of phosphate chemistry. Proc Natl Acad Sci USA 45:1389–1397 Turian G, Rivara-Minten E (2001) Prebiotic phosphoramidation of nucleobases by Mg2+-triggered decyclization of trimetaphosphate. Arch des Sciences 54:233–238 Van Wazer JR (1958) Phosphorus and its compounds, vol 1. Interscience, New York, 954pp Waehneldt TV, Fox S (1967) Phosphorylation of nucleosides with polyphosphoric acid. Biochim Biophys Acta 134:9–16 Wagner E, Xiang YB, Baumann K, Guck J, Eschenmoser A (1990) Chemistry of a-aminonitrilesaziridine-2-carbonitrile, a source of racemic O3-phosphoserinenitrile and glycolaldehyde phosphate. Helvetica Chimica Acta 73:1391–1409 Walling C (1975) Fenton’s reagent revisited. Acc Chem Res 8:125–131 Weber AL (1981) Formation of pyrophosphate, tripolyphosphate, and phosphorylimidazole with the thioester, N, S-diacetylcysteamine, as the condensing agent. J Mol Evol 18:24–29 Weber AL (1982) Formation of pyrophosphate on hydroxyapatite with thioesters as condensing agents. Biosystems 15:183–189 Westheimer FH (1987) Why nature chose phosphate. Science 235:1173–1178 White HB (1976) Coenzymes as fossils of an earlier metabolic state. J Mol Evol 7:101–104 Wolfe-Simon F, Davies PCW, Anbar AD (2009) Did nature also choose arsenic? Int J Astrobiol 8:69–74 Yamagata Y (1999) Prebiotic formation of ADP and ATP from AMP, calcium phosphates and cyanate in aqueous solution. Orig Life Evol Biosph 29:511–520

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Yamagata Y, Inomata K (1997) Condensation of glycylglycine to oligoglycines with trimetaphosphate in aqueous solution II: catalytic effect of the magnesium ion. Orig Life Evol Biosph 27:339–344 Yamagata Y, Mohri T (1982) Formation of cyanate and carbamyl phosphate by electric discharges of model primitive gas. Orig Life Evol Biosph 12:41–44 Yamagata Y, Matsukawa T, Mohri T, Inomata K (1979) Phosphorylation of adenosine in aqueoussolution by electric-discharges. Nature 282:284–286 Yamagata Y, Mohri T, Yamakoshi M, Inomata K (1981) Constant AMP synthesis in aqueous solution by electric discharges. Orig Life Evol Biosph 11:233–235 Yamagata Y, Watanabe H, Saitoh M, Namba T (1991) Volcanic production of polyphosphates and its relevance to prebiotic evolution. Nature 352:516–519

Chapter 4

Abiotic Photosynthesis: From Prebiotic Chemistry to Metabolism Marcelo I. Guzman

Abstract The basic concepts necessary to understand the origin of life are presented. The existing models conducting to the emergence of biochemistry from geochemistry on Earth are introduced. The chapter is focused on abiotic photosynthesis as a way to capture energy and generate the core of central metabolism. Universality in intermediate metabolism, mineral catalysis, hydrothermal vents, and the iron-sulfur proposals are discussed. Special emphasis is given to the model of non-enzymatic prebiotic metabolism that can potentially connect the RNA world and the compartmentalization in protocells approach. Catalysis in mineral surfaces is suggested as the main mechanism for the possible origin of the central metabolic pathways. Candidate semiconductor minerals such as zinc sulfide could have driven this prebiotic chemistry in the young planet. A shallow-water hydrothermal vent system is presented as a model environment where the first microorganisms on Earth used the suggested non-enzymatic chemical reactions as a pioneer mechanism for carbon dioxide fixation and energy storage that resulted in prebiotic metabolism.

4.1

Introduction

The origin of metabolism has been one of the most challenging and controversial issues in the origin of life. Many different proposals have been suggested, each with its own assumptions, and each with evidence supporting a particular mechanism (Cairns-Smith et al. 1992; Guzman and Martin 2008, 2009, 2010; Morowitz et al. 2000; Mulkidjanian 2009; Mulkidjanian and Galperin 2009; W€achtersh€auser 1988, 1990a, b, 2007; Zhang and Martin 2006). An example is the origin of photosynthesis; there are several current publications that advance the discussion of the first photosynthetic organisms (Mulkidjanian 2009; Sadekar et al. 2006; Tice and Lowe

M.I. Guzman (*) Department of Chemistry, University of Kentucky, Lexington, KY 40506-0055, USA e-mail: [email protected] R. Egel et al. (eds.), Origins of Life: The Primal Self-Organization, DOI 10.1007/978-3-642-21625-1_4, # Springer-Verlag Berlin Heidelberg 2011

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2006). In this chapter, a summary of possibilities for the emergence of abiotic metabolism is presented. The proposed model incorporates the chronological events occurring on early Earth conducting to the origin of life. Special emphasis is given to the model of nonenzymatic metabolism. The origin of life is a very difficult problem to approach because no one really knows how and where life emerged. It is also extremely complex to define what life is; however, it is generally accepted that life as we know it on Earth is the result of Darwinian evolution. Present life species must have evolved from previously existent ones during evolution. The concept of a common ancestor for all life was born more than 150 years ago (Darwin 1859). Bacteria, eukaryotes and archaea (Woese and Fox 1977) share the basic structural constituents of the cell and the most fundamental metabolic reactions because all life is based on common biochemical schemes (Kornberg 2000). This hypothetical ancestral organism to all living beings received different names. The most extended and used are the last common ancestor (Fitch and Upper 1987), the last universal cellular ancestor, and the Last Universal Common Ancestor or LUCA (Forterre and Philippe 1999). Known life forms have at least one LUCA1 which emerged from inanimate matter thanks to pure chemical evolution occurring on Earth or elsewhere in the universe. Perhaps, the last universal cellular ancestor was a single organism, an individual cell that existed at a given time and that possessed most of the features (and genes encoding them) that are common to all contemporary organisms. From this single last universal cellular ancestor, the different domains of life would have emerged. All previous hypotheses idealize a first living organism that should have been structurally composed and related to present day cells. Although, the previous unicellular model is generally accepted by the vast majority of scholars in the field, it needs not be the most natural mode of linking the bottom-up approach from geochemical reactor conditions to the top-down extrapolation from existing life, as we know it. On the contrary, a different and completely valid scenario is that the entities that gave rise to the lineages of the three different domains of life were actually the result of independent cellularisation events in an evolving population of precells (Kandler 1994). However, the concept of a distinct LUCA becomes ambiguous in this case, and as a consequence it is more appropriate to speak of the Last Universal Common Ancestral State (LUCAS) (Koonin 2009). Kandler’s scenario may be extended to consider that the last common ancestor was not necessarily organized in a cell-like structure, as modeled by modern unicellular entities. For example, a plasmodial or syncytial organization of life-like intermediates should be a viable alternative, which is heavily underexplored. Independent of the model preferred to describe the tree of life, its three branches indeed depend on one another.2 The assumption

1

While viruses deserve further consideration, they are not considered to be alive in this chapter. All modern eukaryotic photosynthesis is based on the contribution of prokaryote-derived endosymbionts, making in consequence the analysis of extant eukaryotes irrelevant to the origin of photosynthesis. 2

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for this chapter is that LUCA or the precells were not exactly cells but had some degree of similarity to them. Here the term protocell is preferred to refer to the predecessor of LUCA or the precells. Due to the already mentioned uncertainty about how these first hypothetical protocells were made and their structural composition (Moreira and Lo´pez-Garcı´a 2007), it is assumed that they had the four biomolecules found in a cell nowadays: polynucleotides of RNA and DNA, proteins, lipids, and polysaccharides (Deamer 1986; Morowitz et al. 1988; Szostak et al. 2001).

4.1.1

The Requirements for Life

Based on observations of Earth’s biology, there are some requirements for the origin of life. Life requires the right habitat to originate under a range of physical and chemical conditions (pH, temperature, ionic strength, etc.). Life emerging in an aqueous environment is the most commonly accepted hypothesis. In this environment, the cell membrane is the compartment that contains the internal structure, the cytoplasm, where genetic and metabolic processes occur. A more general scheme also includes the interactions among cells of the same or different systems. Empirically, life consists of organic carbon based cells that are found in aqueous solutions, and are composed of macromolecules. Life is able to produce energy, it possesses the capacity to grow and reproduce, and it responds and adapts to environmental conditions through successive generations. In spite of the empirical description given in the previous paragraph, the definition of life generates a major controversy among researchers in the origin of life field. The complexity of the problem is briefly exemplified with the operative definition of life of the National Aeronautics and Space Agency (NASA) of the United States: “Life is a self-sustained chemical system able to undergo Darwinian evolution.” The NASA definition is extensively used in the origins of life field (Horowitz and Miller 1962; Joyce 1994). The term “able to” refers to the presence of genetic material that behaves in a known way. The NASA definition of life is fully compatible with the following one, structured in more detail: To comprehend the beginnings of life requires that we explain the origin of replication as well as of metabolism synergistically. The genetic aspect of the modern definition of life was first proposed by Muller in 1966: “It is to define as alive any entities that have the properties of multiplication, variation and heredity.” While metabolism supplies the monomers from which the replicators (i.e., genes) are made, replicators alter the kinds of chemical reactions occurring in metabolism. Only then can natural selection, acting on replicators, power the evolution of metabolism (Lankenau 2007). The problem is that as a Darwinian process, the definition can only be applied to a population, which then excludes all individual organisms, chemical compounds, and artificial life forms. A species can be defined as groups of potentially interbreeding natural populations, which are reproductively isolated from other such groups (Mayr 1942). The key requirement for grasping the meaning of this

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Self-sustained internal process

S = boundary P = product A = substrate

Fig. 4.1 Scheme of life adapted from Luisi (2006) in “The emergence of life: from chemical origins to synthetic biology,” Cambridge University Press, Cambridge. The definition of life based on autocatalysis is represented by a physical system that can be said to be living if it is able to transform external matter/energy into an internal process of self-maintenance and self-generation. This system is characterized by two competitive reactions, one that builds the components of the boundary (A ! S, d[S]generation/dt), and another one that destroys them (S ! P, -d[S]decay/dt). According to the relative value of these two reaction rates, the system can be in homeostasis3 (d[S]generation/dt ¼ -d[S]decay/dt), or grow (d[S]generation/dt > -d[S]decay/dt), or die (d[S]generation/ dt < -d[S]decay/dt)

definition is a deep understanding of the term “potentially” as that includes time and micro-geography. Single celled microorganisms – as relevant for the origin of life – that are potentially immortal are defined as autonomous germlines but not biological species (Lankenau 2007). Microbial “populations” are only an analogy to metazoan (sexual) populations. But in fact they are something completely different. That is why Manfred Eigen called the large-numbered-Qb virus populations “quasispecies” and not “biological species” (Eigen 1992). A different definition of life based on autocatalysis (Luisi 2003; Varela et al. 1974) is summarized in Fig. 4.1. In this model, the idea of vesicles, confined structures by a boundary, capable of metabolism is essential: the minimal life form is a self-organizing and self-sustaining system with a semi-permeable boundary that, owing to an inner network of reactions, re-generates all the system’s components. The scheme presents the behavior of a cell with an external boundary and a mechanism of internal organization. In Fig. 4.1, S represents the boundary of the life system that is converted in a product P; however, the system is able to regenerate S by transforming incoming substrate A into S again. This definition of

3 Homeostasis is defined here as a state of dynamic equilibrium that does not modify the identity of the physical system.

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life indicates that a system is alive if it is able to transform external matter/energy during a self-sustained internal process to produce its components. This common sense definition is valid not only to describe a single specimen but also a general case of universal value (Luisi 2006). The previous paragraphs summarize basic knowledge about life and living systems. The next section presents some theories and prebiotic chemistry experiments that try to explain the transition from an abiotic environment toward life.

4.1.2

Geochemical and Biochemical Coevolution

One of the first problems to understand in the origins of life is the emergence of biochemistry from a geochemical abiotic system. In other words, how did evolution start from the only operative chemistry in the environment before the existence of life? The famous experiment of Miller supported the hypothesis of chemical evolution (Miller 1953). Miller started his experiment with simple molecules (H2O, NH3, H2, and CH4) contained in a glass ampoule that represented the prebiotic atmosphere. The reactor had two electrodes and was connected in a closed loop to a second balloon half-full of water that represented the prebiotic ocean. Evaporation from the simulated prebiotic ocean was induced by heating the second balloon, while electric spark discharges represented lightning in the first balloon. The “atmosphere” was cooled down and trickled back to the “ocean” through a condenser. The experiment lasted for 7 days and was run as a continuous cycle. The reaction products identified in the gas phase were, in addition to the initial reactants, a mixture of CO and CO2. In the aqueous phase, several amino acids were identified together with some hydroxy acids, aldehydes, and hydrogen cyanide (HCN). The experimental results supported the hypothesis of Alexander Oparin (1938). Later work, using HCN as one of the starting materials, produced some of the bases needed in the synthesis of nucleic acids (Oro´ and Kimball 1961, 1962). In unrelated experiments, the autocatalytic production of sugars was observed from formaldehyde (H2CO) at 100 C, in the presence of calcium carbonate or hydroxide, or of clays such as Kaolinite. The result after 1 week of running Miller’s apparatus (Miller 1953) was that 15% of the original carbon (CH4) was fixed as organic carbon, and a 2% corresponded to a racemic mixture of amino acids, of which glycine was the most abundant. Later, criticism about the relevance of the experiment emerged for the environmental conditions used. Nowadays, it is actually assumed that the atmosphere was not reducing but neutral. However, recent experiments performed under more neutral conditions (Cleaves et al. 2008) revealed the production of the same molecules. It is considered that this mechanism was one of the main sources of organics on the early Earth. Other possible sources were compared and estimated as minor contributors. More recently, advanced adaptations showed the production of pyrimidines when the synthesis took place in ice (Menor-Salvan et al. 2009). The abiotic production of activated pyrimidine ribonucleotides from arabinose amino-oxazoline

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and anhydronucleoside intermediates has been demonstrated in aqueous solutions. The short reaction sequence bypasses the need to synthesize a glycosidic linkage and produces pyrimidine 20 , 30 -cyclic phosphate nucleotide monomer (Powner et al. 2009). However, it is currently unknown how purine monomers could be efficiently synthesized by abiotic mechanisms. A main limitation arising from Miller’s experiments to generate monomers is that they lack a mechanism for the next evolutionary step to proceed. For example, after the synthesis of amino acids, no production of proteins through polymerization reactions was accomplished. Another major criticism is the inability of the mechanism to generate an organized chemical cycle as observed in the following level of evolutionary complexity (Guzman and Martin 2008, 2009).

4.2

Models to Explain the Origin of Life

There are various models and scenarios that could explain the origin of life. Each model represents a unique vision and consequently constrains the experimental simulations taking place in the laboratory. Here are only three such models: (1) the RNA-first world, (2) the compartmentalistic approach, and (3) the proposal of nonenzymatic metabolism. The three models gather major theoretical and experimental work. More information about the RNA-first world (Gilbert 1986; Orgel 2004) or the compartmentalistic approach (Deamer 1997; Forster and Church 2006; Luisi et al. 2006; Morowitz et al. 1988; Szostak et al. 2001) is available in the literature. Among the many other models to explain the origin of life is the thioester world (de Duve 1991) and the glyoxylate scenario (Eschenmoser 2007). The next section only discusses the chemistry that could have made possible the emergence of nonenzymatic metabolism. The implication is that many other models are not considered here.

4.2.1

The Proposal of Metabolism in the Absence of Enzymes

All life systems have a genetic and a metabolic system. One way to define metabolism is as the totality of chemical reactions and physical changes that occur in life systems, including anabolism and catabolism. Anabolism is the part of metabolism that consumes energy, in which simple substances are transformed into more complex chemicals. Catabolism is any metabolic process that involves the degradation of complex structures into simpler products, including the cleavage of organic complexes that release energy needed by the cell or organism (Cammack 2006). There are a few research groups who investigate whether prebiotic metabolism appeared before the existence of enzymes. There is a possibility that their ongoing

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efforts will in the near future link the RNA-first world and the compartmentalistic approach. Several original ideas are explained below.

4.2.1.1

Universality in Intermediary Metabolism

The original ideas of Morowitz and his coworkers (Morowitz et al. 1991; Morowitz et al. 2000; Smith and Morowitz 2004; Srinivasan and Morowitz 2009) constitute the foundation of prebiotic metabolism. They examined the chemistry of a model system, representing a primitive chemolithotroph. Chemolithotrophs are organisms able to obtain their energy from organic matter and synthesize all their necessary organic compounds from CO2. In their analysis for a chemolithotroph, they considered CO2 as the starting reactant, all organics were made of C, H and O, and the energy was provided by redox pairs. In order to predict all the possible organic molecules that would emerge from such a system, its reaction pathways, and their relationships, they used the database of Beilstein on-line, with more than 3.5 millions of organic molecules. After an exhaustive analysis and applying certain physicochemical constraints, they concluded that only 153 organic molecules would emerge in an abiotic environment in the reaction networks, and among them there is a group of 11 carboxylic acids involved in the reverse tricarboxylic acid (r-TCA) cycle also called the reductive citric acid cycle. Figure 4.2 shows the r-TCA cycle. Several reactions take place in the r-TCA cycle (Fig. 4.2) to convert oxaloacetate (a four-carbon compound, C4) into citrate (C6). The reactions include five reductions, numbered 1 through 5, carboxylations (CO2 incorporation), dehydration and hydrations (loss and gain of water, respectively), and isomerizations. Finally citrate undergoes cleavage to regenerate oxaloacetate and a new molecule of acetate (C2) that has incorporated two CO2 molecules. The pathway to incorporate CO2 into acetate produces pyruvate (C3) that consecutively incorporates another CO2 to generate oxaloacetate in an anaplerotic4 reaction. Anaplerotic reactions are those useful to fill up the cycle with a surplus of particular compounds. This surplus allows the entire system to grow and/or to replenish losses from affiliated pathways. The molecules of alanine, glutamate, and aspartate are simple examples of the possible amino acids obtained during metabolic reactions. The species in uppercase letters represent the kind of biomolecules that can be assembled through metabolic pathways. Malonate is only included as an example to indicate a reaction pathway for fatty acids. The cycle can be considered as an engine to produce the major classes of biomolecules. Nowadays, the cycle commonly runs in the opposite direction, in a catabolic forward or oxidizing direction (the opposite of the one described above) to generate the reduced form of nicotinamide adenine

4

Anaplerosis is the act of replenishing TCA cycle intermediates that have been extracted for biosynthesis in cataplerotic reactions. Cataplerosis describes reactions involved in the disposal of TCA cycle intermediates.

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Fig. 4.2 The reverse tricarboxylic acid (r-TCA) cycle is central to universal metabolism. Categories of species produced and examples of potential metabolic intermediates are marked with upper case letters. Combining aspartate and glutamate-derived amino acids all the nucleic bases are eventually derived from amino acids metabolically (Nelson et al. 2000)

dinucleotide (NADH), the energy carrier in a series of decarboxylation reactions mediated by enzymes. Universal metabolism rationalizes that all the possible metabolic cycles are partially common and converge in the r-TCA cycle through at least one of the intermediates. In consequence, this cycle is proposed to be fundamental to the origin of life. Enzymes, specialized proteins able to catalyze all these metabolic reactions, appeared later in metabolism, speeding up the reactions, and eventually took over the complete control of these networks. The deep consequence is that metabolic networks arose before the origin of macromolecules with highly specialized catalytic properties or enzymes. The model is not absolutely accepted, least so by supporters of the RNA-first world scenario, whose main concern relates to the catalytic properties of the minerals involved in the organization of the chemical system (Orgel 2000). Contrarily, others are skeptical about the RNA-first world model, support a nonenzymatic early metabolism of collectively autocatalytic components and consider practically impossible the perfection needed to create an RNA-first world randomly (Shapiro 1986, 2006).

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Clay Mineral Surfaces and Their Importance in Prebiotic Metabolism

Nonenzymatic metabolism requires overcoming the high activation energies (Ea) for the chemical reactions involved. Enzymes have the ability of catalyzing those reactions by decreasing Ea and making possible many energetically unfavorable reactions. In addition, the active sites where the reactions take place in enzymes have a tridimensional geometry of high order where the actual concentration of reactants is considerably larger than in the surroundings. Cairns-Smith explored an idea first suggested by Bernal. Cairns-Smith showed that clay mineral surfaces, in an aqueous environment, can adsorb organic molecules, enhancing their concentration (Cairns-Smith 1977, 1978; Cairns-Smith et al. 1992). Clay surfaces also behave as a mold for polymerization reactions to take place. Organic molecules adsorbed by the clay minerals are not considered to be dissolved in solution. Catalysis and replication in the origin of life occurred early in the evolution process in our planet as a consequence of the high crystalline order existing in the minerals that were widely distributed. Minerals have a simpler crystalline structure than any relevant organic molecule. Crystallites grow and reproduce when they fracture into smaller crystals that can also grow again. The incorporation of impurities during crystal growth represents a prebiotic kind of mutation. Peptides available in the environment are then incorporated to the model. There is then an increase in complexity that is finally followed by a transition to the world of genetics. Cairns-Smith’s model eventually evolved into the world of vesicles. Similar to Morowitz’s proposal previously presented, it also postulates a kind of prebiotic nonenzymatic metabolism. In spite of the wonderful description presented, there is a large difference between the biochemistry to build proteins and nucleic acids in living systems, and fatty acid assembling into vesicles.

4.2.1.3

The Iron-Sulfur World: Deep Water Hydrothermal Vents and Pyrite

Hydrothermal vents were first discovered in 1977 off the Galapagos Islands by a team of geologists exploring the deep sea rift zones (Corliss et al. 1978; Corliss et al. 1979; Lonsdale 1977). Abundant organic compounds were reported to be abiotically synthesized in hot deep-water hydrothermal vents (Holm et al. 1992). The first hypothesis that related those deep water hydrothermal vents and the origin of life was suggested 30 years ago (Corliss et al. 1981). The hypothesis requires certain geochemical and thermochemical conditions to be satisfied (Russell and Hall 1997): Life in deep hydrothermal vents should have emerged as hot, reduced, alkaline, sulfide-bearing submarine waters interfaced with the colder, more oxidized, more acid, Fe2+ >> Fe3+ -bearing water at deep (ca. 4 km) floors of the Hadean ocean (ca. 4 Giga years ago). The suggested energy source was the gradient of pH (four units), temperature (60 C), and redox potential (500 mV) at the interface of these waters over geological time-scales. The exhalate of the deep

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hydrothermal system at very high pressures and temperatures contained outgassing CO, H2, N2, along with reduced nitrogen (NH3 , CN), reduced carbon (CH3COO, H2CO, short alkyl sulfides), CH4, and HS. Colloidally structured iron monosulfide (FeS) precipitate formed at the ocean floor interface. The sustained redox gradient of about half a volt across the walls of the FeS precipitate provided a persistent source of electrons for prebiotic chemistry in addition to catalytic surfaces (Martin and Russell 2003). Simultaneously and independently, a different model of the origin of life was proposed by W€achtersh€auser, which has some common features with universal metabolism (Sect. 4.2.1.1) and lacks the presence of a genetic system. The model is based on the autotrophic origin of life mediated by a nonenzymatic chemical system of the kind of the r-TCA cycle. Autotrophic organisms are those that “produce their own feedstock” from an inorganic source of carbon (carbon dioxide) and a source of energy. In this model, the synthesis and polymerization of organic compounds occurs on the surface of iron-sulfur mineral clusters located in highly reducing volcanic and hydrothermal environments. In W€achtersh€auser’s model, CO2 (and/or CO) is reduced to organic molecules which participated in an autocatalytic cycle as part of chemical evolution is the process in which a compound induces and accelerates a chemical reaction for its own production. The model first appeared as being mediated by a chemolithotrophic bidimensional metabolic system of pyrite, and was then upgraded to iron-sulfur mineral surfaces in general (Huber and W€achtersh€auser 1997; W€achtersh€auser 1988, 1990a, b, 2007). W€achtersh€auser’s model is based on capturing energy released from the environment during the reaction of pyrite (FeS2) formation. In an environment such as a hydrothermal vent, common reactions include those where H2S reacts with available metals cations. Thus, the cation Fe2+ reacts with H2S and precipitates as FeS, and then the reaction FeS + H2S ! FeS2 + H2 takes place favorably accompanied with a negative free energy change DG ¼ 38.4 kJ mol1. Some authors started to call W€achtersh€auser’s model the iron-sulfur-world as it become strengthened. It was suggested that in deep-water hydrothermal vents, both CO2 and N2 could be reduced to organic molecules thanks to the presence of iron and sulfur. The implication is that surface catalysis in W€achtersh€auser’s model and the deep hydrothermal vents are connected. Figure 4.3 shows an example of the possible reaction pathways in W€achtersh€auser’s model. It is worth pointing out that metabolism here is sustained by the electrostatic interactions between negatively charged solute constituents and the mineral surface positively charged. W€achtersh€auser explained that the experimental goal of this model is to reach large chemical reaction cascades to catalytically feed a nonenzymatic metabolic system from the beginning. To judge on the W€achtersh€auser–Morowitz proposals, there is not enough experimental evidence to prove them right as yet. Some experiments presented promising results (Cody et al. 2000), from simulating deep-water hydrothermal vent conditions at high temperatures (250 C) and pressures (100 atm.). The highest accomplishment was the generation of pyruvate (0.07%) and acetate (0.05%) from formate (HCOOH) using FeS and nonanethiol (CH3-(CH2)8-SH) as reactants. The most exciting result

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Assembling of peptides with a feedback mechanism in the system

8 7 Library of minerals central to the cluster: Iron, Cobalt, Nickel 6

Gases:H2S/CO/CO2

1

2

5 4

3

Fig. 4.3 Reactions in the iron-sulfur world. Solid arrows represent the reactions needed to convert carbon monoxide (CO) into peptides. The individual reaction steps are: (1) The adsorption of CO to the mineral cluster. (2) The reduction of adsorbed CO to a surface-bound carboxylic group (–COOH). The –COOH group can be released as formate (HCOOH). A molecule of HCOOH can re-enter the system as adsorbed CO. (3) The –COOH group undergoes reduction to a methyl (–CH3) moiety that bridges the alternative adsorption of carbon dioxide (CO2) through a methanethiol (CH3–SH) intermediate. Further reactions of the –CH3 group with CH3-SH generate surface-bound acetyl group (–C(O)–CH3). (5) The –C(O)–CH3 group is released as methylthioacetate (CH3–S–C(O)–CH3) or acetic acid (CH3COOH). CH3–S–C(O)–CH3 can also be converted to CH3C(O)OH. (6) Surface mediated processes allow adsorbed –C(O)–CH3 to incorporate a new carbonyl group and form a surface-bound biacetyl moiety (–C(O)–C (O)–CH3). The –C(O)–C(O)–CH3 group is desorbed in a redox process as pyruvic acid (CH3C (O)COOH). (7) CH3C(O)COOH undergoes a mineral-mediated reductive amination reaction to produce alanine (CH3CH(NH2)COOH). (8) Activated CH3CH(NH2)COOH (and other similarly generated amino acids) polymerize on the surface of the mineral and produce a library of peptides. Peptides or a cycle such as the r-TCA cycle have the ability to affect the system under optimal conditions to obtain autocatalysis and evolution (Figure adapted from W€achtersh€auser (2000) where the reaction conditions are reported)

reported by Huber and W€achtersh€auser (Huber and W€achtersh€auser 2006) was the production of a-amino acids and a-hydroxyacids such as lactate in 0.1–1.0 mM concentrations, starting with CO, KCN, CH3SNa, Na2S, Fe2+, and Ni2+, at high temperatures (100 C) and pressures (10 atm.).

4.2.1.4

Metabolism on Semiconductor Surfaces

The model of metabolism on mineral semiconductors rationalizes the facts about the first known life systems, and proposes a new mechanism to simultaneously transfer energy and fix organic matter from an existing prebiotic environment.

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The Early Earth Fossil Record The earliest evidence for biological carbon fixation was dated at about 3.8 Giga years ago (Giga-annum, Ga, Ga ¼ 109 years) (Mojzsis et al. 1996; Schidlowski 1988) from the isotopic composition of sedimentary rocks. The oldest life fossils largely accepted by the scientific community were identified in the Buck Reef Chert, South Africa, to be at least 3.4 Ga old (Tice and Lowe 2004) and they are composed of filamentous structures. Morphological studies were interpreted as oxygen evolving cyanobacteria structures. The facts point to the existence of early bacteria able to fix carbon dioxide. These presumable autotrophs not only fixed CO2, but were widely distributed, and were likely formed in a shallow-water oceanic environment (Allwood et al. 2006), exposed to sunlight irradiation (Guzman and Martin 2008, 2009). A closely related proposal agrees with this model and further advances it into a well structured origin of life model (Mulkidjanian 2009; Mulkidjanian and Galperin 2009). Environmental conditions about 4.5–3.5 Ga ago, should have included a 25% lower luminosity than present levels due to the younger Sun (Cockell 2000) but larger levels of UV radiation at the Earth surface due to the lack of ozone in the atmosphere. The anoxic atmosphere was mainly composed by other gases such as carbon oxides, nitrogen, and argon. Atmospheric models constrain the partial pressure of CO2 in the early atmosphere to levels between 0.1 and 10 atm. (Kasting 1993).

Prebiotic Photosynthesis Chlorophyll-based systems allow present day photosynthetic bacteria, algae, and plants to transform sunlight into chemical potential energy to drive ATP synthesis and reduce CO2 to sugars (Nelson et al. 2000). On the contrary, a main unknown issue about the origin of life is to identify the first energy capture and carbon fixation mechanism used by the primitive organisms that populated the young biosphere (Guzman and Martin 2008). The cyanobacteria, the purple bacteria, and the green sulfur bacteria are examples of the various photosynthetic electron transport schemes existent. To date, there are six known carbon fixation pathways used by living organisms (Huber et al. 2008; Thauer 2007). One of them, the r-TCA cycle is often proposed as the leading candidate to be the first carbon fixation mechanism because it operates in ancient green sulfur bacteria (e.g., Chlorobium). Additionally, all six mechanisms share at least a common intermediate, so they are linked in the model of metabolism in mineral semiconductor through central universal metabolism. The result is the possible combination of different carbon fixations mechanisms, or at least the existence of a simplified mechanism that was fundamental for the origin of life. The implication is that this prebiotic mechanism should have used some of the key organic compounds that participate in central anabolism. Moreover, the system should have also been able to implement the core reactions involved in central metabolism abiotically and nonezymatically. For the previous reason, it relies on sulfur-containing semiconductor minerals to

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provide the required level of organization. The advantage of the semiconductor is to provide free energy available in the prebiotic environment to obtain an autonomous chemical cycle.

Scenario Conducting to Prebiotic Photosynthesis It is thought that after the period of late heavy bombardment, between 4.1 and 3.8 Ga ago, there was a boost in the geothermal and volcanic activity on Earth. Consequently, there were more hydrothermal sources distributed in the ocean than today. Among them, there are, and there were, shallow-water hydrothermal vents, located in the top 200 m of the ocean column. The water temperature in these environments is more temperate (3–30 C) than in deep-water hydrothermal vents. Deep-water hydrothermal vents are those located below 200 m. Figure 4.4 presents a scheme for the model environment of metabolism in semiconductor minerals. The scheme shows an oceanic shallow-water vent, in which there is an enrichment of metal ions (Zn2+, Mn2+, etc.) and reducing gases (H2S, H2, etc.) in comparison to the surroundings waters. It is at the exhaust of this kind of venting system that hydrogen sulfide gases H2S(g) or hydrosulfuric acid H2S(aq) combine with metals to form semiconductor colloidal suspensions. The respective mineral sulfide

Fig. 4.4 Shallow-water hydrothermal vent for life generation. The world of semiconductor driven metabolism illustrates the role of photochemistry over mineral surfaces such as zinc sulfide (ZnS) to allow the reactions involved in the reverse tricarboxylic acid (r-TCA) cycle. The output of the r-TCA cycle (see Fig. 4.2) is summarized as carbohydrates (CH), fatty acids, terpenoids, and lipids (FTL), amino acids (AA), and nucleobases (NB) (Adapted from Guzman and Martin (2009))

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semiconductor, such as ZnS, MnS, etc., precipitates due to their very low solubility. The newly formed semiconductor particles can remain in suspension or eventually accumulate as deposits over the bottom of the shallow sea. This scenario provides environmental conditions such as those needed for the sunlight promoted photoactivation of semiconductor catalysts. Sunlight penetrates the shallow water column, transferring energy to dissolved CO2 and/or to organic compounds mediated by the photocatalyst. Organics were provided to the system by any of the relevant prebiotic synthesis such as the Miller experiments (Miller 1987), the mechanism herein described (Guzman and Martin 2008, 2009, 2010; Zhang et al. 2004; Zhang et al. 2007), or by extraterrestrial input from carbonaceous chondrites (Chyba and Sagan 1992). The new scenario not only provides sites for adsorption of organics in sulfide minerals, it also has the main advantage of allowing sunlight energy to be transferred through semiconductor minerals. The unbeatable potential of this proposal is the ability to convert otherwise prevented metabolic-like reactions into viable ones. Some examples were discussed in the literature for the reduction reactions of the r-TCA cycle (Guzman and Martin 2008).

Photocatalysis Applied to Prebiotic Metabolism The photocatalytic principle consists in the absorption of a photon in the wavelength band of absorption of the mineral. The most extensively studied mineral for this application is sphalerite, the cubic form of zinc sulfide. Sphalerite has a bandgap, the energy difference between the valence and the conduction bands, of DE ZnS ¼ 3.6 eV. This DE ZnS corresponds to absorption of radiation at 344 nm. Upon absorption of a photon by the semiconductor, an electron is promoted from the valence band to the conduction band (Fig. 4.5). An oxidizing hole is simultaneously generated in the valence band. The highly reducing electrons located in the conduction band have a standard reduction potential of 1.04 V vs. NHE and can reduce e.g., CO2 to formate (HCOO) via the CO2 radical anion. Hydrogen sulfide

Fig. 4.5 Photodriven reduction and oxidation reactions on colloidal semiconductor particles. Example 1: Photoreduction of carbon dioxide to formate using a conduction-band (CB) electron is shown; the corresponding oxidation of HS- by a valence-band (VB) hole is shown. Example 2: Photoreduction of pyruvate to lactate and photooxidation of lactate to pyruvate

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H2S(g) evolving from the hydrothermal source (or other reducing species) acts as a chemical bridge to close the electrical circuit when reacting with the oxidizing holes (+2.56 V vs NHE) to produce polysulfoxy anions. The novelty of this mechanism is to provide complementary oxidation and reduction reactions that occur at the same time in the tiny nano- to micro-meter diameter scale colloidal particles. Some relevant materials such as ZnS are better reducing agents, while others such as TiO2 are more efficient for oxidation reactions. Experimental efforts, to simulate the possible way that metabolism emerged, try to mimic as closely as possible the scenario described in the preceding section. The photochemical reactor employed is provided with a medium pressure mercury lamp, irradiating UV light (200–400 nm). The colloidal suspension of ZnS is generated in situ upon mixing of stoichiometric amounts of fresh and degassed solutions of sodium sulfide and zinc sulfate to a final load of 2.3 g/l. For the synthesis, stirring is provided while an inert gas such as nitrogen is continuously bubbled to maintain an anoxic atmosphere. The synthesized ZnS represents a model for the semiconductors present in shallow-water hydrothermal vents. The temperature, pH, and the gas phase atmospheric composition, including the concentration of CO2 can be adjusted. ZnS was found to catalyze the simultaneous reduction of CO2 to yield formic acid and the oxidation of sulfite to yield sulfate has been reported with a quantum efficiency of 80% (Henglein 1984; Henglein et al. 1984). Recent experimental work with ZnS semiconductor catalyst shows that when sodium lactate and CO2 are added to the system, under the presence of sodium bisulfide (pH ¼ 7.0), a chain of reactions starts. The time series of chromatographic analyses showed the oxidative generation of pyruvate first, followed by the formation of succinate, glutarate, a-ketoglutarate, and isocitrate. Considerably high yields are reported for several reactions. Figure 4.6 presents a scheme including all the observed molecules in laboratory simulations as indicated with bold case letters (Guzman and Martin 2008, 2009, 2010; Zhang and Martin 2006). The result of experiments and controls demonstrate the heterogeneous photo-generation of the organic products. Another experiment showing the formation of lactate (15% yield) from glyoxylate requires a mechanism that is driven by photochemistry promoted on ZnS surfaces (Guzman and Martin 2010). Oxalate and glycolate are formed as first-generation C2 coupling products of CO2 as well as by the oxidation and the reduction of glyoxylate, respectively. These steps have been demonstrated individually. Also marked in Fig. 4.6 are more complex conversions observed for lactate and pyruvate, such as the one-step production of succinate (12% yield), a-ketoglutarate (50%), and isocitrate (11%). Complex conversions for other compounds include the reduction of oxaloacetate to malate (75%) and of fumarate to succinate (95%) (Guzman and Martin 2008; Zhang and Martin 2006). The carboxylation of a-ketoglutarate to oxalosuccinate (2.5%) has also been demonstrated (Zhang and Martin 2006). The reaction channel of pyruvate to malate via oxaloacetate corresponds to a carboxylation that, although has not been observed, was suggested as possible at pH 7.0 below 25.0 C in a kinetic study with variable temperature if equilibrium conditions were reached (Guzman and Martin 2008).

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Fig. 4.6 Abiotic metabolism in the presence of illuminated ZnS and CO2. (a) The r-TCA cycle with the metabolites observed during the laboratory simulations. (b) Anaplerotic-like synthesis of metabolites feeding the r-TCA cycle. Species in bold letter have been observed. G&Ma Guzman and Martin 2008, G&Mb Guzman and Martin 2009, G&Mc Guzman and Martin 2010, Z&M Zhang and Martin 2006, ERM Eggins, Robertson, Murphy, et al. 1998

The pathway to CO2 into intermediates of central metabolism via ZnS photocatalysis is under continuous progress. The model presented uses UV light as the energy source captured by mineral semiconductors and is able to start key

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metabolic-like reactions from CO2 and organics, which would be otherwise unviable reactions. An important accomplishment is the generation of a-ketoglutarate, an energetically expensive compound. Simpler reductions such as fumarate conversion to succinate are also reported. There are some steps involving dehydration, hydration, isomerization, and fragmentation reactions remaining to close the cycle completely. Primitive carbon fixation as postulated by this model should have undergone further evolution, resulting in present day metabolism. Such evolution took place through an abiotic metabolic cycle that developed in the environment previously to the existence of enzymes. Does it mean that semiconductor driven metabolism preceded the RNA-first world and the existence of enzymes? What is known, based on the observation of chemoautotrophic life, and as discussed in the introduction of this chapter (Sect. 4.1), is that there was at least one original common ancestor of this kind of cellular life forms in the tree of life (Woese et al. 1990) that gathered simultaneously a genetic and a metabolic system (see Sect. 4.1.1). How is mineral photocatalysis connected with prebiotic photosynthesis? A possible connection between mineral photocatalysis and prebiotic photosynthesis is the spontaneous abiotic formation of vesicles on the prebiotic Earth. Vesicles may have provided the compartment for the origin of life. An hypothesis is that this original cell should have been a protocell taking advantage of this mechanism of energy transduction (Guzman and Martin 2008, 2009; Summers et al. 2009), without using ATP (Kee and Pacek 2011). The chemistry of the system later evolved to allow more complex chemistry and to include polymerization reactions. A further interpretation of current results suggests that encapsulated semiconductor particles are a potential engine to drive this chemistry in protocells. The second way to conceive this relationship between photocatalysis and prebiotic photosynthesis is based on the finding that lipids can attach to and surround mineral grains (Hanczyc et al. 2003). Lipids should not only separate inside from outside, as of importance for emerging cells, they should likewise, and perhaps more importantly in a prebiotic setting, insulate the mineral grain from the water solution in an electro-chemical sense. The consequence may be an effective separation of charge, e.g., if electrons are absorbed inside by photocatalytic minerals, such as ZnS, and protons can pass the hydrophobic layer more easily than larger ions. In a lipid–peptide heterogeneous system with hidden polar groups, the system can potentially rearrange itself in gatable configurations. In this way, the immediate recombination of electron–proton pairs would be prevented in the vicinity of the mineral grains, and proton gating could gradually be optimized, so as to allow chemical energy to be extracted at the outside of the lipid-enclosed mineral cavity. The oxidizing hole remaining in the mineral center could subsequently be assisted by organic pigments at or in the membrane. Eventually, a general organic takeover of primordial mineral functions in proto-biogenesis takes place. In this second and alternative way to connect mineral photocatalysis with prebiotic photosynthesis, photocatalytic vesicles were not directly precursory to later protocells. Instead, much of the following prebiotic evolution occurred at

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the outside of these primary vesicles, somewhat in line with the “inside out-cell” or “obcell model” (Cavalier-Smith 2001). In conclusion, the model of photocatalysis applied to prebiotic metabolism: (1) identifies the proper environmental scenario, based on the fossil record of life on Earth, to synthesize mineral photocatalysts, (2) allows persistant carbon fixation on early Earth, (3) constitutes an organized mechanism to capture sunlight energy in semiconductor mineral particles for the production of organic compounds central to metabolism, (4) attributes to natural chemical selection the existence of molecules central to metabolism, (5) unifies universality in intermediary metabolism and surface metabolism with metabolism on mineral photocatalysts, and (6) connects the metabolism-first and the RNA-first models of the origin of life.

References Allwood AC, Walter MR et al (2006) Stromatolite reef from the early archaean era of Australia. Nature 441(7094):714–718 Cairns-Smith AG (1977) Takeover mechanisms and early biochemical evolution. Biosystems 9(2–3):105–109 Cairns-Smith AG (1978) Precambrian solution photochemistry, inverse segregation, and banded iron formations. Nature 276(5690):807–808 Cairns-Smith AG, Hall AJ et al (1992) Mineral theories of the origin of lie and an iron sulfide example. Orig Life Evol Biosph 22(1–4):161–180 Cammack R (2006) Oxford dictionary of biochemistry and molecular biology. Oxford University Press, Oxford Cavalier-Smith T (2001) Obcells as proto-organisms: membrane heredity, lithophosphorylation, and the origins of the genetic code, the first cells, and photosynthesis. J Mol Evol 53(4): 555–595 Chyba C, Sagan C (1992) Endogenous production, exogenous delivery and impact-shock synthesis of organic molecules: an inventory for the origins of life. Nature 355:125–132 Cleaves HJ, Chalmers JH et al (2008) A reassessment of prebiotic organic synthesis in neutral planetary atmospheres. Orig Life Evol Biosph 38(2):105–115 Cockell CS (2000) The ultraviolet history of the terrestrial planets – implications for biological evolution. Planet Space Sci 48(2–3):203–214 Cody GD, Boctor NZ et al (2000) Primordial carbonylated iron-sulfur compounds and the synthesis of pyruvate. Science 289(5483):1337–1340 Corliss JB, Lyle M et al (1978) The chemistry of hydrothermal mounds near the Galapagos Rift. Earth Planet Sci Lett 40(1):12–24 Corliss JB, Dymond J et al (1979) Submarine thermal springs on the Gala´pagos Rift. Science 203(4385):1073–1083 Corliss JB, Baross JA et al (1981) An hypothesis concerning the relationship between submarine hot springs and the origin of life on earth. Oceanol Acta 4(Supplement):56–69 Darwin C (1859) The origin of species by means of natural selection. John Murray, London deDuve C (1991) Blueprint for a cell: the nature and the origin of life. Carolina Biological Co, Burlington Deamer DW (1986) Role of amphiphilic compounds in the evolution of membrane-structure on the early Earth. Orig Life Evol Biosph 17(1):3–25 Deamer DW (1997) The first living systems: a bioenergtic perspective. Microbiol Mol Biol Rev 61(2):239–261

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Part II

Facets of an Ancestral Peptide World

Chapter 5

Salt-Induced Peptide Formation in Chemical Evolution: Building Blocks Before RNA – Potential of Peptide Splicing Reactions Daniel Fitz, Thomas Jakschitz, and Bernd M. Rode

Abstract From a chemical point of view, it seems likely that peptides and smaller proteins were the first biomolecules which may have formed on the prebiotic Earth. In the presence of sodium chloride and copper ions, amino acids are readily connected to oligomers via the Salt-Induced Peptide Formation (SIPF) reaction mechanism in aqueous solution under locally conceivable primitive Earth conditions. The SIPF reaction shows some specific properties suggesting a close relationship to modern life forms, like a preference for a-amino acids and even stereospecific differentiation in favour of the L-forms of some amino acids. Furthermore, the amino acid sequences which are preferably formed by this reaction can still be found with a probability much above average in proteins of still existing life forms, like archaea and other prokaryotic cells. Once formed, even short peptides have a number of highly interesting abilities pointing towards possible further evolutionary pathways: chain elongation on the surface of clay minerals, formation of nanovesicles with membrane-like structure, autocatalytic self-replication from fragments, stabilisation of phosphate ions against precipitation, etc. When at a later stage of chemical evolution the RNA/DNA based replication mechanism began to establish, initially it would probably just have reproduced and gradually replaced peptides and proteins which had existed before and already had exerted some biochemical impact on the environment. By splicing preexisting peptides to larger proteins, the complexity and diversity of biomolecules could have undergone a tremendous progress at that period of evolution towards the highly complex life forms populating the Earth nowadays.

D. Fitz • T. Jakschitz • B.M. Rode (*) Division of Theoretical Chemistry, Institute of General, Inorganic and Theoretical Chemistry, University of Innsbruck, Innrain 52a, 6020 Innsbruck, Austria e-mail: [email protected] R. Egel et al. (eds.), Origins of Life: The Primal Self-Organization, DOI 10.1007/978-3-642-21625-1_5, # Springer-Verlag Berlin Heidelberg 2011

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Introduction

The origin of life on earth around 3.8 billion years ago took place in an environment that seems rather hostile to most modern life forms. After the end of the heavy meteoritic bombardment phase around 3.9 billion years ago, the existence of a stable liquid hydrosphere, which is usually considered as a precondition for the formation of biomolecules, is indicated by sedimentary rocks formed at that time (Nutman et al. 1996). Nevertheless, a number of physical factors made the chemical evolution a challenging task: a hot temperature slightly below the boiling point of water (60–90 C), strong UV irradiation due to the absence of an ozone layer, high inorganic salt concentrations in the primordial ocean (Spitzer and Poolman 2009), volcanic eruptions, shockwaves, etc. A similar scenario with an at least temporarily available liquid hydrosphere most likely even existed some hundred million years earlier (Wilde et al. 2001; Sleep 2010), but the frequent impact of meteorites until around 3.9 billion years ago might well have repeatedly evaporated oceans that had condensed in the meantime, thereby probably annihilating any biochemical systems that may have been established before. In today’s life forms, DNA and RNA are actually the central molecules as they carry all the basic genetic information. When it was found that certain RNA molecules, besides their function as carriers of information, also act as catalysts for a number of biochemical reactions, it seemed obvious that life on earth might have started as an RNA world. In this context, a number of possible formation reactions for the RNA building blocks have been proposed so far. The ribose sugar is formed as a minor sideproduct of the formose reaction discovered by Butlerow in the nineteenth century (Butlerow 1861; Mizuno and Weiss 1974; Breslow 1959; Decker et al. 1982; Shapiro 1988) from formaldehyde catalysed by calcium hydroxide under basic conditions. A possible pathway for the formation of the nucleobases was proposed by Oro in the 1950s when he produced adenine by refluxing solutions of hydrocyanic acid or ammonium cyanide (Oro 1960, 1961a, 1961b; Oro and Kimball 1961, 1962). Similar reactions under varied conditions resulted in the formation of further nucleobases (Joyce 1989; Ferris and Orgel 1965, 1966; Ferris et al. 1972; Ferris and Hagan 1984; Levy et al. 1999; Ferris 1999; Barks et al. 2010). None of these formation reactions, however, seems to be compatible with any realistic prebiotic earth scenario, since rather high concentrations of formaldehyde, formamide or HCN are required for these syntheses. A further problem is the primordial availability of phosphate, since any dissolved phosphate would instantly precipitate in the presence of Ca2+ or similar metal ions. Even if the three building blocks of RNA (ribose, nucleobases and phosphate) would have been available on the prebiotic earth, their assembly and polymerisation in a correct regioselective way is again a serious obstacle even under pure laboratory conditions (Gibson 1993; Joyce et al. 1987; Eschenmoser 1999). It has been proposed that this problem may be overcome by reaction sequences bypassing free ribose and nucleobases and directly leading to complete nucleotides

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from possible prebiotic feedstock molecules, such as cyanamide, cyanoacetylene, glycolaldehyde, glyceraldehyde, and inorganic phosphate (Powner et al. 2009). Furthermore, RNA molecules are much more unstable than for example amino acids and peptides against high temperatures, salinity, and UV irradiation (Larralde et al. 1995; Lazcano and Miller 1996; Levy and Miller 1998; Shapiro 1995; Cleaves and Miller 1998), which were all abundant at the time when chemical evolution started. From an information theoretical approach (Dyson 1999), a set of around ten different building blocks (for example, ten amino acids) is much more tolerant against errors and appropriate for a stepwise production of more and more complex systems than a set of only four different nucleobases. On the other hand, computer simulations of Eigen’s hypercycles (Eigen and Schuster 1977; Niesert et al. 1981) have shown that they would quickly lead to catastrophic events like short circuits and a population collapse including a loss of catalytic activity of the system. All in all, an RNA world as the first step of chemical evolution seems rather implausible from a chemical point of view. In the next sections, we will see that an amino acid and peptide world would be a much better and more realistic candidate as a starting phase of chemical evolution towards life. The capability of peptides to replicate from smaller fragments (Isaac and Chmielewski 2002; Lee et al. 1996; Yao et al. 1998) gives them all the necessary properties for a very primordial form of life. After such a metabolism-first scenario, RNA and DNA may have come into play at a later stage of evolution, thereby making replication much more efficient but probably only gradually replacing peptides and proteins that had already existed before.

5.2

Primordial Formation of Amino Acids

Miller and Urey showed with their famous experiment in the 1950s (Miller 1953, 1955; Bada and Lazcano 2003) that amino acids, along with a variety of other small organic molecules, can easily be formed in atmospheric processes when a reducing gas mixture (H2, CH4, NH3, water vapour) is exposed to electric discharges simulating lightning events. In a neutral primordial atmosphere consisting mainly of carbon dioxide, nitrogen and water vapour which is supported by modern geochemical findings (Levine et al. 1982; Delano 2001; Holland 1984), however, organic molecules are only formed in much lower yields, but a number of amino acids could still be detected in such a scenario (Plankensteiner et al. 2004a, 2006). Considering the prebiotic atmosphere as a large ‘reaction chamber’ and the long time scale available, substantial amounts of amino acids should have been provided for further evolutionary processes. A second source of amino acids were meteorites of the carbonaceous chondrite type. Most of these meteorites were formed together with the solar system, and some of them still crash onto earth nowadays like, for example, the Murchison meteorite that fell down in Australia in 1969. This extensively investigated

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meteorite (Kvenvolden et al. 1970, 1971; Krishnamurthy et al. 1992; Pizzarello et al. 2004) contains numerous classes of organic molecules including more than 70 different proteinogenic and non-proteinogenic amino acids. Furthermore, amino acids can also be formed in special hydrothermal vent environments on the surface of pyrite (FeS2) when high partial pressures of carbon monoxide are present (Wa¨chtersha¨user 1990, 2000; Cody et al. 2000; Huber and W€achtersh€auser 1997). Such scenarios are quite rare on earth today but may have been more widespread in former times.

5.3

Primordial Formation of Peptides

Peptide formation from amino acids in aqueous solution faces two major problems. First of all, it is a thermodynamically unfavourable process with the equilibrium far on the side of free amino acids, especially in aqueous solution. Secondly, on the kinetic side at almost any pH value, the unfavourable protonation status of the amino group (protonated at lower pH values) and carboxy group (deprotonated at higher pH values) provides a high activation energy barrier. To overcome these difficulties. it has been proposed to start from activated amino acids like amino acid esters (Brack et al. 1975) or to use a number of condensation reagents like cyanates (Flores and Leckie 1973), trimetaphosphates (Chung et al. 1971), imidazole (Weber et al. 1977; Sawai and Orgel 1975, Sawai et al. 1975), cyanamides (Steinman et al. 1964; Steinman and Cole 1967) and others, but none of these pathways is actually plausible under primordial earth conditions. Also, melting experiments of dry amino acids have been reported, where at elevated temperatures up to 180 C water molecules released by condensation reactions quickly evaporate and spherule-like polymers (‘proteinoids’) were formed when an excess of amino acids with acidic or basic side chain were used (Fox and Harada 1958, 1960; Harada and Fox 1958). These ‘proteinoids’, however, have later been found to contain mainly ester-like bonds between the side chains and have, therefore, little in common with real peptides or proteins (Andini et al. 1975). One should keep in mind that bond formations of this kind between reactive groups of the amino acid’s side chains may well have impurified or ‘poisoned’ peptides that probably were formed in similar dehydrating scenarios, if a high fraction of amino acids with reactive side chains was present. Only the most simple amino acid, glycine, can be oligomerised rather easily, for example in a flow reactor in which an aqueous solution of glycine is alternately heated to 200 C in a high-pressure chamber and then cooled down again (Imai et al. 1999), with higher yields and longer oligoglycine peptides formed when some CuCl2 is also added. Under similar high-temperature and high-pressure conditions occurring in hydrothermal vent systems in an aqueous slurry of (Ni,Fe)S and in the presence of CO and H2S or CH3SH as a catalyst and condensation agent, the formation of smaller peptides like Phe-Phe could also be achieved (Huber and W€achtersh€auser 1998; Huber et al. 2003). Such conditions, however, also increase

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hydrolysis rates of existing peptides and racemisation of amino acids. Also sphalerite (ZnS) has been shown to efficiently catalyse peptide formation, at least from glycine (Ohara and Cody 2010). Peptide formation from simple amino acids like glycine and to a smaller extent alanine is also catalysed on the surface and edges of clay minerals such as kaolinite or montmorillonite (Lahav et al. 1978; Flores and Bonner 1974; Bujdak et al. 1994, 1995) and smectites, especially when rich in Fe(II) (Bujdak and Rode 1996). As this mechanism provides only low yields and is not suitable for more complex amino acids, the main role of surface adsorption on clay minerals could rather be to stabilise existing peptides against hydrolysis, to concentrate them and to promote chain elongation, which is catalysed more efficiently than the formation of dipeptides from amino acids (Bujdak et al. 1994). Also, silica and alumina show similar if not better properties in this regard, in comparison with composite natural minerals (Basiuk et al. 1990; Bujdak and Rode 1997, 1999, 2001, 2003).

5.4

The Salt-Induced Peptide Formation (SIPF) Reaction

In the context of chemical evolution, the main focus, of course, is on organic molecules and biomolecules, whereas the possible role or influence of inorganic substances like minerals or dissolved salts, which were certainly abundant on the primitive earth, is often neglected. Such dissolved salts, however, play key roles in the Salt-Induced Peptide Formation (SIPF) reaction, which probably is the easiest and most universal way to form small peptides under locally possible primordial earth conditions in aqueous solution. Many organic molecules like amino acids form complexes with di- and trivalent metal cations which leads to some interesting effects. Such a complex containing two amino acids would bring them into close vicinity and the complexation of an amino acid’s amino group prohibits protonation even at acidic pH values, thereby retaining its nucleophilic character. These two properties could drastically lower the activation energy barrier for the formation of a peptide bond. On the other hand, the primordial ocean contained similar if not higher concentrations of sodium chloride in comparison with today’s oceans (500 mM). In dilute solution, the sodium cations are on average surrounded by six water molecules in their first hydration shell. At higher concentrations, this six-fold coordination cannot be maintained any longer, which was demonstrated in Monte Carlo simulations of solutions with different sodium chloride concentrations (Limtrakul and Rode 1985; Limtrakul et al. 1985). This phenomenon provides a strong dehydrating effect at higher sodium chloride concentrations, and it can push the equilibrium constant of amino acids and peptides decisively towards the condensed polymers, depending on the sodium chloride concentration. These considerations were, for the first time, experimentally tested in the late 1980s when solutions containing glycine, sodium chloride, and different di- and trivalent metal cations were heated to 85 C for a few days (Schwendinger and

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Rode 1989; Rode and Schwendinger 1990). Among the tested metal ions, (Mg(II), Ca(II), Cr(III), Mn(II), Co(II), Ni(II), Cu(II), Zn(II), Cd(II), Al(III), Fe(II) and Mo(VI)), only Cu(II) provided a substantial formation of di- and also triglycine if the sodium chloride concentration was at least 3 mol/l, while Mo(VI), Co(II), Ni(II) and Fe(II) provided only traces of diglycine and the other metal ions no detectable peptide formation at all. This outstanding property of the Cu(II) ion may likely be explained by its special position in the Irving-Williams series of the first row transition metal ions, according to which Cu(II) forms the most stable complexes of these metal ions with organic ligands (Irving and Williams 1948). Nowadays, copper is one of the most abundant transition metal ions found in sea water (Quigley and Vernon 1996), where the more soluble Cu(II) is preferentially complexed with bisulphide (Cu(HS)+) and/or organic ligands (Al-Farawati and van den Berg 1999). In modern aerobic life, it also plays a role in protein and enzyme chemistry as the metal centre of a number of important enzymes. The anoxic state of the primordial ocean, however, makes the early availability of Cu(II) in bulk more problematic (Ochiai 1978, 1983). On the other hand, large amounts of copper sulphide minerals can be found in precambrian rock formations (‘greenstone belts’), where photooxidation or other modes of atmospheric weathering could have made copper(II) ions available on the primordial earth. In contrast to most of the anoxic prebiotic hydrosphere, the early atmosphere is estimated to have already acquired some oxygen (Levine and Augustson 1982), as mainly energised by sunlight irradiation. This was potentially high enough to keep Cu(II), once mobilised, in the active divalent state, especially if these ions were sequestered by chelation in organic layers dominated by carboxylic acids. Not inconceivably, therefore, the SIPF reaction can have played an important role in the very early steps of chemical evolution, like the formation of the first peptides in a locally favourable surface environment. To reach the required concentrations of sodium chloride, the Salt-Induced Peptide Formation reaction could have taken place in the form of evaporation cycles in coastal lagoons, puddles or salt lakes where the water evaporated rather fast due to the high temperatures. After the reaction has taken place in the upconcentrated residue, these places were filled up again by high tide or by rain and the next cycle could start. Such a scenario provides even higher peptide yields than the constant volume experiments mentioned before (Saetia et al. 1993).

5.4.1

Reaction Mechanism of the SIPF Reaction

The crucial species for peptide formation in the SIPF reaction is a Cu(II) complex containing two amino acids (or an amino acid and a peptide), one chloride ligand, and two water molecules staying weakly bound at elongated distances due to the Jahn-Teller distortion of copper (see Fig. 5.1). One amino acid is bonded twice to the copper ion, via its carboxy and amino groups. The coordination of the amino group prevents protonation, although the solution has a rather acidic pH value

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Fig. 5.1 Active SIPF complex with one chelating L-alanine ligand, one L-alanine bound end-on via its carboxy group, one chloride ligand and two water molecules at elongated distances. The complex was geometry optimised with the ab initio Hartree Fock method with triple-zeta basis sets (double-zeta for the copper ion) including polarisation and diffuse functions and with the polarisable continuum model (PCM) to account for hydration effects

around 3 under the reaction conditions because of the Lewis acid behaviour of the Cu(II) ions. Thereby, the nucleophilic character of the amino group is preserved to enable the formation of a peptide bond with the second amino acid (or peptide), which is only coordinated end-on via its carboxy group. The main role of the chloride ligand is to prevent the second amino acid from also chelating the Cu(II) centre, which would result in a stable but unreactive complex. Furthermore, the relatively large chloride ion pushes the peptide bond forming reaction centres closer together. Neutron diffraction measurements and Monte Carlo simulations (Texler et al. 1998) of solutions containing 0.5 M CuCl2 and 5 M NaCl support that, under the concentration conditions where the SIPF reaction takes place, each copper ion is on average coordinated with one chloride ligand. When chloride is replaced by other anions like nitrate, sulphate or fluoride, peptide formation is strongly decreased or inhibited (Rode and Schwendinger 1990). Only bromide was recently found to provide similar or even better peptide yields in some cases, but in the primordial scenario chloride was surely much more abundant than bromide anions. The dehydrating effect could also be mediated by other cations than sodium. Divalent ions with a higher hydration enthalpy, such as Mg(II), Ca(II), and Ba(II), would even produce peptides more readily, but they also shift the pH to lower values and, thereby, increase proton-mediated hydrolysis rates of preexisting peptides and shift the Cu(II) complex species distribution away from its optimum

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(Eder and Rode 1994). Potassium and also NH4+ ions provide a comparable peptide formation effect, but sodium ions seem to be the best compromise for the highest peptide yields after longer reaction times, besides having been abundant in the primordial hydrosphere with highest probability among all cations. When clay minerals are simultaneously present in SIPF experiments, peptide formation tends to result in a higher fraction of longer peptides (up to, at least, hexamers in the case of glycine), what underlines the compatibility and complementarity of these two mechanisms (Son et al. 1998; Rode et al. 1999b). When clays like calcium montmorillonite, hectorite, and also silica or alumina are added to the SIPF scenario, obtained yields of Gly5 and Gly6, when starting from diglycine or a mixture of diglycine and glycine, but also of Ala3 and Ala4 when starting from dialanine/alanine, are in most cases clearly higher than in the absence of these minerals. This mechanism seems to show a clear indication for a synergy leading to higher peptides and stabilising them against hydrolysis by adsorption to clay surfaces.

5.4.2

General and Specific Properties of the SIPF Reaction

The SIPF reaction has been experimentally performed with most of the proteinogenic amino acids so far and has been found to produce peptides from each of them, although to a varying extent, with yields going up to more than 10% in the case of proline after only a few evaporation cycles (Plankensteiner et al. 2005c). Peptide yields also strongly depend on the amino acid starting concentration and the amino acid to Cu(II) ratio. For most amino acids, peptide formation is most efficient with an amino acid to copper starting ratio of 2:1 (for example 80 mM amino acid, 40 mM CuCl2 and 500 mM NaCl in evaporation cycle experiments). An interesting phenomenon in the SIPF reaction is mutual amino acid catalysis. In the presence of glycine, diglycine or histidine peptide formation from other amino acids that provide only rather low peptide yields when present alone is drastically increased (Plankensteiner et al. 2002, 2005a, c; Suwannachot and Rode 1998; Reiner et al. 2006; Fitz et al. 2008; Li et al. 2008, 2010). After intermediate formation of mixed longer peptides including the catalytically active species, the catalyst and a peptide consisting of the other amino acid are set free again by hydrolysis, which is a common side reaction under the acidic SIPF conditions and reduces peptide yields in some cases. The highest catalytic effect is achieved when the concentration of glycine or histidine is 1/8 of the other amino acid’s starting concentration and this effect is for some amino acids more pronounced at lower starting concentrations and lower amino acid to Cu(II) ratios (e.g. alanine) and for some other amino acids at higher starting concentrations (e.g. arginine). An overview of the catalytic effects of glycine, L-, and D-histidine is given in Table 5.1. Some specific properties of the SIPF reaction indicate a very close connection to peptides and proteins still existing in present life forms. First of all, the SIPF

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Table 5.1 Overview of the catalytic properties of glycine, L- and D-histidine in the SIPF reaction on the formation of dipeptides from other amino acids for different starting concentrations after 1, 4 and 7 evaporation cycles Starting concentration 20 mM 40 mM 80 mM Evaporation cycles 1 4 7 1 4 7 1 4 7 Amino acid Catalyst L-Val Gly +++ ++ ++ +++ +++ +++ 0 ++ ++ D-Val Gly +++ +++ +++ +++ +++ +++ +++ +++ +++ L-Leu Gly + ++ ++ ++ +++ +++ +++ +++ +++ D-Leu Gly ++ ++ ++ ++ ++ +++ +++ +++ +++ L-Trp Gly + 0 0 ++ + + D-Trp Gly 0 0 0 L-Pro Gly + 0 0 0 0 0 + D-Pro Gly + 0 0 0 0 0 0 L-Ser Gly ++ ++ ++ ++ ++ ++ ++ +++ +++ D-Ser Gly ++ ++ + +++ ++ ++ ++ +++ +++ L-Met Gly + + 0 0 0 0 0 0 D-Met Gly + + + + + 0 + + L-Lys Gly + + + + + 0 + + 0 D-Lys Gly + + 0 + 0 0 + + 0 L-Arg Gly + 0 0 + + + + ++ ++ L-Ala L-His +++ +++ +++ + +++ +++ 0 D-Ala L-His +++ +++ +++ ++ +++ +++ L-Val L-His + ++ ++ ++ +++ +++ + ++ ++ D-Val L-His +++ +++ +++ +++ +++ +++ +++ +++ +++ L-Leu L-His + ++ +++ ++ +++ +++ ++ +++ +++ D-Leu L-His + ++ ++ ++ ++ +++ ++ +++ +++ L-Pro L-His 0 0 0 + + ++ ++ + D-Pro L-His 0 0 + ++ L-Ser L-His + 0 0 0 D-Ser L-His + 0 + L-Met L-His + + 0 + 0 0 0 0 + D-Met L-His ++ + + + 0 0 0 0 + L-Lys L-His ++ + + ++ + + + ++ 0 D-Lys L-His 0 + + ++ ++ ++ ++ ++ + L-Arg L-His + ++ + ++ ++ + +++ +++ +++ L-Ala D-His +++ +++ +++ ++ +++ +++ + 0 D-Ala D-His +++ +++ +++ ++ +++ +++ 0 L-Leu D-His ++ ++ ++ ++ +++ +++ ++ +++ +++ D-Leu D-His ++ +++ ++ ++ ++ +++ +++ +++ +++ L-Met D-His ++ + + + 0 0 0 + D-Met D-His ++ + + + 0 0 0 0 + L-Lys D-His ++ + + + ++ + ++ ++ + D-Lys D-His 0 + + +++ ++ ++ +++ ++ + L-Arg D-His + + + ++ + + +++ +++ +++ The concentration of the catalytic amino acid is 1/8 of the other amino acids concentration (2.5, 5 or 10 mM). The symbols display the catalytic factor P (dipeptide yield with catalyst / dipeptide yield without catalyst). []: P < 0.8 (decreasing yield in the presence of a catalyst); [0]: 0.8 < P < 1.2 (almost no catalytic effect); [+]: 1.2 < P < 3 (weak catalytic effect); [++]: 3 < P < 10 (medium catalytic effect); [+++]: P > 10 (strong catalytic effect)

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reaction prefers a-amino acids occurring in proteins over their b-analogues (Schwendinger et al. 1995), which can be explained by the more favourable fivemembered ring of an a-amino acid chelated to the Cu(II) ion compared to the less stable six-membered conformation with a b-amino acid. As mentioned before, the SIPF reaction forms different dipeptide sequences with varying efficiency. The corresponding peptide yields depend on the complex formation constants of the different amino acids, the nucleophilic and electrophilic properties of the complexed species, the mobility of the amino acids in the highly polar salt solution, the stability of resulting peptides, and possibly other factors. In a study (Rode et al. 1997; Rode 1999) the SIPF reaction has been performed with nine of the most common amino acids as single and binary amino acid systems under the same reaction conditions (500 mM NaCl, 40 mM CuCl2, 80 mM amino acids in single or 40 mM amino acids each in binary systems, five evaporation cycles). The dipeptides formed with the highest yields match astonishingly well with the amino acid sequences occurring most frequently in ribosomal proteins of archaea (probability of incidental coincidence ~1016) which are among the most ancient life forms still existing on earth (see Table 5.2) and only slightly worse with a selection of 400 different ribosomal proteins of other prokaryotic cells (probability of incidental coincidence ~1014). The coincidence is also very high when the Table 5.2 Comparison of the most frequently occurring amino acid sequences in ribosomal proteins of archaea and dipeptides produced in the highest yields by the SIPF reaction and the number of coincidences (Coinc.) Most frequent AB-linkages Amino acid A Source

Amino acid B

Most frequent BA-linkages Coinc. Amino acid B

Source

Archaea ala, asp, glu, val SIPF lys, asp, ala, gly glu, asp, ala, Archaea gly ¼ val SIPF glu, gly, ala, asp Archaea ala, leu, gly, glu SIPF gly, ala, pro, val Archaea gly, val, ala, leu SIPF gly, ala

Amino acid A

Coinc.

Asp

Archaea glu, val, asp, ala SIPF gly, lys, asp, ala

2

Asp

Glu

glu, leu, val, asp glu, gly, asp glu, lys, val, gly gly, leu, val, lys glu, gly, ala, leu ala, gly, leu, val ala, lys, glu ¼ val, gly Archaea SIPF asp, gly, ala lys, gly, ala, Archaea val ¼ asp SIPF val, his, ala, gly ala, asp ¼ glu, gly, leu Archaea

2

Glu

3

Gly

3

Pro

2

Lys

Archaea gly, lys, glu, leu SIPF gly, ala, asp

1

3

His

Archaea glu, lys, gly, ala SIPF ala, gly, val, his

2

2

Ala

3

2

Leu

Archaea ala, glu, leu, val ala, pro, gly, SIPF leu ¼ val Archaea glu, ala, asp, val SIPF gly, pro, ala, his

2

Val

Gly Pro

Lys

His

Ala

Leu

Val

Archaea SIPF Archaea SIPF Archaea SIPF

SIPF ala, gly, val, his Archaea ala, gly, glu, lys SIPF gly, val, ala, his ala, glu, asp, leu ¼ val Archaea gly, his, ala, leu SIPF

Archaea asp, glu, ala, gly SIPF gly, his, ala, leu

2

4 2 2

1

2

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preferred SIPF products are compared to a selection of human and animal prions, which are believed to be an old relic of evolution (Rode et al. 1999a). This obvious fingerprint of the SIPF reaction in proteins of the oldest still living organisms seems to be a crucial argument for its involvement in their formation and indicates that after the establishment of an RNA/DNA based information storage and replication process at a later stage of chemical evolution, this new and more efficient mechanism would mainly have reproduced peptides and proteins that had already existed before. A third specific property that associates the SIPF reaction with modern proteins is its stereospecific preference in favour of the L-form of some amino acids. When starting from L-alanine, dialanine yields are reproducibly around 10% higher than from D-alanine under various starting conditions and reaction times (Plankensteiner et al. 2004b; Fitz et al. 2008). Even more pronounced is the stereoselective discrimination in the cases of valine (Plankensteiner et al. 2005b) and isoleucine, where the L-forms produce several-fold higher peptide yields than their D-analogues. Amino acids with aliphatic side chains seem to take a special position here, since such a general enantiospecific preference could not so far be detected for any other amino acid. This stereospecific behaviour is difficult to explain in the context of classical chemistry as no additional chiral reagents (except the amino acids themselves) are used. One possible explanation is based on parity violation in weak nuclear interactions (Lee and Yang 1956; Wu et al. 1957). This effect leads to marginal ground state energy differences, often called ‘parity violating energy differences’ (PVEDs) (Tranter 1985; Berger and Quack 2000; Wesendrup et al. 2003; Laerdahl et al. 2000), between enantiomers. It is the only natural mechanism known to date that could lead to a general preference for one enantiomeric form, without any further classical chiral influence, such as a chiral surface or circularly polarised radiation. This effect, however, is immeasurably small for organic molecules like amino acids. Nevertheless, complex quantum mechanical calculations suggest that PVEDs are approximately proportional to Z6, Z being the atomic number of the heaviest element of the molecule. In the active SIPF complex, the central copper ion is heaviest (Z ¼ 29). It is also a chiral centre, because of the different ligands coordinating to it. Thus, one could expect much higher PVEDs – by several orders of magnitude – comparing a copper complex containing L-amino acids and its D-counterpart on the one hand and the free amino acids on the other. The combination of the normal chemical chirality and the inherent chirality provoked by parity violation in weak nuclear interactions at the relatively heavy copper centre could actually lead to a diastereomer-like behaviour of an L- and a D-amino acid SIPF complex providing different chemical properties and reactivities (Fitz et al. 2007). Quantum mechanical calculations of the geometries of active SIPF complexes with different amino acids have shown an interesting correlation (Fitz et al. 2007). For those amino acids where a stereospecific preference in favour of the L-form in the SIPF reaction has been detected experimentally, the equatorial ‘plane’ of the amino acids’ coordination sites to the Cu(II) centre of the SIPF complex is considerably more distorted towards a tetrahedral conformation than for other

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amino acids without an enantiomeric preference. This could mean that the measurable stereoselectivity of SIPF for only a few amino acids is due to cumulative interaction of the more or less chirally distorted SIPF complex centre with the inherent chirality of the copper ion, as provided by parity violation in weak interactions. Nevertheless, if the SIPF reaction is able to trigger peptide formation to a slight excess of at least some L-amino acids, this would finally lead to a generally L-amino acid dominated peptide world because of stabilisation effects like helix or b-sheet formation, which are only effectively possible for homochiral polymers, yet indispensable for a proper and reproducible chemical reactivity and the functioning of a peptide or protein.

5.5

Further Properties and Evolution of Peptides

It seems unlikely that the SIPF reaction could have produced larger peptides or proteins by itself as hydrolysis of peptide bonds is a common side reaction under typical SIPF conditions. However, if the peptides were stabilised, for example by adsorption to clay surfaces or mutual aggregation, the copper ions could well have promoted further chain elongation (in addition to condensation of peptides catalysed by the clay surface itself) as long as fresh amino acids and smaller peptides and drying/wetting cycles were available. While proteins of most modern life forms are mostly denatured in highly concentrated sodium chloride solution, proteins of extremely halophilic bacteria behave the opposite way: salt concentrations of several mol. per litre are required for their proper activity. Investigations on their amino acid distribution (Lanyi 1974) revealed that halophilic proteins are in many cases more negatively charged than their analogues in other bacteria, which provides better stabilisation effects in the presence of high concentrations of sodium counter ions. For this purpose, they contain higher ratios of glutamic and aspartic acid at the expense of strongly hydrophobic amino acid residues, like valine, leucine, isoleucine and phenylalanine. As the SIPF reaction takes place under similarly salt-rich conditions, it sounds reasonable that the main population of peptides in early chemical evolution could have been somehow similar in composition to proteins of contemporary halophilic bacteria due to superior stabilisation of their structures and, therefore, more reproducible properties and activities, while other peptides would have been hydrolysed or otherwise degraded more quickly. How could a peptide world look like once the first peptides have been formed on the primordial earth? A number of recent investigations show that a peptide world would by no means be a dead-end-street and that even smaller peptides have some astonishing properties and features for performing functions that are mostly carried out by other classes of biomolecules in modern life forms. In the absence of RNA/DNA as carriers of information, it is of special interest that some specific peptides are able to catalyse their own self-replication from peptide fragments (Isaac and Chmielewski 2002; Lee et al. 1996; Yao et al. 1998),

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even in a stereo-selective way, thereby combining functions that are performed by a combination of DNA, RNA and proteins in today’s organisms. Furthermore, peptides consisting of only a few amino acids with one hydrophilic end made up of one or two aspartic acids, for example, and one hydrophobic part (glycines, valines, leucines, . . .) easily form nanovesicles with membrane-like structure, similar to that of biological phospholipids, in aqueous solution (Carny and Gazit 2005; Vauthey et al. 2002; Zhang et al. 1993; Santoso et al. 2002). In such an encapsulated and, thereby, protected environment it would be much easier for less stable biomolecules to develop as the inside of these spherules or nanotubes might provide more favourable conditions like lower salinity, for example. Furthermore, these membranes might adsorb other organic molecules and, in combination with these, provide some catalytic effects for biochemical reactions on their surface. As a positive side effect, such an aggregation of peptides would also stabilise the membrane-forming oligomers themselves against hydrolysis and other degrading reactions. The central selectivity filters of channel proteins for cations, protons or water are made up of short strands of amino acids (e.g. four strands four amino acids long in case of the potassium channel) (Milner-White and Russell 2008) which presumably can have been incorporated in a simple peptide membrane and could have decisively influenced the conditions inside such a compartment by selectively letting pass only certain substances. As mentioned earlier, phosphate ions required for the formation of RNA/DNA would rapidly precipitate in the presence of Ca2+ or some other metal cations. In this context, the P-loop occurring in a wide variety of intracellular enzymes nowadays might be of special interest (Milner-White and Russell 2008). It contains a nest made of five main chain NH groups which wrap themselves around a phosphate anion. A small prebiotic peptide including such a nest could explain how phosphate could have been stabilised and kept in solution for RNA synthesis.

5.6

Considerations About Peptide Splicing Reactions

After a number of peptides having a variety of different features had evolved as explained in the previous section, an expedient next step would be to connect some of these features into one single biomolecule by splicing of peptides, thereby markedly increasing the complexity and evolutionary options of the system. Such peptide splicing reactions could initially have taken place on the surface of clay minerals, for example, or peptides similar to those showing autocatalytic effects might have adopted the ability to connect also other peptides in a more and more efficient way. When at a later stage of chemical evolution the RNA/DNA based replication mechanism began to establish, i.e. the genetic take over, this probably happened in a more moderate environment (either inside cell-like compartments or in a generally more moderate surrounding) because of the instability of these polymers against high temperatures and high salinity. Such conditions, on the other hand,

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are quite unfavourable for the peptide producing SIPF reaction. Extending these considerations it sounds reasonable that, initially, the RNA/DNA based replication process would mainly have reproduced shorter peptides that had been produced by the SIPF reaction before and that have already proven their usefulness for several purposes. It sounds implausible that the newly established RNA/DNA mechanism instantly would have been able to reproduce the whole variety of peptides, proteins or other biomolecules that had evolved so far and, hence, other mechanisms to combine or splice shorter peptides to more complex biomolecules had most probably already been developed before. Such a pathway of stepwise evolution can explain very well the astonishing concordance of the main products of the SIPF reaction and still existing proteins (Sect. 5.4.2; Table 5.2). The nature of primitive peptide splicing reactions in early chemical evolution can only be guessed, as peptide splicing in modern life forms is not a very common process any more and because its mechanisms today are surely very different and much more complex than in early chemical evolution. For biochemical reactions outside cell-like compartments, clay minerals with their abundant availability and large variety of catalytic properties, especially for peptide chain elongation, might have played a crucial role but also specialised peptides similar to the self-replicating ones could probably have developed very early in chemical evolution and might have been key-molecules for the evolution of the first highly complex proteins.

5.7

General Conclusion

The Salt-Induced Peptide Formation (SIPF) reaction provides a suitable pathway to connect amino acids to shorter and, in combination with the catalytic effect of clay minerals, maybe also longer peptides under plausible prebiotic Earth conditions in an aqueous environment. No similarly easy and straightforward way for an establishment of a pure RNA world could be detected to date, what makes an amino acid/ peptide world as a very early step of chemical evolution towards living systems more likely from a chemical point of view. Even short peptides have been found to possess astonishing properties and abilities, which indicate that such a peptide world would have been by no means a dead end street, and that many features and characteristics of life as we know it could, at least to a certain extent, well have evolved and developed in such a scenario.

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Plankensteiner K, Reiner H, Schranz B, Rode BM (2004a) Prebiotic formation of amino acids in a neutral atmosphere by electric discharge. Angew Chem Int Ed 43:1886–1888 Plankensteiner K, Righi A, Rode BM, Gargallo R, Jaumot J, Tauler R (2004b) Indications towards a stereoselectivity of the salt-induced peptide formation reaction. Inorg Chim Acta 357:649–656 Plankensteiner K, Reiner H, Rode BM (2005a) Catalytically increased prebiotic peptide formation: ditryptophan, dilysine, and diserine. Orig Life Evol Biosph 35:411–419 Plankensteiner K, Reiner H, Rode BM (2005b) Stereoselective differentiation in the salt-induced peptide formation reaction and its relevance for the origin of life. Peptides 26:535–541 Plankensteiner K, Reiner H, Rode BM (2005c) Catalytic effects of glycine on prebiotic divaline and diproline formation. Peptides 26:1109–1112 Plankensteiner K, Reiner H, Rode BM (2006) Amino acids on the rampant primordial earth: electric discharges and the hot salty ocean. Mol Divers 10:3–7 Powner MW, Gerland B, Sutherland JD (2009) Synthesis of activated pyrimidine ribonucleotides in prebiotically plausible conditions. Nature 459:239–242 Quigley MN, Vernon F (1996) Determination of trace metal ion concentrations in seawater. J Chem Educ 73:671–675 Reiner H, Plankensteiner K, Fitz D, Rode BM (2006) The possible influence of L-histidine on the origin of the first peptides on the primordial earth. Chem Biodivers 3:611–621 Rode BM (1999) Peptides and the origin of life. Peptides 20:773–786 Rode BM, Schwendinger MG (1990) Copper-catalyzed amino acid condensation in water – a simple possible way of prebiotic peptide formation. Orig Life Evol Biosph 20:401–410 Rode BM, Eder AH, Yongyai Y (1997) Amino acid sequence preferences of the salt-induced peptide formation reaction in comparison to archaic cell protein composition. Inorg Chim Acta 254:309–314 Rode BM, Flader W, Sotriffer C, Righi A (1999a) Are prions a relic of an early stage of peptide evolution? Peptides 20:1513–1516 Rode BM, Son HL, Suwannachot Y, Bujdak J (1999b) The combination of salt induced peptide formation reaction and clay catalysis: a way to higher peptides under primitive earth conditions. Orig Life Evol Biosph 29:273–286 Saetia S, Liedl KR, Eder AH, Rode BM (1993) Evaporation cycle experiments – a simulation of salt-induced peptide synthesis under possible prebiotic conditions. Orig Life Evol Biosph 23:167–176 Santoso S, Hwang W, Hartman H, Zhang S (2002) Self-assembly of surfactant-like peptides with variable glycine tails to form nanotubes and nanovesicles. Nano Lett 2:687–691 Sawai H, Orgel LE (1975) Prebiotic peptide-formation in the solid state. J Mol Evol 6:185–197 Sawai H, Lohrmann R, Orgel LE (1975) Prebiotic peptide-formation in the solid state. II. Reaction of glycine with adenosine 50 -triphosphate and P1, P2-diadenosine-pyrophosphate. J Mol Evol 6:165–184 Schwendinger MG, Rode BM (1989) Possible role of copper and sodium chloride in prebiotic evolution of peptides. Anal Sci 5:411–414 Schwendinger MG, Tauler R, Saetia S, Liedl KR, Kroemer RT, Rode BM (1995) Salt induced peptide formation: on the selectivity of the copper induced peptide formation under possible prebiotic conditions. Inorg Chim Acta 228:207–214 Shapiro R (1988) Prebiotic ribose synthesis: a critical analysis. Orig Life Evol Biosph 18:71–85 Shapiro R (1995) The prebiotic role of adenine: a critical analysis. Orig Life Evol Biosph 25:83–98 Sleep NH (2010) The Hadean-Archaean environment. Cold Spring Harb Perspect Biol 2:a002527 Son HL, Suwannachot Y, Bujdak J, Rode BM (1998) Salt-induced peptide formation from amino acids in the presence of clays and related catalysts. Inorg Chim Acta 272:89–94 Spitzer J, Poolman B (2009) The role of biomacromolecular crowding, ionic strength, and physicochemical gradients in the complexities of life’s emergence. Microbiol Mol Biol Rev 73:371–388

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Steinman G, Cole MN (1967) Synthesis of biologically pertinent peptides under possible primordial conditions. Proc Natl Acad Sci USA 58:735–742 Steinman G, Lemmon RM, Calvin M (1964) Cyanamide: a possible key compound in chemical evolution. Proc Natl Acad Sci USA 52:27–30 Suwannachot Y, Rode BM (1998) Catalysis of dialanine formation by glycine in the salt-induced peptide formation reaction. Orig Life Evol Biosph 28:79–90 Texler NR, Holdway S, Neilson GW, Rode BM (1998) Monte Carlo simulations and neutron diffraction studies of the peptide forming system 0.5 mol kg1 CuCl2-5 mol kg1 NaCl-H2O at 293 and 353 K. J Chem Soc. Faraday Trans 94:59–65 Tranter GE (1985) The parity violating energy differences between the enantiomers of a-amino acids. Mol Phys 56:825–838 Vauthey S, Santoso S, Gong H, Watson N, Zhang S (2002) Molecular self-assembly of surfactantlike peptides to form nanotubes and nanovesicles. Proc Natl Acad Sci USA 99:5355–5360 W€achtersh€auser G (1990) Evolution of the first metabolic cycles. Proc Natl Acad Sci USA 87:200–204 W€achtersh€auser G (2000) Origin of life. Life as we don’t know it. Science 289:1307–1308 Weber LA, Caroon JM, Warden JT, Lemmon RM, Calvin M (1977) Simultaneous peptide and oligonucleotide formation in mixtures of amino acid, nucleoside triphosphate, imidazole, and magnesium ion. Biosystems 8:277–286 Wesendrup R, Laerdahl JK, Compton RN, Schwerdtfeger P (2003) Biomolecular homochirality and electroweak interactions. I. The Yamagata hypothesis. J Phys Chem A 107:6668–6673 Wilde SA, Valley JW, Peck WH, Graham CM (2001) Evidence from detrital zircons for the existence of continental crust and oceans on the Earth 4.4 Gyr ago. Nature 409:175–178 Wu CS, Ambler E, Hayward R, Hoppes D, Hudson RP (1957) Experimental test of parity conservation in beta decay. Phys Rev 105:1413–1415 Yao S, Ghosh I, Zutshi R, Chmielewski J (1998) Selective amplification by auto- and crosscatalysis in a replicating peptide system. Nature 396:447–450 Zhang S, Holmes T, Lockshin C, Rich A (1993) Spontaneous assembly of a self-complementary oligopeptide to form a stable macroscopic membrane. Proc Natl Acad Sci USA 90:3334–3338

Chapter 6

Scenario of the Primary Pump: Emergence and Operation of an Automatic Engine to Generate Primordial Peptides and Beyond Nucleic Acids Auguste Commeyras

Abstract The emergence of peptides and nucleic acids is a major concern in prebiotic chemistry. Using water as solvent, the most practical way to form peptides and nucleic acids by dehydration reactions is to use amino acids and nucleotides in their activated forms. One is then faced with activated compounds considered so sensitive to hydrolysis that their prebiotic relevance is frequently questioned. Thus, the prebiotic synthesis of such macromolecules remained a wide open problem. By analyzing how living organisms work, one finds that the peptides and nucleic acids are, in fact, synthesized in aqueous medium from activated amino acids and nucleotides. Here, it is crucial that the living world is constantly kept out of equilibrium. We then asked, according to the “principle of evolutionary continuity,” whether the environment of early Earth could have favored the spontaneous emergence of a particular chemical non-equilibrium system, which could have been the source of macro-chemical evolution. We have identified such a system, together with operating conditions necessary to keep it out of equilibrium. We have called this energizing process the “primary pump.” We detail its operating mechanism and evaluate its credibility. We show how this primitive pump was running continuously for a long geological period in which it could maintain disequilibrium in a particular chemical system of complex interactions. We show how the amino acids could be concentrated, how they were condensed in a dehydration reaction, how the primary pump has been able to select certain monomers during the condensation steps, and how the primary pump may

The online version of this chapter (doi: 10.1007/978-3-642-21625-1_6) contains electronic supplementary material in form of two animated files, which are available to authorized users. A. Commeyras (*) Institut des Biomole´cules Max Mousseron (IBMM), UMR 5247, CNRS, groupe Dynamique des Syste`mes Biomole´culaires Complexes (DSBC), Universite´ Montpellier 1&2, Place E Bataillon, Montpellier 34095, France COLCOM, Cap Alpha, Avenue de l’Europe, Clapiers, 34940 Montpellier Cedex 9, France e-mail: [email protected]; [email protected] R. Egel et al. (eds.), Origins of Life: The Primal Self-Organization, DOI 10.1007/978-3-642-21625-1_6, # Springer-Verlag Berlin Heidelberg 2011

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have caused the evolution of the peptides formed. We point out how the primary pump could have transferred its energy to other reactions, such as taking part in the appearance and evolution of nucleic acids. The “scenario of the primary pump” is a dynamic process through which peptides could have emerged and, perhaps, co-evolved with nucleic acids – two interactive macromolecules fundamental to life.

6.1 6.1.1

Why a Primary Pump? The Principle of Continuity Requires a “Primary Pump”

Life is a chemical system out of equilibrium, a dynamic system that continuously receives energy. This energy is the driving force of evolution. On the primitive Earth, the elementary molecules (CO2, H2O, N2, . . .) have been gradually transformed into essential-for-life molecules (amino acids, nucleotides, . . .) through energy provided by sunlight, lightning (in thunder storms and volcanic plumes),1 and entry of meteorites into the atmosphere (Commeyras et al. 2005a, b). In the primary pump scenario, we postulate that essential molecules could have been assembled, organized, and maintained out of equilibrium by a constant supply of energy. We believe that such a dependable power pack had spontaneously emerged on the “primitive Earth.” We have called this permanent provider of energy the “Primary pump,” in reference to its primordial origin and function. The purpose of this chapter is to seek a process, compatible with the environment of the early Earth, which could gather all the requirements desired.

6.1.2

The Primary Pump

A pump is a machine that transports material uphill against an energy difference. It is characterized by: Energy consumption, a movement (rotation, flap), and a nonreturn valve. Life is associated with many pumps. A beating heart is life (hearts have non-return valves). If the heart stops beating, it means death. In a pump, energy is provided by oil, by electricity, by food, and, ultimately, by ATP in

1

The energy of sunlight is unable to dissociate, N2 into radicals N. This energy is only found in lightning (in thunder storms and volcanic plumes) or entry of meteorites. The radicals N then give nitric oxide (NO) in the excited state (singlet). The transition, singlet state to normal state, emits the light of lightning and meteorite trails. In volcanic plumes, NO production is very high due to direct reaction and almost continuous volcanic lightning (Navarro-Gonzalez et al. 1998; Mather et al. 2004). On the primitive earth (with a small amount of O2), the nitrosating agents, once formed, were stable. Today, with a large amount of oxygen in the atmosphere, NO is quickly transformed into NO3H.

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biological pumps. Obviously a primary pump has been very simple, but what could have been its source of power, its principle of operation?

6.1.3

Darwin and Evolution

Life has a history. Today, the Earth numbers between 5 and 100 million different organisms. One billion years ago, life was certainly very primitive and deregulated, and essentially all the organisms should have been “interbreeding.” Scientists believe that 2.5 or 3.5 billion years ago, there should have lived a peculiar species, the “Last Universal Common Ancestor” (LUCA), from which all modern organisms have descended. It is well before LUCA that we must ask the question of the origin of life. With the appearance of the stage of LUCA, Darwinian evolution2 was under way. Long before LUCA, the inert material was progressively becoming more and more complex until reaching the LUCA stage. When inert matter had been able to reproduce, it jumped into a new world – unknown and extraordinary. What is the origin of this common ancestor? We believe that LUCA, like modern organisms, necessarily depended on a chemical energy pool that allowed it to survive and evolve. Whether the heart of this chemical energy currency was ATP is still an open question (Gargaud et al. 2009). If the pumps for LUCA were similar to those that enable the life of bacteria, they should have been very effective. Indeed, to stay alive, bacteria concentrate millions or even billions of times the nutrients of their environment. Long before LUCA, the concentration of reactants in the primitive ocean was extremely low (Sect. 6.2.1). Accordingly, for the primary pump to be effective (taken as a machinery to complexify the inert prebiotic matter), it had to implement very efficient processes to concentrate its reagents. Searching for an appropriate concentration process was one of our concerns throughout this work.

6.1.4

What is the Environment that Led to the Birth of LUCA?

Very briefly, the environment of the pre-LUCA era can be summarized as follows. The Earth was born 4.56 billion years ago. The Earth’s crust (floating on the upper mantle) was formed very early (between 4.55 and 4.3 billion years ago). The rocks of the crust were mainly basaltic and very hot (Tessalina et al. 2010). The water (remaining from planetesimals and brought in by comets) was vaporized initially (clouds). The atmosphere was reductive and rich in CO2 (~10 bars). The water in the clouds was acid (carbonic acid H2CO3 pH 3.5). During the cooling phase, oceans

2 There is no reason to believe that LUCA as such was the start of Darwinian evolution. It just happens to be the stage from which more than a single line of descendents have survived to modern times (Forterre 2005). We simply have no way of knowing how many other lineages from preLUCA stages have all gone extinct.

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were formed. Alkali metals (e.g., sodium) were easily extracted from basaltic rocks. The pH was then controlled by the buffer carbonic acid/sodium bicarbonate. When subsequently calcium was extracted, limestone precipitated and concentration of CO2 decreased in the atmosphere, yet stabilized at intermediate levels.3 Just like today, the oceans did not cover young continents completely (Gargaud et al. 2009), although the total mass of continental crust was much smaller early on. Eighty million years after its formation (that is to say, very early), Protoearth collided with the protoplanet Theia, a Mars sized protoplanet in the same orbit. This was a kind of cosmic reset. The Moon was created by this collision. The Moon was originally closer to the Earth of about 70,000 km (today it moves away 1 cm/year). It was revolving around the Earth, not in 28 days as present, but in about 17 days. With the Moon nearer, Earth’s tides were greater than today. With a rotation speed faster (day length was ~50–75% its present value), the frequency of the tides was higher than today. The early oceans, probably washed with high efficiency, young continents, pulling their soluble portion.The early Earth resembled a gigantic washing machine (Lathe 2004; Varga et al. 2006). The high impact rate of cosmic bolides after the Moon-forming event subsided rather slowly, and even reached peak values once again during the so-called late heavy bombardment phase (Gomes et al. 2005). Life has emerged in such an energized environment.

6.2 6.2.1

Primary Pump: The Challenges Peptide Synthesis

Although knowledge in this area is not definitive, it is considered that the primitive earth might have carried about hundred racemic amino acids. Their concentration did not exceed a few mg/l4 in an ocean with a pH between 5 and 6 (Commeyras

3

Only after Life and oxygenic photosynthesis had emerged, could CO2 concentration drop to modern levels, as driven by both organic carbon sequestration and biogenic acceleration of limestone precipitation, mainly due to pH increase in the oceans. 4 An exhaustive inventory of the origins of all the organic molecules occurring on the primitive Earth 4 Giga years ago was carried out by Chyba and Sagan (1992). Three probable sources were distinguished: exogenous contribution, endogenous synthesis associated with impact of meteorites, endogenous production associated with other available energy sources (solar UV, lightning).The total contribution of these three sources would have been heavily dependent on the composition of the primitive atmosphere. The primitive oceans could have contained (at a steady state) between ca. 0.4 10–3 g/l of total organic matter in the case of a neutral atmosphere and 0.4 g/l in the case of a reductive atmosphere. The organic matter carried out by carbonaceous chondrite was mainly (>60%) macromolecular, practically insoluble, and characterized as PAHs with few or no impact on the origin of life, but who knows? (Deamer, 1992). The soluble part (<40%) is a complex mixture of molecular organic compounds in which the following have been characterized: acids (carboxylic, dicarboxylic, hydroxyl, sulphonic, phosphonic); amines; amides; nitrogenous heterocycles including purines and pyrimidines; alcohols; sugars; carbonyl

6 Scenario of the Primary Pump: Emergence and Operation of an Automatic Engine Miror

R

H

primitive ocean pH 5-6

H

R

CO2H HO2C

H 2N L

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R NH2

D

Etc spontaneous reaction

Initial conditions: About 100 differents α-aminoacids (50% L + 50% D), total amount 10mg/l

N H

H

R

H C N O H

C N O H

R

H C OH O

+ n H2O

n L

Target: homochiral peptide with 20 α-aminoacids

Fig. 6.1 Homochiral peptide from initial conditions

et al. 2005a, b). Taking into account all of these initial conditions, no previous work on the emergence of prebiotic peptides (Fig. 6.1) has been undertaken, certainly because these conditions have been a priori considered to be very unfavorable to such syntheses. If we venture in this direction, the first classic reflex is to decompose the problem into a series of basic questions such as: 1. Is it necessary to concentrate the amino acids in order to condense them? 2. Is it necessary to activate the amino acids, and if so how and where to be able to condense them? 3. If peptides are formed, could they evolve via a reaction loop: hydrolysiscondensation? 4. Can we imagine a prebiotic process to repeat such a cycle? 5. Can we consider that the above cycle could be responsible for chemo- and enantio-selection? 6. What process can we imagine to transform a racemic mixture of a-amino acids into a homochiral peptide? 7. How can the emergence of catalytic properties of peptides be imagined? Thus, we are faced with a series of questions, which are difficult to consider independently. It is indeed implausible to imagine that processes as diverse as those involved in the questions above, have worked separately. Had that been the case, the emergence of homochiral peptides would have been solved by chance. Our group did not want to follow this reductionist approach. We have instead preferred to be interested in a holistic approach, focusing on the scenario, rather than the actors. Like the chemical energy that keeps the cells alive and out of equilibrium with a capability of evolution, we devised a primitive machinery that could have provided an answer to all of the issues raised by the emergence of peptides.

compounds; aliphatic and aromatic hydrocarbons; amino acids; and even hydantoins. The average mass of extracted amino acids was only 0.6 mg per gram of carbonaceous meteorite. Only the amino acids are taken into account in the first part of this document.

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It is this machinery that we have called “primary pump” (Commeyras et al. 2002). We designed it as a reaction cycle, repeated continuously. The primary pump has two different aspects: chemical reactions and mechanical/material transport.

6.2.2

The Primary Pump: Chemical Part

To see the primary pump in its environment go to Sect.. 6.3. The following four reactions are the chemical components of the primary pump. Step 1: Activation of amino acids, Step 2: Polymerization of activated amino acids to create peptides, Step 3: Epimerization of the last amino acid incorporated into the peptide chain to give the configuration of the preceding amino acid, Step 4: Depolymerization of the peptide chain formed to split it into shorter peptides. The potentially prebiotic qualifications of these reactions are discussed below. Step 1: Activation of a-amino acids The reaction of a gaseous mixture NO/O25 on anhydrous N-carbamoyl-amino acid gives equimolar amounts of N-carboxyanhydride of a-amino acids (NCAs), water, and nitrous acid (Collet et al. 1996). The NCAs are activated a-amino acids. They are potentially prebiotic (Pascal et al. 2005). The gas mixture NO/O2, as generated by energetic processes in the atmosphere, is also potentially prebiotic (Commeyras et al. 2002). The N-carbamoyl-amino acids can prebiotically be obtained in two ways (Fig. 6.2), by hydration of hydantoins or carbamoylation of a-amino acids (Commeyras et al. 2005a, b). Step 2: Polymerization of NCAs The polymerization reaction of NCAs to provide peptides is known to work. We have shown that this reaction may be performed in water as solvent, provided that the pH is above 4, which could have been the case of the primitive ocean (Commeyras et al. 2005a, b; Pascal et al. 2005; Collet et al. 2010).

5

In prebiotic conditions, the abiotic oxygen, at very low partial pressure, was essentially formed by photo dissociation of CO2 in upper atmosphere. Nitric oxide (NO), at much higher partial pressure than O2, was formed by lightning and meteoritic impact in lower atmosphere. The meeting of NO and O2 gives (N2O3, NO2, N2O4) in equilibrium. The O2 concentration is low in the mix NO/O2 (at all likelihood that was the case in prebiotic conditions) while the concentration in N2O3 is strong. And precisely, N2O3 is the powerful nitrosating agent. It reacts with N-carbamoyl amino acids in dry phase with a very high kinetic constant (kN2O3 ¼ 1.0 108s1) (Lagrille et al. 2007), to give NCA, water, and HNO2 in equivalent amounts. Under the same conditions, N2O3 causes decarbamoylation of peptides (see animations 1 and 2).

6 Scenario of the Primary Pump: Emergence and Operation of an Automatic Engine R

Strecker H2O

O R

HO

R CN

HO

CO2H N-carbamoylα−amino acid

α−hydroxy acid

NH3

+

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R

R

R

HNCO

HCN

H 2N

H 2N

CN

CO2H

α−amino acid

CO 2

R

R

Bücherer-Bergs

CO2H

O NH2 H2 O

R HN CN CO2 - H +

HN

HN

NH

O O

N C O

NH2 O

R O NH O

HN

Fig. 6.2 N-carbamoyl-amino acids from two different pathways, Strecker in extraterrestrial conditions, B€ucherer-Bergs on early Earth

Step 3: Epimerization6 Epimerization of the last amino acid incorporated into a peptide chain to give the configuration of the previous amino acid is less documented than the steps 1 and 2. It is a catalytic reaction (Honnoraty et al. 1995, Plasson et al. 2004). It could be effective when the peptide chain adopts the helical conformation, that is to say beyond a length of about 10 mer. Further work must be done to develop knowledge in this field. Step 4: Depolymerization The depolymerization reaction of a peptide chain is slow (Radzicka and Wolfenden 1996, Smith and Hansen 1998). In water at pH between 4 and 7, the half-life time of this reaction is 400 years, compatible with prebiotic conditions and the primary pump scenario. If the hydrolysis rate is faster than the rate of formation of peptides, the process to be considered is counterproductive. If instead, the rate of hydrolysis is slower than the rate of elongation and preferentially occurs in unfolded regions, the process leads to the accumulation of foldable peptides in the reaction medium. In consequence of partial hydrolysis and a lengthening of fragments, the peptides formed were forced to evolve (Commeyras et al. 2005a, b). In particular, if the rate of chain elongation is slightly autocatalytic, requiring a certain length to be most effective, partial hydrolysis of the longest peptides could increase the number of growing chains substantially. During these cycles, while some amino acids are incorporated more rapidly than others, chemo-selection results in the peptide chain (Commeyras et al. 2002, 2004a,b). Similarly, if one of the two D- or L-enantiomers is more rapidly incorporated than the other in the peptide chain, this is resulting in enantioselection (Commeyras et al. 2004a, b). The enantiomer not incorporated in the

6

For the definition of this term, see wikipedia.

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chain racemizes slowly in the primitive ocean (Csapo et al. 2004) and the process continues. The result is a total transformation of the initial stock of racemic amino acids into a homochiral peptide. To understand these phenomena of chemoselection, enantio-selection, and deracemization, we refer the reader to the work of Raphael Plasson who has developed experimental and kinetic models which describe the dynamic operation of the primary pump (Plasson 2008; Plasson and Bersini 2009; Plasson and Brandenburg 2010; Plasson et al. 2004, 2007, 2010).

6.2.3

The Primary Pump: Mechanical Part

As mentioned above, the concentration of a-amino acids in the early oceans was very low, certainly lower than 10 mg/l (Commeyras et al. 2005a). In water and at such low concentrations, the dehydration reactions are impossible. For the primary pump to operate, the following conditions must be met: • The N-carbamoyl-amino acids must be synthesized preferentially (Taillades et al. 1998, Commeyras et al. 2005a,b), somewhere in a patchy environment. • The N-carbamoyl-amino acids must be concentrated to become anhydrous. • The anhydrous N-carbamoyl-amino acids must be subjected to the action of a gaseous mixture NO/O2 (N2O3) to form N-carboxyanhydrides (NCAs), water, and nitrous acid whose pH ~ 1.5. • The pH of the environment of NCAs must be suddenly changed to become less acidic (pH > 4). • All reactions of this cycle must occur under natural conditions. • All reactions of this cycle must be repeated continuously. For such a logical sequence to operate effectively, the primary pump must have a mechanical component. The mechanical means of interest must be compatible with the natural environment of the primitive Earth. Indeed, all these conditions could have been observed at surfaces close to the triple junction of ocean, continental crust, and atmosphere. That is to say on the “beaches” of a primitive continent, and nowhere else. Figure 6.3 summarizes these conditions. The dotted line represents the boundary between the ocean and continents. • In the ocean at pH 5, N-carbamoyl-amino acids were formed in two different ways as we have seen previously. • At high tide, the water inundates the land surface with a batch of diluted material. Upon withdrawal, evaporation concentrates the N-carbamoyl-amino acids to the crystallization point. Thus, evaporation could have been the first natural technique to concentrate the starting material of the primary pump.

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First cycle Atmosphere

NO + O2

N2 + H2O + CO2 HNO2

Emerged land

H NH2CO N

O

COOH

O

R

N H

R H NH2CO N

O

COOH R

Ocean

R

NCA O

O NCA O

N H

HNCO COOH R

NH2

H2N

R2 H N

COOH

O R1 H2N

O R2 H N COOH N R H O R 3

1

tetra HNCO

Peptides Fig. 6.3 Diagram of the primary pump – first cycle, idealized

• The atmospheric gas mixture (NO-O2) reacts on the anhydrous N-carbamoylamino acids, and transforms them into NCAs (Commeyras et al. 2004a). The acidic environment due to nitrous acid (pH ~ 1.5) stabilizes the NCAs. • At the next high tide, the NCAs are suddenly covered by the ocean at pH > 4. They polymerize to give peptides. • All reactions of this cycle are natural. • This cycle is repeated continuously with the frequency of the tides.

6.2.4

Details Giving Credit to the Primary Pump

Isocyanic acid (HNCO) can react on the emerging peptides to give N-carbamoylpeptides (H2NCO-Peptide). In this way, the peptides are protected. With such protection, peptides cannot be elongated and the primary pump loses its prebiotic credibility. For this not to occur, the protection must be removed. In considering this question, we showed experimentally that in the presence of the gas mixture NO/O2 the protection is immediately removed. This reaction produces nitrogen and carbon dioxide (Collet et al. 1999). We noted that the conditions to remove this protection are strictly identical to those that allow

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Second cycle and following

Atmosphere

N2O3

N2 + H2O + CO2 HNO2

NH2CO

H N

NH2CO

H N

COOH

H2NCO-Peptides

O O NCA R N O n Peptides H

COOH

H2NCO-Peptides

O O NCA R N O n+mPeptides H

R

Emerged land

R

Ocean

HNCO COOH R

NH2

H2N

R2 H N O

COOH R1

O

H2N R3

N H

R2 H N O

COOH R1

tetra HNCO

H2NCO-Peptides

Peptides

Fig. 6.4 Diagram of the primary pump – recurrent cycling

N-carbamoyl-amino acids to give the NCAs, so the free peptide obtained must be elongated during the same cycle, and the primary pump can continue to run (Fig. 6.4). The conditions for this deprotection, and the total recycling of matter, appeared to enhance the credibility of this scenario.

6.2.5

The Primary Pump: Myth or Reality?

The reaction cycle of the primary pump can only turn in one direction. (The main steps are indeed irreversible). This reaction cycle draws on a stock of racemic a-amino acids and (ideally) assembles them into a stock of homochiral peptides. With a high speed (Two cycle per ancestral day), homochiral peptides accumulate in the environment. The energetic pressure of the primary pump maintains the “homochiral peptides” out of equilibrium (Plasson and Bersini 2009). For the stock of homochiral peptides to return to equilibrium, the only possibility would be to stop rotation of the earth, thus eliminating the mechanical component of the primary pump. The new equilibrium, after several hundred years (Csapo et al. 2004), would be characterized only by the presence of racemic amino acid and no

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peptides. Obviously, such a scenario is impossible. This could mean that, after having started, the evolutionary process had been irreversible. In summary, the primary pump appears to be a scientifically acceptable tool to study how inert matter could have been complexified, even as some gray areas will remain to be clarified. The operation of the “primary pump” is visualized by the following cartoon. Animated scheme. Primary pump in motion {link: Animation 1}

6.2.6

How Many Peptides Possible?

The number of peptides that the primary pump can assemble is given by the relation 2 * YX, where 2 are the D- and L-enantiomers of a-amino acids, Y is the number of available amino acids, and X is the number of monomer in the peptides. Initially, with about one hundred a-amino acids and two enantiomers the number of peptides that the primary pump could make was huge. After chemo- and enantio-selection, this number was lower (1 * 20 X) but nevertheless significant. Insofar as the reaction cycle (pump) has played a role in the history of the Earth, it took a creative power, and selection, unparalleled in the world of chemistry. Modern life has retained a number of peptides much lower than theoretical possibilities. Nothing is known of the selection processes that have been implemented.

6.2.7

The Primary Pump Yesterday and Today

The primitive Earth atmosphere was reducing (Gargaud et al. 2009). Traces of oxygen were formed in the upper atmosphere (Commeyras et al. 2002). The activation of the lower atmosphere, by photoactivation, lightning, volcanic plumes, and meteor entry, produced nitric oxide (NO), which together with traces of oxygen gave nitrosating agents such as N2O3 (Lagrille et al. 2007). These nitrosating agents, with isocyanic acid, were the source of chemical energy for the primary pump. To this chemical energy was added the mechanical energy of ocean tides (cosmic energy). Today, the Earth’s atmosphere contains 21% oxygen. This oxygen instantly transforms nitric oxide into nitric acid. The nitrosating activity is then immediately lost. The primary pump cannot work anymore. We must therefore consider that the primary pump ceased working with the gradual arrival of the oxygen in the atmosphere, accordingly with the emergence of life (the “takeover”) and biogenic photosynthesis. Life has created the poison (O2) which, in turn, could make it disappear. Fortunately, evolution was saved by allowing living cells to adapt, so as

140

A. Commeyras

to operate at relatively high proportions of oxygen. Darwin’s principle (i.e., natural selection) was, of course, already present from the start.

6.3

The Primary Pump in its Environment

6.3.1

The Primary Pump has not Remained Isolated in its Environment

The experimental information presented below shows how the primary pump has shared its energy with its environment. In summary of these experimental data, Fig. 6.5 locates the primary pump at the heart of an idealized device that can describe how the original organic material could have continued to evolve. We have represented the Sun, the Earth with its atmosphere, and the Moon. The atmospheric layer of about 10 km thickness played a crucial role in promoting the emergence of life.

Origin of life. Primary pump Scenario.

Sun h ν, Meteorites, Sparks Elementary molecules Essential molecules

EARTH Primary pump N2 + H2O + CO2 NO + εO2 Moon

NDP

Energetic coupling O OR OP OH NH2 O

n Aactivated

NH2CO A

Extension of the Primary pump

H 2PO 4-

nA

NMP NTP S P - PPP+-+ + ++ +P +S + PP- ++P S ++ +P P -+ + + S P -+ + + P + P- + + + + + P S P P PS P+ +H N NH3 NH + + 3 3 + H 3 O +H3N NHNH + H NH3N NH O O NH + O+NH3N 3 +H + + H3N3 + N HNOHNONH 3HN + O +3H NH3 3N O + O+ +HHN 3N O HN + + HN O NH 3 O NH 3 HN3NH3 + 3 ONHHN O NHHN +NH H O 3 HN3+ O H NH NHO3NH HNH HN O O NH + + NH + O HNONOH +H HN3N ONH NHNH3+ HNO 3NO HNON HNO O 3 HNOHNO HNOONH3 HN OONH NH HNO H NH +H O + NH O 3N O NH 3 3+ NH N + HHN + + O HN + O O3NH NHO3NH 3N NHNH NH3O + NH + NH NH +H NO NH + O NH +3 3NH 3 +NH 3+O3NH + N H O H3N O NH NH 3 NH + 3 +H N + +3 + NH ONHNH + NH 3 3 3 3 3

N H

+ ++ + ++ + + + + + + + + + + + + + + + ++ ++

- - -

N H

NH3+ NH + + + 3 3 NH + H NH3N +H N 3 H O NH 3 ONH + O NH 3+ 3N O NH3N + + N O O H3N + +O + H N H3 + HN HN NH3 HN NH3 + 3 OH3N + + 3 + O HN O NH +O NH + O NH H3NHNO NH 3 NH3 HN NH3 O 3HN HN HN3+ O NHO+ O NH H O NH NH HN O + + N O NH H 3 NH HO OH +H H HNON O NH NH3 3 NH3+NOHN O + 3N O HN ON HNO HN HN O HNO O NH3 NH HN O H NH + HN O ONH +H O O 3N NHN NH 3 3+ NH HN O + NH O HNO +NH H3N NHNH O3+ NHO NH3+O 3 + + NH O NH + + O NH 3 NH +3 +O H3N+NH NH NH 3 NH NH + 3 3 H 3N O NH 3 O + NH + 3 +H N + NH NH + NH3 3 + O H3N NH 3 3 +H

A = Amino acids NMP = nucleotide mono phosphate NDP = nucleotide di phosphate NTP = nucleotide tri phosphate

Concentration of NTP at the surface of the dendritic peptides

Dendrigrafted peptides

The dendritic peptides could have been the first template.

S PPP-

S PP- P-

RNA RNA synthesis are in project

Fig. 6.5 Overall presentation of the primary pump scenario: Activation of the primitive atmosphere gives activated molecules. These activated molecules give the molecules essential to life. The primary pump, continuous turn’s amino acids into amino acids activated (NCAs). Parts of NCAs give dendritic peptides. Another part reacts with phosphoric acid and then with nucleosides to form the NTPs. The NTPs concentrate and stabilize with the dendritic peptides. Beyond that, the first oligonucleotide may have formed

6 Scenario of the Primary Pump: Emergence and Operation of an Automatic Engine

6.3.2

141

From Elementary Components to Essential Bio-Molecules

The primitive atmosphere (N2, CO2, CH4, H2O, rare gases), excited by solar radiation, lightning, volcanic plumes, and meteor entry, has produced energy-rich molecules HNCO, HCN, NO, O2, HCC-CN H2N-CN, RCHO, HOCH2CHO, HOCH2CHOHCHO . . . (Gargaud et al. 2009). These activated precursors have led to essential bio-molecules, such as amino acids or nucleotides (Commeyras et al. 2005a, b; Powner et al. 2009).

6.3.3

a-Amino Acids

Seventy-four different a-amino acids have been identified in the Murchison meteorite (Cronin and Pizzarello 1983, 1997); they were formed via the Strecker reaction (Commeyras et al. 2005a, b), so it is reasonable to think that some amino acids on the primitive Earth were of exogenous origin; but we believe that a much higher proportion of a-amino acids was probably formed on the primitive Earth itself, through a B€ucherer-Bergs reaction (Taillades et al. 1998, Commeyras et al. 2005a, b). The difference between the two processes comes from the presence of high proportions of CO2 in the primitive atmosphere. Strecker reaction: RCHO + HCN + NH37 ! a-amino acids. B€ucherer-Bergs reaction: RCHO + HCN +CO3(NH4)2 ! Hydantoin ! N-carbamoyl amino acid ! NCA ! peptides. These two mechanisms have a common part, and then they diverge (Fig. 6.2). We represent below, in motion, the production of peptides from activated molecules. Animated scheme. Peptide synthesis from activated molecules {link: Animation 2} Through the B€ ucherer-Bergs reaction, the first stable products formed are the hydantoins. The hydration of hydantoins gives N-carbamoyl-amino acids. Then the reaction of the gas mixture NO/O2 on N-carbamoyl-amino acids gives the NCAs. In water at pH > 4, NCAs lead to peptides. During the formation of peptides, CO2 is regenerated and returns to the atmosphere. This animation shows the existence of a “short link” between the primitive atmosphere and peptides. In a supportive environment, a primitive atmosphere leads to peptides with minimal steps. All responses are geologically rapid (some days), and these reactions are repeated every day. The synthesis of peptides on the primitive Earth might therefore have been very fast.

7

We must note that ammonia (NH3) was probably formed in the oceans by NO reduction by Fe2 + (Summers and Chang 1993).

142

6.3.4

A. Commeyras

Structure of the First Peptides, and Their Potential Role in Evolution

The first emerging peptides from polymerization of NCAs cannot have been encoded for particular sequences, since the genetic code had not yet emerged. The likely presence of polyamine or thio-amino acids in the reaction medium was probably used as cross-linking agents and led to the formation of dendrigraft amino acid condensates.8 Such globular structures have polycationic surfaces. Depending on their hydrophilic/hydrophobic balance, they remained soluble in water up to very large molecular weight (~106 Da) (Collet et al. 2010). Some of them have necessarily been adsorbed to the polyanionic surface of rocks and finer grains of sediment. These dendrigraft peptides, soluble or adsorbed, could have played the role of micro-reactors in chemical evolution. We will discuss this aspect below.

6.4 6.4.1

Emergence of Oligonucleotides Prebiotic Synthesis of Nucleotides

Similar to peptide formation, the synthesis of nucleic acids from nucleotides is a dehydration reaction (Fig. 6.6). This applies to modern metabolism and prebiotic considerations alike. John Sutherland and colleagues have recently shown that it is possible to synthesize pyrimidine ribonucleotides from activated elementary molecules (Fig. 6.7) (Powner et al. 2009). O -O P O O-

B O -O P O O-

O

H H HOH H OH or H HO-

O -O P O O-

+

H H

H H OH or H

B O H H HOH H OH or H

B O

O O

B P O

-O

O H H HOH H OH or H

Fig. 6.6 Formation of sugar phosphate bonds, formally by a dehydration reaction

8

Dendrigraft polymers are a class of highly branched macromolecules belonging to the dendritic polymer family. Multiple branching levels characterize the architecture of these molecules, in analogy to dendrimers and hyperbranched polymers (Teertstra and Gauthier 2004).

6 Scenario of the Primary Pump: Emergence and Operation of an Automatic Engine

HO

O

N N

Cyanamide

- H2O

NH2

O

N

O

O

NH2

N

H2N N

N

HO O HO

HO

O

P

O

N

NH2+

N

O N N

NH2

Guanine

OH O-

NH2

HO N

Thymine

N

N

Adenine

HC C C N

Cyanoacetylene

O

Purine

N O

HO

N

O

Uracil

Cytosine HO

NH

NH

N

Glycéraldehyde

O

O

NH2

OH

Glycolaldehyde H2N C N

pyrimidine

O

OH

143

O

N

N O

- H2O

O

O P O O-

activated Pyrimidine

Fig. 6.7 Synthesis of activated pyrimidine ribonucleotides in prebiotically plausible conditions

Extrapolating from these results, we here assume that a group of ribonucleotides could have been formed on the primitive Earth. We will not discuss the order of appearance of RNA or DNA, but only how nucleic acids could emerge. The authors involved in this research (Fernando and von Kiedrowski 2007) concluded that at least three conditions are necessary to form these macromolecules: 1. The nucleotides must be activated. 2. The activated nucleotides must be concentrated. 3. The activated and concentrated nucleotides must be positioned on templates, to promote their dehydration. We will only take an interest in finding potentially “prebiotic” solutions to these three requirements.

6.4.2

Activation of Nucleotides

6.4.2.1

What Activation Mode for Nucleotides?

The activation via the triphosphate form (NTP) is used by living systems to produce nucleic acids. These syntheses are enzymatically catalyzed. Under abiotic conditions, attempts to oligomerize the Adenosine TriPhosphate (ATP) failed, the rate of hydrolysis of ATP being faster than its condensation. Using imidazole as

144

A. Commeyras

activating agent and at concentrations at least equal to 0.015 M, oligonucleotides up to 40 monomer units were obtained in the presence of montmorillonite (Prabahar et al. 1994). The prebiotic relevance of such a mode of activation of ATP has been seriously questioned. (Shapiro 2006). Following the primary pump scenario, we have revisited this issue. Two sets of accepted data jumped to our mind: 1. ATP is universally used by the living world. 2. The living world uses a variety of metabolic pathways to produce ATP. These data may give rise two different interpretations: 1. Perhaps, triphosphates were the first form of activation of nucleotides, and later during evolution, production modes of triphosphates have diversified (i.e., lost and gained independently). 2. Alternatively, primitive nucleotides have not been activated as triphosphates in the beginning, but in a different way which remains to be discovered. Subsequent evolution has repeatedly discovered (exapted9) triphosphates as a means of activating nucleotides; this is why the modes of triphosphate production are different from one species to another. What looks illogical in the second interpretation is to imagine that evolution has universally imposed this mode of activation with a retroactive effect. We therefore considered the first interpretation as potentially more reasonable and scrutinized the scenario of the primary pump for potential impact on additional reactions: 1. A primitive process to form nucleoside triphosphate (NTP) 2. A primitive process to concentrate NTP 3. A primitive process to condense NTP, giving the first strands of RNA and (or) DNA

6.4.2.2

The Primary Pump and the Synthesis of NTPs

As we have seen before, peptides are formed when the NCAs produced in dry phase are poured into water at pH > 4. If phosphoric acid is present, at pH between 4 and 8, amino acyl phosphates are obtained as is shown in Fig. 6.8 (Biron and Pascal 2004). The amino acyl phosphates obtained in 15 min hydrolyse completely in 1.5 h. This hydrolysis leads back to amino acids and phosphoric acid. However, if alcohols or acids are present (in the same range of pH 4–8), these compounds are phosphorylated by aminoacylphosphate (Fig. 6.9). For example, methanol gives methyl phosphate. Nucleoside Mono Phosphate (NMP) gives Nucleoside Di-Phosphate (NDP). Nucleoside Di-Phosphate (NDP) gives Nucleoside Tri-Phosphate. The ATP formed has been characterized by photon emission in

9

Exaptation: see wikipedia.

6 Scenario of the Primary Pump: Emergence and Operation of an Automatic Engine R HN

3

2

O

1 O

H

4 5

H2PO4O

HN

R H

R H

CO2

H2N

C O OH O OP O OH

O

NCA

145

O O

OP O OH

Aminoacylphosphates

Fig 6.8 Amino acid N-carboxyanhydrides as phosphate-activating agents in aqueous solution

R1 O

HN O

O

R1

O

+

-

O P OH

H 3N

+

O-

phosphate

NCA

O O

P

R1

R2OH

O O-

O O-

H 3N

+

O-

+

R2 O P O O-

O

trans-phosphorylated compound

aminoacyl phosphate NH2 N

O -

O P O O-

H H

R1

O

R1

O

HN

H3N+

O

phosphate buffer pH 5.7

O O P OOO

O

OH

N

N N

NH2

H

O

H OH

O

N

O

-

O P O P O P O OOO-

AMP

ADP

H H

O

OH

aminoacyl phosphate

N

N N

H H OH

ATP

NCA

Fig. 6.9 Alcohols as AMP or ADP can be phosphorylated by aminoacyl phosphate

the presence of the enzymatic Luciferin/luciferase system (A. Commeyras, L. Garrelly, H. Collet unpublished data). In this chapter, and by extrapolation, we have assumed that the different nucleoside triphosphates (NTP) have been obtained by the same reaction. This reaction shows that the primary pump may share its energy with phosphates. This property illustrates the vision of prebiotic reactions by Prof. Albert Eschenmoser (Eschenmoser 2007), that is to say: The less “robust” (constitutionally unidirectional) chemical reactions in a given environment are, the more sensitive and responsive to catalytic acceleration and inhibition, and the higher will be their chances to become assisted and eventually steered by catalysts that may contingently be present or be emerging in the environment.

In this scenario, the primary pump is a special device that continuously produces a family of energetic molecules, the NCAs. A fraction of these NCAs are used to form peptides. Others react with phosphates, which are distributed in many directions. We illustrate below how a possible pathway for the generation of nucleic acids could have emerged.

146

A. Commeyras

6.4.3

Concentration and Stabilization of Nucleoside Triphosphate (NTP)

6.4.3.1

Dendrigraft a-Amino Acids

In developing other goals of using the primary pump concept, we have shown (Collet et al. 2010) that it is possible to produce dendrigraft lysine (DGL), polymerizing lysine NCA in water at pH between 5 and 7, that is to say in potentially prebiotic conditions. Successive generations of DGL are obtained using as an initiator for the generation (n) the product of the generation (n – 1). The molar mass of these dendrigraft lysines grows exponentially. Table 6.1 gives some characteristics of DGL through five generations. Figure 6.10 idealizes the structure of DGL-G3. Such a structure is a nano-sphere of 7 nm in diameter fully soluble in water. The amine functional groups of these nano-particles are localized at the surface of the nano-sphere. In water at pH 5–6, the amine functional groups are fully protonated, DGL particles are polycationic. Under prebiotic conditions, of course, other amino acids would be interspersed stochastically and the degree of branching would be less compact. On the beaches of the primitive continents, such dendrigraft amino acids (DGA) could have been produced in a few weeks through wet and dry cycles. If this was the case, obviously strong interactions have existed between these polycationic DGA, and NTP, which under the same conditions are polyanionic (3 negative charges per NTP molecule). 6.4.3.2

Concentration of NTP at the Surface of DendriGraft Amino Acids (DGA)

The polycation–polyanion interactions have been studied in the case of DGL-G3 and ATP. It is shown that the affinity constant between DGL-G3 and ATP is equal to 106 1 M at pH 7.4 at 8 mM ionic strength (Zou et al. 2010). The complex between these two molecules is composed of 25 ATP per molecule of DGL-G3. Assuming that ATP is mainly localized to the surface of DGL, the structure of the complex can be visualized by Fig. 6.11. If the ATP molecule has a thickness of 1 nm, the local ATP concentration can range from 0.2 to 0.3 moles per liter. These high values are largely beyond the minimum required. Table 6.1 Some characteristics of dendrigraft lysines of generations 1–5 Generation 1 2 3 4 1,450 8,600 22,000 65,300 Mn (g/mol) N (number of lysines) 8 48 123 365 1 2 3.5 4.5 Rh (nm) at pH 7

5 172,300 963 6

6 Scenario of the Primary Pump: Emergence and Operation of an Automatic Engine

N

2

HN

HN

NH2

H2N

NH

O

O H

H2

O NH 2

NH

H2

2

NH2

O

O

NH

NH2

2

2

O

NH2

NH

N H2

O

HN

NH

2

2

H2N

NH

2 NH

NH2

HN

HN

NH

O

2 NH

H2 N

NH2

O

N

2

N H2

H2 N NH2

HN

O O

O

O

O

NH

HN O

NH2 NH2

NH

NH

NH

HN

HN NH

O

O

NH2

NH2

O

H2N

O

N H

NH2

O HN

O

O

O HN HN

N

N

HN

HN

NH

NH

2

NH2

N H

O NH

NH2

NH O

O

NH

N OH

H2N

NH2

HN

NH2

O

N H2

2

H2 N

O HN

NH

O

HN

O

NH

O

H N

O

NH

H2 N

H2N

O

NH

O O

NH2 NH2

H N

H2 N

N H2

O

H N

O

O

H N

NH

O

NH2

NH2

N H

NH2

N H

HN

NH2

NH

NH O O

O

O

N H

O

NH

N H

O

2

O

N H2

O H N

O

N H

O

N H

N O H

HN

H N

O NH

HN H2 N

O

NH

O

NH2 HN

O

HN

NH2

NH2

NH

N H2

2

H N

O H2 N

NH

NH

NH H N

NH

O

O

O

O O HN

NH2

O

NH

H2N

N H

2

HN

NH2

O

NH

O

HN NH2 O

O

DGL G3 123 Lys

N H2

O OH

H2N

NH

O

O

H2 N

NH2

2 NH O

H

O

H2N

O HN

O

H2 N

H O N

O

NH

O

HN

O

O

H O N NH

O

O

H2 N

HN

H N

2

NH

NH 2

HN

O

NH

NH

N H

NH

NH2 O

NH

H N

N

O

NH

H2N

O

HN

H N

O HN

NH

O

NH

HN

H2N O

O

2

NH O

O

NH2

NH2

O NH

O

HN

NH2 H2N

H

H2 N

2 NH O

HN

O

N H2

H N

O O

O

NH2 HN

NH O

O

N H

NH

HN

H2N

O

N H2 N H2

NH H2 N

H N

O HN

O

N H O

H2 N

HN

O

N H2

O

N

2

N H2

O

H2N

O

H

N H2

N H

NH2

NH HN

H2N

NH

O

O

NH

NH O

HN

2

NH2

NH

NH

O O

O

NH

H2 N

N H2

O

HN

HN

HN

O

O

2

NH

O NH

NH O

O

H2N

H2 N

H2 N

N H2

NH

O

H2 N

H2N NH2

H2N

O

N

2

NH H 2N

N H2

N H2

N H2

N H2

NH2

H

N H2 H2 N

147

NH2

H N

NH

2

2

Fig. 6.10 Idealized structure of DGL-G3

6.4.3.3

Stabilization of ATP (NTP) in the Complex DGL-G3/ATP

By measuring the rate of hydrolysis of ATP in the absence and presence of DGL, we observed that the presence of DGL significantly slowed the hydrolysis of ATP so that at temperatures of 30 C or below, ATP is stable for months. This stability in the complex DGL-ATP does not prevent the ATP to be active in the presence of a modern enzyme system. We have indeed shown that it is possible to determine the total ATP content in the complex DGL-ATP using the reaction of ATP on the enzymatic system Luciferase/ Luciferin. (A. Kovalova, L. Garrelly, A. Commeyras unpublished data). This accessibility of ATP to enzyme systems may have been of evolutionary importance. This result can certainly be extended to any kind of complex NTP/polycations. We ought, however, not to forget that in living organisms, the NTP and beyond nucleic acids are mainly associated with peptides. The study of interactions polyanions/polycations is difficult but of great interest.

148

A. Commeyras H2 N

NH2

N

N

N N

N

+

NH2 N

N

+

3 NH

+

3 N NH + H

+

3 NH O

O

N H

NH

O

NH2

NH3+

3 NH

N

O

+

3 NH

+

-

+

+

N

O

3 NH

NH O

O

+H

O

NH

+

3 NH

HO H H HO H H O

-

O

O

P

O P

NH2

OH P O O

O

-

+

H3

+ 3

N H3

NH3 +°

N

N

NH3+

NH

NH

N

O NH3 +

+

O

OHN

HN

3 NH

NH O

NH3 + NH3

HN NH O

O

N

+

O

NH+ NH

+ 3

O

O

N N

N

+

3 NH

+ 3

+

NH

N

N N

NH3 + OH N O N O H H NH3+ O P O O O O P O N NH H O O NHO O P O NH2 O NH3 + N O + H NH3 NH3+ HOH H H O NH3 +H N H N HO H O O N OHN H

O

N H

H H HO O H HO H NH3+ NH3 +

HN

O

NH3 +

HN ON H

O HN

NH2

H N

O

NH3 +

NH

O-

O P O O P O O O P O O

O

O

HN

NH3 + O

O NH3 + + H3 N NH HN HN HN O O HN NH O O HN P O HN NH O O O O HN P O NH2 H N O + 2 HN O O NH3 NH3 + P NH3+ NH+ + O OH H OH OH HN + + H N NH H 3 H 3 N O H OO O- ON OH N O P O O OP P O P HO HOH OH H O N O P O O H H O O O H2 N P O O O N N

O

NH3 + +HN

NH3+° NH

O

O

O

NH

HN

-

NH

NH3+

O

NH

O

O N

+H 3

H3 N

NH HO N

O NH

H3 N

HO

N + H +3 3 NH

+

N OH

HN

HN O

NH +

+ NH3 + NH3 NHO O H NH N O

O

HN O O NH HN O OH O DGL G3 HN 123 Lys O O O NH NH O HN N N H OH O HN N H O HN NH O

NH

NH O

N

HN

O

NH

O

N

N

+H 3

N

+H3

O NH NH3+HN

H N

H3N

NH2

N

+ 3

O

HN

NH O

H3 N

O

+

+ 3

+

O

NH

N H3

O

O

NH

H N

HO N

O

NH

+

O

NH

N

O

H N

O

N

+H3

NH

H O ON P O O OP O O O + O - H3 N + H3 N P O OH + H3N H OH N N H OH N O H2N H H N

O

+

NH3+O

NH

N

H H O HO H NH3 +HO H

H3 N

+ NH3HN

NH

+

HN

OHN

+H3

H3N

O

+

O

HN

NH3 +

N

OH H H

N

HOH

+H 3 +

H O

NH

N

N

H3 N

+H3

+

+H3

O

N

NH

O

O P O O O P O O O P O O

HN

O

NH

H N

N H

NH

O

N N

+H3

O

O

HN O

N

+H3

N H2 N

O

HN

+ O O H3 N O O P O + HN NHO NH O O P O +H3 N+ O H3N O HO O N NH O P + H3N OH

N

O

+

OHN

H N

+

O

HN

H 3N

HN

HN

O HN

O

O

N H

-

NH3 +

HN

+

O +

O

O

N H

O

NH3 ° O HN NH O O HN NH NH

H3 N

NH O HN

+

HN

H3N

+H3

3 NH

O

N

HO

+

+

+ H N NH2 3 NH

O HN

H N

+

N

O

O NH

H OH H

N

H3 N

H3N

NH

NH

NHO N H + H3N

N

N

N

+H 3

O

H3N O NH O O

H3 N

NH3 + +H3 N

NH HN

+

O N

+H 3

+

+H

H H OH

N

N

H3 N

N

P O HO

N

H 2N

+H 3

O

N

+

O

+

O

+

N

O H H OH H + H OH H3 N +H3

O O P O O P O O

+H

N

N

+H 3

N

+H 3

O O O OP O P O O OP HO O-

N

N

O H H H OH H OH +H3

H2 N

N

O N O O O O P O P H H HO P O OO H H O OH OH

N NH2

N NH2

Fig. 6.11 Idealized complex DGL-G3/ATP. Interactions polyanions/polycations concentrate and stabilize the peptides on dendritic NTPs. These could serve as a template to promote the condensation of the NTPs.for the polymerization of activated nucleotides. In the scenario of the primary pump the NTPs are formed every day but certainly at very low concentrations. In diluted solutions they can hydrolyse, or be concentrated in the complex DGA/NTP. – The question is then to know if NTPs must have been stabilized or not in the complex DGA/NTP

6.4.4

The Primary Pump and Nucleic Acid Synthesis

No experiment has yet been done on whether the NTP concentrated and stored in the complex DGL-NTP can be condensed. The following is therefore purely speculative. One can only wonder: • Whether this condensation is possible or not? • If it is possible, does it take place in the aqueous phase, or rather in dry phase only? • Whether the pH played a role in condensation?

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• Whether a catalyst was needed or not? • If catalysis was needed, were catalysts present beforehand or later-emerging in the environment? Indeed we have seen, in the scenario of the primary pump, that the primary structure of peptides is constantly changing. It is also known that catalytic activities are present in non-coded dendrigraft amino acids (Darbre and Reymond 2006). Therefore, it is reasonable to assume that catalytic activities able to promote the polymerization of NTP may have emerged from such environments. And to go beyond, if nucleic acids are formed, they will hydrolyse slowly, and a new cycle reaction will appear. This cycle will be focused on nucleic acids synthesis, with activation, condensation of nucleotides, partial hydrolysis of nucleic acids, new activation, and so forth. The template of this new cycle will always consist of dendrigraft peptides that will gradually become homochiral. These templates will select chiral nucleic acids, and so on. This new cycle will work through the constant supply of energy provided by the primary pump – comprising the heart of the primordial evolutionary engine.

6.5

In Brief

1. The primary pump is a driving force of a global system. 2. Every day, the primary pump built the NCA from which dendrigraft amino acids are formed. 3. The structure of these peptides are evolving. 4. Via trial and error the most likely molecules are subject to chemo- and enantioselection. 5. The chemical reactions are assisted by catalysts present, or potentially emerging in the environment 6. Concentration processes used are simple and natural. 7. In the scenario of the primary pump, reaction pathways are short. Matter and time are saved. The key molecules are recycled. 8. The primary pump shares its energy with phosphoric acid. 9. This distribution of energy leads to a permanent activation of nucleotides into nucleotide triphosphate (NTP). 10. The primary pump, via dendrigraft peptides, concentrates and stabilizes NTP. 11. NTP concentrated and stabilized in a complex of Dendrigraft Amino acids (DGA)-NTP may have led to nucleic acids. The scenario of the primary pump appears to be co-evolutionary. This means that a chemical energetic system pushes the peptides and potentially nucleic acids to co-emerge and co-evolve in the same environment.

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How Long has the Primary Pump Worked, and What Conditions Stopped its Operation?

The primary pump may have worked as long as the proportion of NO in the mixture NO/O2 remained high (>4). This could correspond to the initial conditions of the primitive Earth in which oxygen was scarce. When the proportion of oxygen increased, the concentration of nitrosating agents decreased dramatically and the pump gradually stopped working. Primitive life has then adapted to the increased concentration of oxygen when the role of NO decreased. But the role of NO may not have completely disappeared, since in modern life NO is a neurotransmitter of all living organisms. One wonders whether it would be a fossil remnant of early times or it would have played a role in the complexification of the organic matter. This potentiality could be an additional element to our thinking.

6.7

General Conclusion

The primary pump scenario is a tool (like the “intuition pump” according to Daniel Dennett) that allows us to understand the transition from inert to living matter. By continuous pumping, the primary pump could have led to something new and significant. This should be satisfying to the unstoppable reasonist Shadok (French Cartoon) in his relentless determination to continue pumping (Fig. 6.12), “It is better to pump even if nothing happens, rather than risk something worse happening if we do not pump.”

Fig. 6.12 Shadok’s motto: “It is better to pump even if nothing happens, rather than risk something worse happening if we do not pump”

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«Devises Shadok – Il vaut mieux pomper meˆme s’il ne se passe rien que risquer qu’il se passe quelque chose de pire en ne pompant pas.» (Jacques Rouxel) Acknowledgments For their fruitful collaboration to this work, we are indebted to our colleagues from Montpellier: He´le`ne Collet, Jacques Taillades, Louis Mion, Robert Pascal, Herve´ Cottet, Odile Vandenabeele, Laurent Boiteau, Jean-Christophe Rossi, Jean-Philippe Biron, Raphae¨l Plasson, as well as to Herve´ Martin from the laboratory Magmas & Volcans at the University of Clermont-Ferrand (France), and to Michel Dobrijevic and Franck Selsis from the Observatoire Aquitain des Sciences de l’Univers (Bordeaux, France). I also thank the students who, directly or indirectly, participated in this work. We are also indebted to Laurent Garrelly, Fabien Granier, and Anna Kovalova from COLCOM for their recent participation. The University Montpellier 2 for its ongoing support and assistance provided to the company COLCOM. The Centre National de la Recherche Scientifique (CNRS), especially its Chemical Science Department and the Institut National des Sciences de l’Univers (INSU). The Exobiology Research Group of the Centre National d’Etudes Spatiales (CNES). The European Community through the COST D27 action (prebiotic chemistry), for their support. I also thank the descendants of the late Jacques Rouxel for permission to quote a small part of the genius of Shadok’s Dad to finish with a smile this serious discussion.

References Biron J-P, Pascal R (2004) Amino acid N-carboxyanhydrides: activated peptide monomers behaving as phosphate-activating agents in aqueous solution. J Am Chem Soc 126:9198–9199 Chyba C, Sagan C (1992) Endogenous production, exogenous delivery and impact-shock synthesis of organic molecules: an inventory for the origins of life. Nature 355:125–132 Collet H, Bied C, Mion L, Taillades J, Commeyras A (1996) A new simple and quantitative synthesis of a-amino acid-N-carboxyanhydrides (oxazolidines-2,5-dione). Tetrahedron Lett 37:9043–9046 Collet H, Boiteau L, Taillades J, Commeyras A (1999) Solid phase decarbamoylation of N-carbamoylpeptides and monoalkylureas using gaseous NOx: a new simple deprotection reaction with minimum waste. Tetrahedron Lett 40:3355–3358 Collet H, Souaid E, Cottet H, Deratani A, Boiteau L, Dessalces G, Rossi J-C, Commeyras A, Pascal R (2010) An expeditious multigram-scale synthesis of lysine dendrigraft (DGL) polymers by aqueous N-carboxyanhydride polycondensation. Chem Eur J 16:2309–2316 Commeyras A, Collet H, Boiteau L, Taillades J, Vandenabeele-Trambouze O, Cottet H, Biron J-P J-P, Plasson R, Mion L, Lagrille O, Martin H, Selsis F, Dobrijevic M (2002) Prebiotic synthesis of sequential peptides on the Hadean beach by a molecular engine working with nitrogen oxides as energy sources. Polym Int 51:661–665 Commeyras A, Taillades J, Collet H, Boiteau L, Vandenabeele-Trambouze O, Pascal R, Cottet H, Plasson R, Biron J-P, Souaid E, Garrel L, Lagrille O, Danger G, Rossi J-C, Selsis F, Dobrije´vic M, Martin H (2004a) Molecular origins of life: homochirality as a consequence of the dynamic co-emergence and co-evolution of peptides and chemical energetics. In: Pa´lyi G, Zucchi C, Caglioti L (eds) Progress in biological chirality. Elsevier, Amsterdam Commeyras A, Taillades J, Collet H, Boiteau L, Vandenabeele-Trambouze O, Pascal R, Rousset A, Garrel L, Rossi J-C, Biron J-P, Lagrille O, Plasson R, Souaid E, Danger G, Selsis F, Dobrije´vic M, Martin H (2004b) Dynamic co-evolution of peptides and chemical energetics, a gateway to the emergence of homochirality and the catalytic activity of peptides. Orig Life Evol Biosph 34:35–55

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Commeyras A, Boiteau L, Vandenabeelle-Trambouze O, Selsis F (2005a) Peptide emergence, evolution and selection on the primitive Earth. I. Convergent formation of N-carbamoyl amino acids rather than free alpha-amino acids? In: Lectures in astrobiology, vol 1. Springer, Berlin/ Heidelberg Commeyras A, Boiteau L, Vandenabeele-Trambouze O, Selsis F (2005b) Peptide emergence, evolution and selection on the primitive Earth. II. The primary pump scenario. In: Lectures in astrobiology, vol 1. Springer, Berlin/Heidelberg Cronin J, Pizzarello S (1983) Amino acids in meteorites. Adv Space Res 3:5–18 Cronin J, Pizzarello S (1997) Enantiomeric excesses in meteoritic amino acids. Science 275:951–955 Csapo J, Collins M, Csapo-Kiss Z, Varga-Visi E, Pohn G, Csapo J Jr (2004) Use of amino acids and aminoacid racemization for age determination in archaeometry. In: Pa´lyi G, Zucchi C, Caglioti L (eds) Progress in biological chirality. Elsevier, Amsterdam Darbre T, Reymond J-L (2006) Peptide dendrimers as artificial enzymes, receptors, and drugdelivery agents. Acc Chem Res 39:925–934 Deamer DW (1992) Polycyclic aromatic hydrocarbons: primitive pigment systems in the prebiotic environment. Adv Space Res 12(4):183–189 Eschenmoser A (2007) On a hypothetical generational relationship between HCN and constituents of the reductive citric acid cycle. Chem Biodivers 4:554–573 Fernando C, von Kiedrowski G (2007) A stochastic model of nonenzymatic nucleic acid replication: “Elongators” sequester replicators. J Mol Evol 64:572–585 Forterre P (2005) The two ages of the RNA world, and the transition to the DNA world: a story of viruses and cells. Biochimie 87:793–803 Gargaud M, Martin H, Lopez-Garcia P, Montmerle T, Pascal R (2009) Le Soleil, la Terre. . . la vie. La queˆte des origines. Belin, Paris Gomes R, Levison HF, Tsiganis K, Morbidelli A (2005) Origin of the cataclysmic Late Heavy Bombardment period of the terrestrial planets. Nature 435:466–469 Honnoraty A-M, Mion L, Collet H, Teissedre R, Commeyras A (1995) Deracemization process of a-amino acids via pyridoxal. I. Synthesis and activity of polymerizable forms of pyridoxal. Bull Soc Chim Fr 132:709–720 Lagrille O, Taillades J, Boiteau L, Commeyras A (2007) Investigation of N-carbamoylamino acid nitrosation by NO + O2 in the solid-gas phase. Effects of NOx speciation and kinetic evidence for a multiple-stage process. J Phys Org Chem 20:271–284 Lathe R (2004) Fast tidal cycling and the origin of life. Icarus 168:18–22 Mather TA, Pyle DM, Allen AG (2004) Volcanic source for fixed nitrogen in the early Earth’s atmosphere. Geology 32:905–908 Navarro-Gonzalez R, Molina M, Molina L (1998) Nitrogen fixation by volcanic lightning in the early Earth. Geophys Res Lett 25:3123–3126 Pascal R, Boiteau L, Commeyras A (2005) From the prebiotic synthesis of a-amino acids towards a primitive translation apparatus for the synthesis of peptides. Top Curr Chem 259:69–122 Plasson R (2008) Comment on “re-examination of reversibility in reaction models for the spontaneous emergence of homochirality”. J Phys Chem B 112:9550–9552 Plasson R, Bersini H (2009) Energetic and entropic analysis of mirror symmetry breaking processes in a recycled microreversible chemical system. J Phys Chem B 113:3477–3490 Plasson R, Brandenburg A (2010) Homochirality and the need for energy. Orig Life Evol Biosph 40:93–110 Plasson R, Bersini H, Commeyras A (2004) Recycling Frank: spontaneous emergence of homochirality in noncatalytic systems. Proc Natl Acad Sci USA 101:16733–16738 Plasson R, Kondepudi DK, Bersini H, Commeyras A, Asakura K (2007) Emergence of homochirality in far-from-equilibrium systems: mechanisms and role in prebiotic chemistry. Chirality 19:589–600 Plasson R, Brandenburg A, Jullien L, Bersini H (2010) Autocatalyses. Artif Life 12:4–11

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Powner MW, Gerland B, Sutherland J (2009) Synthesis of activated pyrimidine ribonucleotides in prebiotically plausible conditions. Nature 459:239–242 Prabahar KJ, Cole TD, Ferris JP (1994) Effect of phosphate activating group on oligonucleotide formation on montmorillonite: the regioselective formation of 30 ,50 -linked oligoadenylates. J Am Chem Soc 116:10914–10920 Radzicka A, Wolfenden R (1996) Rates of uncatalysed peptide bond hydrolysis in neutral solution and the transition state affinities of proteases. J Am Chem Soc 118:6105–6109 Shapiro R (2006) Small molecule interactions were central to the origin of life. Q Rev Biol 81:105–125 Smith RM, Hansen DE (1998) The pH-rate profile for the hydrolysis of a peptide bond. J Am Chem Soc 120(35):8910–8913 Summers DP, Chang S (1993) Prebiotic ammonia from reduction of nitrite by iron(II) on the early earth. Nature 365(6447):630–633 Taillades J, Beuzelin I, Garrel L, Tabacik V, Bied C, Commeyras A (1998) N-carbamoyl-a-amino acids rather than free a-amino acids formation in the primitive hydrosphere: a novel proposal for the emergence of prebiotic peptides. Orig Life Evol Biosph 28(1):61–77 Teertstra SJ, Gauthier M (2004) Dendrigraft polymers: macromolecular engineering on a mesoscopic scale. Prog Polym Sci 29(4):277–327 Tessalina SG, Bourdon B, van Kranendonk M, Birck J-L, Philippot P (2010) Influence of Hadean crust evident in basalts and cherts from the Pilbara Craton. Nat Geosci 3:214–217 Varga P, Rybicki KR, Denis C (2006) Comment on the paper “Fast tidal cycling and the origin of life” by Richard Lathe. Icarus 180:274–276 Zou T, Oukacine F, Le Saux T, Cottet H (2010) Neutral coatings for the study of polycation/ polyanion interactions by capillary electrophoresis: application to dendrigraft poly-L-lysines with negatively multicharged molecules. Anal Chem 82(17):7362–7368

Chapter 7

The Relevance of Peptides That Bind FeS Clusters, Phosphate Groups, Cations or Anions for Prebiotic Evolution E. James Milner-White

Abstract The focus of this chapter is on the significance for evolution of threedimensional peptide motifs made from main chain atoms of a few amino acid residues. The CONH group of every peptide bond is a polar group with the oxygen having substantial fractional negative charge and the hydrogen having significant partial positive charge. Appropriate conformations of polypeptides create binding sites for either anions or cations by bridging of the form NH–anion–HN or CO–cation–OC between non-adjacent CONH groups. These motifs are more common in proteins than is generally realized. About 8% of amino acid residues in native folded proteins belong to anion-binding motifs and another 8% belong to cation-binding motifs. Examination of native and synthetic polypeptides suggests these figures were even higher for peptides occurring during early evolution. The most common cation-binding motif is one named the niche, while the most common anion-binding motif is called the nest. Some nests bind single atoms and some bind groups like FeS clusters and phosphates. The P-loop, which is the commonest ATP/GTP-binding feature in proteins, as well as being one of the most ancient features of proteins in general, incorporates a phosphate-binding nest within its active site. If di- or triphosphates were the major sources of instant energy in the earliest forms of metabolism, phosphate-binding nests can be said to have retained the structure required for energy generation and thus be one of the most ancient molecular relics in existence.

E.J. Milner-White (*) Professor of Structural Bioinformatics, Institute of Biomedical and Life Sciences, Glasgow University, Glasgow G128QQ, UK e-mail: [email protected] R. Egel et al. (eds.), Origins of Life: The Primal Self-Organization, DOI 10.1007/978-3-642-21625-1_7, # Springer-Verlag Berlin Heidelberg 2011

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Introduction

In the prebiotic world, it seems at first sight hard to imagine how proteins and nucleic acids evolved from simple molecules. Cairns-Smith (1982) and de Duve (1987) have suggested that, in systems of medium-sized molecules continually being synthesized and degraded, any molecules with properties that encourage their own synthesis or prevent degradation grow faster than the others and so are, to a limited extent, self-replicating. Furthermore, in mixtures of quite different molecules being synthesized, one set of molecules, A, may assist the synthesis of another set, B, and set B may also assist synthesis of set A. Such mutually synergistic effects can be imagined to be quite powerful and a possible example is given in Sect. 7.2.2. Effects of this sort are not especially unusual in a chemistry laboratory and, the more the matter is considered, certain molecules are expected to be more successful than others, and can be said to have some degree of selfreplicating properties. The realization that nucleic acids, notably those at the centre of the ribosome, are capable of catalysis, as well being inherently self-replicating molecules, has led to the idea of the RNA world (Wolf and Koonin 2007) where RNA was the central controlling macromolecule of early biological systems. However, nucleotides are more complex and difficult to synthesize than simple amino acids. Amino acids such as glycine appear readily in artificial prebiotic soups (Hennet et al. 1992) whereas nucleotides do not. Some of the properties in the expected early peptides, as will be shown, lend themselves to assisting the development of prebiotic systems in ways that are hard to envisage for polynucleotides. Claims have also been made that some protein features are the most ancient conserved macromolecular entities that exist (Lupas et al. 2001; Brakoulias and Jackson 2004; Ma et al. 2008; Volbeda et al. 2010; Kurland 2010). I shall proceed on the basis that at the very earliest stage in evolution, a period occurred when proteins or peptides were major biomolecules in the sense that a synergy existed between proteins and peptides on the one hand and metabolic entities on the other. This idea by no means precludes the existence of an RNA or protein/RNA world before the emergence of DNA, but the premise is that any such era came later. The earliest uncoded proteins were probably not composed of large domains of tightly folded polypeptide chains like the proteins we are familiar with. They would have been small, simple and heterochiral in nature. Without a genetic code as we know it, different polypeptide molecules would probably have had different compositions and sequences and lacked defined three-dimensional structures at ˚ or so) most present-day proteins exhibit. While they were the large scale (25 A not limited to the 20 amino acids in current proteins, they were probably limited by their ease of synthesis, with a preponderance of glycines and a few others such as alanine and aspartate. These others were almost certainly heterochiral at least initially. The homochirality of present-day amino acids has a great effect on the structures they adopt (Ramakrishnan et al. 2006) and the a-helix, especially, is only favoured in homochiral peptides. These factors result in early peptides having been

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more exposed to solvent water and variable and motile in their 3D structure than present-day evolved proteins. This does not mean they lacked any structure at all, ˚ , many structures do occur. One aspect is as, especially on a smaller scale of say 5 A that the variability and unpredictability of the side chains would have caused main chain, rather than side chain, features to be employed for functional purposes. The small motifs described below are the ones that employ main chain atoms only for various anion or cation binding activities and this aspect would have made them functionally useful in the early prebiotic world.

7.1.1

Web Access to Motifs

A web application, Motivated Proteins (Leader and Milner-White 2009), gives access to a database of high resolution proteins in which the motifs described, as well as other small ones, can be visualized with the help of the ‘jmol’ molecular graphics program. Motifs can be identified and visualized on their own in atomic detail. Alternatively, their situations in relations to the rest of the protein, either in 3D or in relation to the sequence, can be viewed. A glossary provides an introduction to motifs in general.

7.2 7.2.1

Short Peptide Motifs Anion Binding: Nests

In nests, seen in Fig. 7.1a, three consecutive amino acids form a cavity such that the main chain NH groups of the first and third residues bridge, via hydrogen bonding, a negatively charged, or partially negatively charged atom, oxygen atom. The nest is thus an anion binding site (Watson and Milner-White 2002a, b; Pal et al. 2002; Milner-White et al. 2004; Kubik 2009). The feature is common such that 8% of residues in all soluble native folded proteins exhibit it. Two or more nests can overlap to form a larger and wider cavity that can bind an anionic group instead of just a single atom. The NH groups all point approximately to the centre of the curve formed by the polypeptide. One such cavity is observed in the well-known ATP/GTP binding site in proteins: the P-loop in G-proteins, kinases and ATPases, as in Fig. 7.1c. A nest conformation is generated when the f,c angles of two successive amino acid residues are approximately enantiomeric, with the angles in Table 7.1. The f,c angles of the third nest residue do not affect the nest conformation. Nests, defined by these angles, are of two kinds called RL and LR. Overlapping nests can be RLR, RLRL, etc., as in Fig. 7.1b, c. R stands for right-handed (negative f) and L stands for left-handed (positive f). In proteins, 8% are RL and 20% are LR. On the whole, most RL nests are perfect in the sense of the first and third NH groups bridging a

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Fig. 7.1 Motif structures: (a) nest binding a carbonyl oxygen; (b) nest binding a phosphate ion; (c) nest binding a Fe4S4 iron-sulphur centre; (d) covalent Ni+-tetrapeptide complex; (e) two of the four peptides forming the selectivity filter of the potassium channel; (f) the peptide forming the aquaporin channel; (g) catgrip bound to a calcium ion; (h) niche3 bound to a K+ ion; (i) niche4 bound to a K+ ion; (j) vancomycin, which can be regarded as a nest designed to bind a carboxylate group in the bacterial cell wall (the carboxylate of acetate is seen in sticks and the antibiotic is in spacefill); (k) three strands of a-sheet. (Note the similarity in conformation with e and f.) a-sheet, unlike b-sheet, has an inherent polarity, indicated by the partial charges d+ and d. In a–i and k the side chains are omitted. Colours: carbon, green; oxygen, red; nitrogen, blue; sulphur, mustard; potassium, purple; iron, rust; nickel, brown; chlorine, green; calcium, yellow; phosphorus, orange

proteinaceous oxygen atom. A proportion of LR nests are not bridged at all or are bridged by water molecules. In general as a result, LR nests are less concave than RL nests. Vancomycin is a glycopeptide antibiotic, with residues of alternating D- and L-configurations. It acts by binding to a C-terminal D-ala residue, an intermediate in bacterial cell wall synthesis. In the crystal structure of vancomycin-acetate, acetate

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Table 7.1 Dihedral angles and structures of motifs cA fΒ cΒ fC cC Enantiomeric? Motif fA 3 76 22 Nearly RL nest 94 LR nest 74 16 77 20 Nearly RL a-strand 60 60 60 60 Yes 60 60 60 Yes LR a-strand 60 Niche3 91 6 87 141 No 19 94 9 99 134 No Niche4 73 RL catgrip 70 150 70 150 Yes 150 70 150 Yes LR catgrip 70 Covalent metal-peptide 180 0 180 0 Yes Many three-residue motifs are specified by two pairs of f,c angles for two successive amino acids. When its main chain atoms are enantiomeric (fA ¼ fΒ and cA ¼ cΒ) a dipeptide is said to be enantiomeric and may occur as part of a longer enantiomeric peptide. The niche4 is a four-residue motif specified by three pairs of angles. In proteins glycines are common (50–80%) at residues where f > 0

mimics the terminal D-ala carboxylate. Figure 7.1j shows how vancomycin can be regarded as a box with a nest at the bottom for binding the carboxylate of acetate (Milner-White et al. 2004). The peptidic part of vancomycin consists of alternating L and D-amino acids, consistent with heterochiral peptides of this sort forming nests readily. Several other naturally occurring and synthetic peptides are known that form nests (Milner-White et al. 2004; Berkessel et al. 2006; Pajewski et al. 2005). It seems that during early evolution, nests were used for anion binding and remain in several present-day protein and peptide features. Considering phosphate binding, more recently evolved features like protein kinases, IP3 binding and tyrosine phosphates employ positively charged side chains of lys, arg and his for anion binding. Perhaps the functional nests emerged at a time, before the genetic code, when amino acids with side chains were of sporadic and unreliable occurrence, so features relying on main chain atoms were more reproducible. The P-loop, a well-known feature in proteins that binds the b-phosphate of ADP, ATP, GDP or GTP, incorporates an overlapping LRLR nest pentapeptide that may be a relic of the earliest phosphate-binding peptides. Some P-loop nests bind the phosphates of ligands like pyridoxal phosphate as supportive handles, but the majority of P-loop proteins bind nucleotides. FeS proteins where the iron-sulphur centre is bound to nests, discussed later, may also be early enzyme relics. Life is thought by many workers to have evolved in lukewarm hydrothermal vents with alkaline, reduced, H2O, rich in H2, H2S, alkyl sulphides, Fe++, Ni++, emerging at the bottom of an acidulous ocean (Russell and Hall 1997). Since nucleotides are more difficult to make than peptides, a period with peptides but no nucleic acids, would have occurred. Another period, with nucleic acid but before the advent of the genetic code, is also likely to have existed. In experiments simulating early evolution, the amino acids made are glycine-rich and amino acids with side chains are heterochiral. Examination of the structures of small peptides with such amino acid compositions reveals that they tend to form nests readily (Milner-White et al. 2004).

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The iron and sulphide-rich fluid emerging from lukewarm hydrothermal vents at the ocean floor forms precipitates of iron sulphide in the form of bubbles or froth and it is here that life may have developed (Russell and Martin 2004). In the light of this proposal, it is intriguing that 50% of iron-sulphur centres in present-day proteins are bound to nests, often overlapping ones. If all the sulphur atoms are included, the net charge of any iron sulphur centre is negative, whether the iron is Fe2+ or Fe3+. So, they can be regarded as anionic. In some cases, there are four overlapping nests as in the example of the iron-sulphur centre in Fig. 7.1c. Ironsulphur centres are potentially valuable for catalysis via oxidation-reduction reactions since they can exist as Fe2+ or Fe3+ and the ability to stabilize and sequester these centres as peptide nests must have been, and still is, an advantage. As well as sequestration, the NH groups would tend to stabilize the reduced rather than the oxidized forms. Originally, such iron-sulphur centres would have incorporated sulphur atoms from alkyl sulphides present in hydrothermal vent fluid rather than from cysteine side chains in present-day proteins (Milner-White and Russell 2005, 2008).

7.2.2

Possible Synergy Between Nests and Polyphosphates

As well as binding phosphate, many P-loop proteins catalyse the transfer of a phosphate of ATP or GTP to HOH (ATPase, GTPase) or to a substrate (kinase). Some primitive anaerobic organisms use pyrophosphate, PPi, instead of ATP as the

a

Fig. 7.2 Pyrophosphatase mechanism. How the main chain atoms of a short peptide may catalyse: (a) the pyrophosphatase reaction PPi ! Pi + Pi; and (b) the reverse reaction, pyrophosphate synthase. Although octaglycine is presented, other combinations of amino acid residues would be effective

b

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high energy molecule. It has been suggested that the earliest P-loop proteins made use of this energy by catalysing the transfer of a PPi phosphate to HOH (PPase) as in Fig. 7.2a, and also the reverse reaction of pyrophosphate synthase as in Fig. 7.2b (Milner-White and Russell 2010). In the figure, it is supposed that the catalytic functions currently performed by side-chain groups were originally carried out by main chain atoms. The C-terminal carboxylate acts as a base catalyst in the PPase reaction and an acid catalyst in the synthase reaction. A present day protein that catalyses the pyrophosphatase reaction is the integral membrane protein H+-pyrophosphatase (Baltscheffsky et al. 1999). It harnesses a proton gradient for pyrophosphate synthesis. This protein incorporates a sequence signature characteristic of a P-loop, so probably has a phosphate-binding nest resembling that of other P-loop proteins (Hedlund et al. 2006). Accordingly, the phosphate-binding part of this pyrophosphatase might qualify as being a very ancient relic. A body of evidence (Rabinowitz et al. 1969; Chung et al. 1971; Yamanaka et al. 1988; Ni et al. 2009) reveals that, when polyphosphates are mixed with L-amino acids, the amino acids become phosphorylated and one of the end products is a dipeptide. These molecules in turn react similarly, forming tripeptides and, eventually, longer peptides emerge. This chemical reaction occurring simultaneously with the enzymic reaction of Hþ- pyrophosphatase, which might have been a short peptide as in Fig. 7.2, amounts to synergy: polyphosphates react with amino acids to form peptides and the peptides catalyse the formation of polyphosphates (MilnerWhite and Russell 2010). The polyphosphates in question could be pyrophosphate or they could be triphosphates by analogy with ATP. The synergy between these two reactions seems a plausible way in which an early metabolism got started by mutual selection for the two sets of molecules, polyphosphates and peptides, that give rise to the metabolism.

7.2.3

Alpha-Sheet and Amyloid

Considering the distribution of nests, not all have the same concave shape. Some have little or no concavity and are flattened. Such peptides are similar to that expected in a-sheet, as seen in Fig. 7.1k, a structure predicted by Pauling and Corey in 1951. However, crystal structures of native proteins revealed little and a-sheet was largely ignored. Although flattened nests are relatively common, they are only rarely organized into a-sheet in proteins. Recent work has suggested that a-sheet, shown in Fig. 7.1k, may be the material of the toxic amyloid precursor and that it converts itself into the somewhat more stable and non-toxic b-sheet of mature amyloid by the process of peptide plane flipping (Armen et al. 2004; Daggett 2006; Milner-White et al. 2006; Hayward and Milner-White 2008; Grillo-Bosch et al. 2009). If the amyloid precursor is indeed the material of amyloid that is extremely

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toxic to cells, the evolution of cells would have selected against its occurrence, explaining why it is relatively rare in native folded proteins. Amyloid is well known as the proteinaceous substance that is the causative agent of diseases such as Alzheimer’s, Parkinson’s, Huntington’s, type II diabetes and CJD. These so-called amyloidoses are not due to microorganisms but rather due to harmful misfolded proteins called amyloid. Recently, it has been shown that a propensity to form amyloid is not just the property of a few specialized proteins but that ordinary proteins such as myoglobin can also form amyloid in appropriately denaturing environments (Fandrich et al. 2001). Mature amyloid is composed of multiple layers of large b-sheets. However, it is the amyloid precursor, rather than the mature form, that is toxic. The nature of this precursor is controversial, but evidence is accumulating suggesting it is a-sheet. Amyloid (whether made of a- or b-sheet) is a sticky and gelatinous substance. It has been suggested that it was the material that formed a primitive cell membrane with a degree of impermeability during early evolution (Chernoff 2004; Milner-White and Russell 2008). Such cells might have been hardly more than blobs of amyloid gel adhering with interstices between them forming the intracellular medium. However, once phospholipid cell membranes evolved, amyloid would have become, not just redundant, but harmful, because its toxicity is expressed by damage to phospholipid cell membranes (Stefani and Dobson 2003). Amyloid is typically formed from identical polypeptides rather than from polypeptides with different sequences. This involves self-recognition which can only occur via the identical side chains lining up and recognizing each other (hydrophobic–hydrophobic and hydrogen bonding–hydrogen bonding). Hence amyloid has a fairly high degree of self-replicating propensity. This property could have been useful during early evolution before the advent of cells with phospholipid membranes, perhaps less so in present-day proteins because of its toxicity giving rise to various amyloid diseases.

7.2.4

Cation Binding: Covalent Metal-Peptide Complexes

Copper, nickel, cobalt and iron cations readily complex with peptides in alkaline solutions by binding to successive main chain amide nitrogen atoms (Harford and Sarkar 1999). Amide NH protons are displaced by the metal during the formation of these tight complexes in which the main chain atoms adopt a flat conformation as seen in Fig. 7.1d. The angles are in Table 7.1. Such complexes, with up to four amide protons substituted by metals, also occur naturally within a number of native proteins (prion protein of CJD) and enzymes (acetyl CoA synthase), sometimes at active sites. The Ni tetraglycine complex of Fig. 7.1d exhibits a remarkable similarity to present day macrocyclic tetrapyrrole cofactors. Also, the analogous Co-tetraglycine resembles a corrinoid group. In these types of complex, the metal has octahedral coordination to four planar nitrogens with two positions free to assist in catalytic reactions. The peptide ones could thus have performed much the same

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catalytic functions as hemes and corrinoids, before being largely, but not entirely, superceded by them.

7.2.5

Cation Binding: Niches

The niche (Torrance et al. 2009) is a three to four residue motif with the characteristic f,c angles given in Table 7.1. It is by far the commonest feature where main chain carbonyl groups bridge metals or partial positive groups. The niche accommodates atoms or groups that offer partial positive charges, including water molecules or metal ions, as well as amines, guanidines, and other NH2 groups. Seven percent of all residues in an average soluble protein belong to a niche; another 7% have the niche conformation but no obvious bridging group. Fifty-five percent of niches occur either following a type 1 b-turn or at the C-termini of a-helices, and niches turn out to be the most common C-terminal features of ahelices. 3/10 helices also frequently terminate in niches. Niches that bind K+, Na+ or Ca2+ occur in some functional contexts: in the cyclic peptides valinomycin and antamanide; in several enzymes that are allosterically activated by Na+ or K+; and in the calcium pump, where a niche is involved in the ion transport. Niches are of two sorts, the niche3 with three residues as in Fig. 7.1h and the niche4 with four as in Fig. 7.1i. Unlike the other motifs described here, these peptide motifs are not enantiomeric and cannot overlap.

7.2.6

Cation Binding: catgrips

Another enantiomeric conformation is called the catgrip (Watson and MilnerWhite 2002b) where alternating main chain CO groups bind Ca++ or other cations in a ring-shaped conformation as in Fig. 7.1g. The main-chain CO groups point into the ring; this is employed for specific Ca++ ion binding in the annexin, phospholipase A2, and subtilisin loops, and the regularly arranged b-roll loops of the serralysin protease family. Apart from their role in calcium binding, catgrips are relatively common in proteins though their numbers are 10% or less of those of nests or niches.

7.2.7

Cation Binding: Channels

Potassium channels possess a characteristic GYG signature sequence that folds to a similar three-dimensional structure; some of these transporters are about 10,000 times more permeable to potassium and rubidium ions than to sodium ions. Crystal structures (Zhou et al. 2001) of these integral membrane proteins reveal a row of

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main chain carbonyl groups from adjacent GYG residues arranged in four-fold symmetry around the potassium ions in the narrow part of the channel as in Fig. 7.1e. The feature is called the selectivity filter. The most decidedly linear part of this row of carbonyl groups is formed by the main chain conformation of the GY residues. The conformation is like that of overlapping nests, but extended, so that the nest concavity is lost. Another channel which includes a functional peptide of this sort is the aquaporin channel (Sui et al. 2001), illustrated in Fig. 7.1f. Here, the channel has a single peptide and its function is to allow a row of water molecules to be transported across the membrane. These flattened nest conformations are relatively unusual in native proteins but their conformation is similar to that of the peptides within the a-sheet discussed earlier.

7.3

Conclusions

Anion and cation binding tends to be regarded as mediated mainly by the charged side chains of aspartate, glutamate, lysine, arginine and histidine. This work shows that, in spite of the main chain atoms of peptides not being portrayed conventionally as having a formal charge, they are none the less often employed for binding anions and cations in current proteins. There are indications this was even more common in the earliest peptides when the side chains were presumably not genetically encoded and thus amino acids with charged side chains could not be relied upon to occur. The anion-binding motif expected to have been particularly common in early evolution is the nest. This feature is present as a phosphate binding feature within P-loops, which are well known as the most abundant ATP or GTP-binding motifs in proteins. In present day proteins, P-loops are mostly associated with phosphoryl transfer reactions for energy generation from ATP or GTP and it seems plausible this function is an evolutionary relic of the earliest energy generating systems from di- or triphosphates. Nests are also employed for binding iron-sulphur centres in proteins which would also have been catalytically useful in early evolution. In relation to nests, the a-sheet conformation resembles a flattened version of nests and its significance with respect to amyloid is discussed; a key aspect for early evolution is that amyloid has been suggested as a very early material of cell membranes. The commonest cation-binding motif is the niche and this was probably also present in the peptides during early evolution. Three other functionally useful cation-binding features are also described: catgrips, for calcium binding; peptide channels, for sodium and potassium ion transport across membranes; and covalent metal-peptide complexes where the peptide binds copper, nickel, cobalt or iron metal ions, for catalysis. Knowledge about the conformations of proteins and short polypeptides has changed. Some of the shapes of polypeptides likely to have occurred regularly in developing forms of life in a pre-RNA world can now be guessed. We begin to glimpse how the functional properties of these peptides, especially their facility for either cation or anion binding, would have assisted evolution of the earliest types of

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metabolisms. Of these, phosphate binding peptides appear to have left the most traces as they would seem to have been retained in a large proportion of proteins using ATP and GTP in present day proteins, occurring not only in a binding capacity but also still, arguably, being closely associated with catalysing the energy-generating hydrolysis of these polyphosphates.

References Armen RS, DeMarco ML, Alonso DOV, Daggett V (2004) Pauling and Corey’s a-pleated sheet structure may define the prefibrillar amyloidogenic intermediate in amyloid disease. Proc Natl Acad Sci USA 101:11622–11627 Baltscheffsky M, Schultz A, Baltscheffsky H (1999) H+-proton-pumping inorganic pyrophosphatase: a tightly membrane-bound family. FEBS Lett. 452(3):121–7 Berkessel A, Koch B, Toniolo C, Rainaldi M, Broxterman QB, Kaptein B (2006) Asymmetric enone epoxidation by short solid-phase bound peptides: further evidence for catalyst helicity and catalytic activity of individual peptide strands. Biopolymers 84(1):90–96 Brakoulias A, Jackson RM (2004) Towards a structural classification of phosphate binding sites in protein-nucleotide complexes. Proteins 56:250–260 Cairns-Smith AG (1982) Genetic takeover and the mineral origins of life. Cambridge University Press, UK Chernoff YO (2004) Amyloidogenic domains, prions and structural inheritance: rudiments of early life or recent acquisition? Curr Opin Chem Biol 8:665–671 Chung NM, Lohrmann R, Orgel LE, Rabinowitz J (1971) Mechanism of the trimetaphosphateinduced peptide synthesis. Tetrahedron 27:1205–1210 Daggett V (2006) a-sheet: the toxic conformer in amyloid diseases? Acc Chem Res 39:594–602 De Duve C (1987) Selection by differential molecular survival: a possible mechanism of early chemical evolution. Proc Natl Acad Sci USA 84:8253–8256 Fandrich M, Fletcher MA, Dobson CM (2001) Amyloid fibrils from muscle myoglobin – even an ordinary globular protein can assume a rogue guise if conditions are right. Nature 410:165–166 Grillo-Bosch D, Carulla N, Cruz M, Sanchez L, Pujol-Pina R, Madurga S, Rabanal F, Giralt E (2009) Retro-enantio N-methylated peptides as beta-amyloid aggregation inhibitors. ChemMedChem 4(9):1488–1494 Harford C, Sarkar B (1999) Amino terminal Cu(II) and Ni(II) binding ATCUN motif of proteins and peptides. Acc Chem Res 30:123–130 Hayward S, Milner-White EJ (2008) The geometry of a-sheet: implications for its possible function as amyloid precursor in proteins. Proteins 71:415–425 Hedlund J, Cantoni R, Baltscheffsky M, Baltscheffsky H (2006) Analysis of ancient sequence motifs in the H+-PPase family. FEBS J 273:5183–5193 Hennet RJ-C, Holm NG, Engel MH (1992) Abiotic synthesis of amino acids under hydrothermal conditions and the origin of life: a perpetual phenomenon? Naturwissenschaften 79:361–365 Kubik S (2009) Amino acid containing anion receptors. Chem Soc Rev 38:585–605 Kurland CG (2010) The RNA dreamtime. Bioessays 32:866–871 Leader DP, Milner-White EJ (2009) Motivated proteins: a web application for studying small three-dimensional protein motifs. BMC Bioinformatics 10(1):60–64, http://motif.gla.ac.uk/ Lupas AN, Ponting CP, Russell RB (2001) On the evolution of protein folds: are similar motifs the result of convergence, insertion or relics of an ancient peptide world? J Struct Biol 134:191–203 Ma B-G, Chen L, Ji H-F, Chen Z-H, Yang F-R, Wang L, Qu G, Jiang Y-Y, Ji C, Zhang H-Y (2008) Characters of very ancient proteins. Biochem Biophys Res Com 366:607–611

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Milner-White EJ, Russell MJ (2005) Sites for phosphates and iron-sulfur thiolates in the first membranes: 3 to 6 residue anion binding motifs (nests). Orig Life Evol Biosph 35:19–27 Milner-White EJ, Russell MJ (2008) Predicting the conformations of peptides and proteins in early evolution. Biol Direct 3:3 Milner-White EJ, Russell MJ (2010) Polyphosphate-peptide synergy and the organic takeover at the emergence of life. J Cosmol 10:2 Milner-White EJ, Nissink JW, Allen FH, Duddy WJ (2004) Recurring main-chain anion-binding motifs in short polypeptides: nests. Acta Crystallogr D Biol Crystallogr 60(11):1935–1942 Milner-White EJ, Watson JD, Qi G, Hayward S (2006) Amyloid formation may involve alpha- to beta sheet interconversion via peptide plane flipping. Structure 14(9):1369–1376 Ni F, Gao X, Zhao Z-X, Huang C, Zhao Y-F (2009) On the electrophilicity of cyclic acylphosphoramidates (CAPAs) postulated as intermediates. Eur J Organic Chem 18:3026–3035 Pajewski R, Ferdani R, Pajewska J, Li R, Gokel GW (2005) Cation dependence of chloride ion complexation by open-chained receptor molecules in chloroform solution. J Am Chem Soc 127(51):18281–18295 Pal D, Suehnel J, Weiss M (2002) New principles of protein structure: nests, eggs and what next? Angew Chem 41:4663–4665 Pauling L, Corey RB (1951) The pleated sheet, a new layer configuration of polypeptide chains. Proc Natl Acad Sci USA 37:251–256 Rabinowitz J, Flores R, Krebsback R, Rogers G (1969) Peptide formation in the presence of linear or cyclic polyphosphates. Nature 224:795–796 Ramakrishnan V, Ranbhor R, Kumar A, Durani S (2006) The link between sequence and conformation in protein structures appears to be stereochemically established. J Phys Chem B 110:9314–9326 Russell MJ, Hall AJ (1997) The emergence of life from iron monosulphide bubbles at a submarine hydrothermal redox and pH front. J Geol Soc London 154:377–402 Russell MJ, Martin W (2004) The rocky roots of the acetyl-CoA pathway. Trends Biochem Sci 29:358–363 Stefani M, Dobson CM (2003) Protein aggregation and aggregate toxicity: new insights into protein folding, misfolding diseases and biological evolution. J Mol Med 81:678–699 Sui H, Han B-G, Lee JK, Wallan P, Jap BK (2001) Structural basis of water-specific transport through the AQP1 water channel. Nature 414:872–878 Torrance GM, Leader DP, Gilbert DR, Milner-White EJ (2009) A novel main chain motif in proteins bridged by cationic groups: the niche. J Mol Biol 385(4):1076–1086 Volbeda A, Nicolet Y, Fontecilla-Camps J (2010) An ancient protein fold links metal-based gas reactions with the RNA world. J Cosmol 10:4 Watson JD, Milner-White EJ (2002a) A novel main-chain anion-binding site in proteins: the Nest. A combination of phi,psi values in successive residues gives rise to anion-binding sites that occur commonly and are found at functionally important regions. J Mol Biol 315(2):171–182 Watson JD, Milner-White EJ (2002b) The conformations of polypeptide chains where the mainchain parts of successive residues are enantiomeric. Their occurrence in cation and anionbinding regions of proteins. J Mol Biol 315(2):183–191 Wolf YI, Koonin EV (2007) On the origin of the translation system and the genetic code in the RNA world by means of natural selection, exaptation and subfunctionalization. Biol Direct 2:14 Yamanaka J, Inomata K, Yamagata Y (1988) Condensation of oligoglycines with trimeta- and tetrametaphosphate in aquaeous solutions. Orig Life Evol Biosph 18:165–178 Zhou YF, Morais-Cabral JH, Kaufman A, MacKinnon R (2001) Chemistry of ion coordination and ˚ resolution. Nature 414:43–48 hydration revealed by a K1 channel-Fab complex at 2.0 A

Chapter 8

Peptide-Dominated Vesicles: Bacterial Internal Membrane Compartments as Model Systems for Prebiotic Evolution James N. Sturgis

Abstract It is often presumed that prebiotic lipid membranes were colonized by proteins. In this chapter, I describe what I believe is a reasonable scenario for the emergence of mixed protein-lipid membranes in a pre-biotic setting and the subsequent emergence of the different classes of membrane protein. I suggest that the first pre-biotic membranes could have resembled bacterial internal membranes in their structure, being peptide dominated, and in their function, serving as an energy source. This scenario places hydrophobic and amphiphilic peptides at the center of abiotic evolution and the formation of the first membranes.

8.1

Introduction

The tree of life was first imagined by Darwin (1859) as a series of branchings from a common origin. Today’s organisms form the leaves of this tree and the origin of life is at the base of the trunk. Current visions of the origin of life require the association of three components: genes (carrying information for guiding metabolism), metabolism (building the molecules of life) and membranes (creating a cellular self), the combination of these leading to the formation of autonomous evolving systems (Pereto 2005). Genes are necessary for heritability from generation to generation of the original cells’ characteristics. Membranes are necessary to maintain the association of the genotype and the metabolic phenotype it encodes, once a genetic system exists, and to localize the metabolism. Finally, the metabolism is necessary to fabricate the other two components. This vision of life does not constrain the chemical nature of any of the components, only their functional role.

J.N. Sturgis (*) Laboratoire d’Inge´nierie des Syste`mes Macromole´culaires (LISM), CNRS UPR9027, Institut de Microbiologie de la Me´diterrane´e, Aix-Marseille University, 31 Chemin Joseph Aiguier, 13402 Marseille, France e-mail: [email protected] R. Egel et al. (eds.), Origins of Life: The Primal Self-Organization, DOI 10.1007/978-3-642-21625-1_8, # Springer-Verlag Berlin Heidelberg 2011

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Recent images (Scheuring et al. 2004, 2005; Scheuring and Sturgis 2009) have considerably changed our vision of biological membranes. Until recently, the image of membranes was predominated by the fluid mosaic model (Singer and Nicolson 1972) in which proteins float in a sea of lipids. However, it is now clear that the typical biological membrane has much more protein than in such a vision (Engelman 2005), and in some cases can be viewed as a matrix of protein with a few lipids filling the interstices (Scheuring and Sturgis 2006).

8.1.1

Epochs in Early Evolution

The origin of life is a complex subject and it can be approached in two distinct ways. A top-down approach compares existing life-forms and infers from this comparison the characteristics of the last universal common ancestor (LUCA). The second, a bottom-up approach, is based on geochemistry and uses chemical principles to infer the possible characteristics of the first life-form (origin of life – OOL). These two approaches currently do not quite meet and there is a gray area in between where chemistry slowly becomes biochemistry and biology. In this area, one can venture guesses as to what happened during this period. The period before the formation of the first self-propagating systems and the beginnings of life are often referred to as pre-biotic stages, while after the undeniable appearance of life there are the biological stages of evolution. In between these two moments, the gray area during which life appeared is often referred to as the protobiological stage (Pereto 2005). These events occurred on a geological time scale and are hard to date precisely. However, the approximate dates for LUCA is ~3.75 Ga while the formation of the earth dates to ~4.5 Ga, though conditions on the early earth were probably too harsh before ~4.25 Ga. This suggests that pre- and proto-biological evolution occurred over a period of about 500 Ma (Maher and Stevenson 1988). Various different scenarios exist for the OOL based on different driving forces, different definitions of life and different orders of events. This was well summarized by Pereto (2005) with three dichotomies: heterotrophic or autotrophic origin, that is how complex was the prebiotic soup; genetics first or metabolism first, in which order do selfreplicating polymers and (bio)energetic mechanisms appear; and finally, early or late appearance of cells, that is when do membranes appear during the process. Here, I specifically address the issue of membrane formation, and suggest, in agreement with Bywater (2009), that the early formation of chemically complex peptide-rich membranes is a possibility.

8.1.2

Roles and Properties of Membranes

Biological membranes today are capable of playing multiple roles, and those in ancient life forms were probably capable of playing some or all of these. The simplest role of a membrane is that of a container. This role is probably the

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primordial role of the membrane in the origin of life and during proto-biotic evolution. Today, it is necessary to maintain the association between the genotype and phenotype to allow evolution to proceed. This function is performed particularly well by membrane lipids due to their ability to self-assemble and encapsulate various molecules. This role of containment also results in an effect on mobility and distribution. First, there is a possibility for dispersion without dilution. A liposome, or proto-cell with a membrane, can keep its contents protected from the environment. Thus, there is the appearance of a self that can migrate and disperse to new environments. Conversely, a sticky membrane can result in a sessile existence and the proto-cell can remain where un-contained chemical systems are washed away and dispersed by dilution. Life depends on out of equilibrium chemical systems (Eigen and Schuster 1977). The ability of a container to preserve the content from changes in the environment is an enormous step toward achieving a chemical system able to remain out of equilibrium either for long periods of time or regularly. Such disequilibrium could arise in response to energetic input from outside and/or stochastic differences between the compartments. Another consequence of a container, beyond keeping the inside and outside separate is to create and allow the maintenance of a whole series of transmembrane potentials. These potentials can be used to drive the chemistry of the cell. The creation and maintenance of these potentials, such as osmotic, electric, concentration potentials, make biological membranes today energetically so important. The presence of these potentials associated with the chemistry of biological systems results in the use of vectorial reactions to couple chemistry and the maintenance, or use, of these transmembrane potentials. Thus, the presence of membrane-bounded compartments almost magically results in the creation of a series of potentials driving different molecular species from one side of the membrane to the other. The development and use of these potentials depends on the permeability of the membrane, the more impermeable the membrane the more different concentration gradients can exist. A semi-permeable protomembrane might only constrain a few oligomeric solutes resulting in little effective transmembrane potential. However, today’s lipid membranes are able to strongly diminish the passage of all ions and many uncharged species, resulting in a multiplicity of strong transmembrane potentials. An important aspect of these transmembrane potentials arises because life requires energy in some form, and the various potentials represent a source of energy. Life needs to extract energy from the environment in order to drive to replication. Thus, the association of energy with the existence of proto-cells is important, while the universal use and maintenance of membrane potentials in living systems is suggestive of an early origin. However, the initial energetically important compartmentation is not necessarily that between the cell and its environment but could involve intracellular vesicles. Another consequence of a membrane is to divide space into chemically distinct compartments. This goes beyond the division mentioned above separating the inside and the outside worlds to include the division separating the aqueous,

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hydrophilic, world from the hydrophobic interior world buried within the membrane thickness. There are also as a result two interfacial regions where there are strong gradients of chemical properties. This division will result in the partitioning of different components between the different regions, and possibly the concentration of certain chemical species within a proto-membrane. Below, I will develop first the top-down approach to describe the membranes of LUCA, and then the bottom up approach to examine how proto-membranes can form and evolve toward the inferred membranes of LUCA.

8.2

Membranes of LUCA

Biological membranes today play several important functional roles as the barrier for a cell between self and non-self. First and foremost, they control the influx and efflux of metabolites and so regulate the cellular metabolism and availability of resources. This barrier role also allows them to control the ionic environment of the cell and prevent the escape of genetic material. However, beyond these essentially passive roles, many biological membranes today are biochemically active participating in particular in the bioenergetic metabolism of cells, largely through chemi-osmotic coupling (Mitchel 1961). These functional roles are largely beyond the capabilities of a single component membrane and appear to require the presence of both lipids, to ensure the barrier, and proteins, to permit chemistry and controlled transfer. For this reason, biological membranes today are a complex mosaic of lipids and proteins. These membranes contain multiple different proteins assembled into more or less stable multi-protein complexes and a wide variety of chemically distinct lipid molecules. This mixture varies from lipid-rich membranes or regions where a few proteins float in a sea of lipids (Singer and Nicolson 1972) to much more protein-rich membranes in which a few lipids fill the gaps between tightly packed proteins (Scheuring and Sturgis 2006). Recently, atomic force microscopy (AFM) has allowed the visualization of the protein organization in several biological membranes revealing details of the organization and packing of the different components (Scheuring and Dufreˆne 2010). In Fig. 8.1, images of several different membranes are shown. The panel A illustrates the organization of components in a bacterial photosynthetic membrane from Rhodospirillum photometricum. These are particularly protein-rich membranes in which proteins are closely packed and there is almost no space for lipids. The panel B shows several patches of membrane from Rhodopseudomonas palustris, with rough (protein-rich) and smooth (lipid-rich) regions of membrane coexisting next to each other. These observations underscore the modern view that biological membranes are not homogeneous and that proteins occupy a substantial part of surface area in the structure, much more significant than suggested by older models such as that of Singer and Nicolson (1972). Phylogenetic analysis and comparative biochemistry can lead to inferences about the last ancestor of organisms alive today (LUCA). I will examine here

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Fig. 8.1 AFM images of several native membranes showing high protein density and lateral heterogeneity. (a) Photosynthetic membrane disks of Rhodospirillum photometricum with a high density of proteins, large ring structures are the photosynthetic core complex and the smaller structures are the peripheral lightharvesting complex. (b) Membrane fragments from Rhodopseudomonas palustris showing the juxtaposition of rough protein-rich membranes and smooth lipid-rich membranes

what can be inferred by this top-down approach about the characteristics of the membranes of LUCA. The three major branches of life, eukaryotes, archaea and bacteria, are presumed to be derived from this common ancestor. However, it is not necessarily possible to make inferences this far back. Recently, it has been suggested that elevated levels of horizontal gene transfer after the differentiation of this ancestor make inferences of its characteristics impossible (Glansdorff et al. 2008) and the characters that are inferred are the common characteristics of a population of organisms that had already separated into the various branches of living organisms. Nevertheless, this type of analysis has provided some information about the membranes of LUCA.

8.2.1

Archaeal and Bacterial Membranes

Lipids and amphiphiles are a central component of biological membranes. In Fig. 8.2, I show the structure of two typical membrane lipids, one of an archaeal type (archaetidyl ethanolamine) (A) and the other of a bacterial type (palmitoyloleyl-phosphatidyl choline) (B). In eukarya, the membrane lipids are of the bacterial type. These amphiphilic molecules both contain a hydrophilic head-group based on phospho-glycerol and a hydrophobic tail 16–18 carbons long; these are common features of the lipids that make up from biological membranes. An amazing property of such molecules is that they are able to self-assemble to form

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Fig. 8.2 Lipid structures showing conserved organization of hydrophobic tail and phosphoglycerol based head group. (a) An archaeal phospholipid – Archaetidyl ethanolamine (2,3 di-OGeranylgeranyl sn glycerol-1-phospho ethanolamine), (b) palmitoyl oleyl phosphatidyl choline, a bacterial type glycero-phospholipid . For each structure (a, b) I show a space-filling model (c and d respectively – hydrogen atoms are not shown). (e) Structural model of one of the classes of membrane protein believed to be represented in LUCA showing the organization in multiple transmembrane a-helices in the membrane; the example is the membrane embedded part of an ABC transporter

a bilayer structure, such as the biological membrane, giving a thin hydrophobic barrier between two aqueous compartments. This self assembly process is routinely used in laboratories to form liposomes of various sizes. These self-assembled bilayers are remarkably impermeable to ions and macromolecules though small uncharged molecules (for example oxygen) can traverse them relatively easily. It is noteworthy in this context that almost all common metabolites are polar and as such effectively contained by lipid bilayers. Although the two structures shown in Fig. 8.2 share a similar architecture, a closer look at the chemical structures shows several important differences between archaeal and bacterial lipids suggesting that their evolution post-dates LUCA and calling into question the types of lipids present in early cell membranes.

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The differences occur at three different levels. The first concerns the chemical nature of the hydrophobic part of the molecule. In bacterial lipids (Fig. 8.2b, d), these are generally linear fatty acid chains synthesized by the polymerization of acetyl groups. In contrast, archaeal membranes lipids (Fig. 8.2a, c) have polyprenyl hydrophobic groups synthesized by the isoprenoid pathway. The presence of many branched methyl groups gives the molecular model a distinctly spiky appearance (Fig. 8.2c). Second, the linkage between the hydrophobic part of the molecule and the central glycerol is different; in archaeal lipids, this is typically an ether linkage as the result of the condensation of a poly-prenyl alcohol and a glycerol molecule, while bacterial lipids have an ester linkage formed between the fatty acyl chain and a glycerol molecule. Finally and most intriguingly, the stereo-chemistry of the central glycerol molecule is different; in the bacterial type lipids, it is the L-enantiomer built from sn-glycerol-3-phosphate and in archaeal type lipids, it is the D-enantiomer built from sn-glycerol-1-phosphate. These differences pose serious questions as to the nature of the membranes in the LUCA: did it have lipid membranes, if so what was their chemical structure. These differences have given rise to considerable discussion (Pereto et al. 2004). However, a certain consensus appears to be emerging. Some of the characteristics in particular the presence of isoprenoid hydrophobic chains and an ether linkage may be associated with the adaptation of archaea to thermophilic environments and thus perhaps these differences are secondary. In contrast, the differences in the glycerol stereo chemistry is relatively important in biochemical terms and may suggest that the membrane lipids of LUCA were not glycero-phospholipids or perhaps, though less likely, were a racemic mixture containing a mixture of the two enantiomers. Overall, the comparison leads to the conclusion that we can infer very little for sure about the membrane lipids of LUCA.

8.2.2

Membrane Proteins of LUCA

If the lipids differ considerably between bacterial and archaeal membranes, there are many membrane proteins that appear to antedate LUCA. Indeed in the predicted genome of LUCA, there are many important integral membrane proteins (Ouzounis et al. 2006). In particular, the major functional categories of membrane proteins are all represented in this group of archaic proteins. This in itself provides strong evidence that LUCA possessed a lipid-based membrane system containing multiple membrane proteins. The conserved membrane proteins all belong to the a-helical class of membrane proteins. These proteins typically have one or several transmembrane a-helical segments embedded in the lipid membrane (Fig. 8.2e) connected by loops on the cytoplasmic and periplasmic side of the membrane. The various families conserved between archaea, bacteria and eukarya (Table 8.1) include the major cellular bioenergetic systems, transporters, channels, F-type ATP synthases and ATPases of the V- and P- types. This multiplicity of functional proteins suggests an ancestor

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Table 8.1 Classes of membrane protein identified in LUCA (Ouzounis et al. 2006) Transporters

Enzymes Receptors

ABC transporters Antiporters (major facilitator superfamily) V- and P-type ATP’ase Arsenite-Antimonite (ArsAB) eflux family ion channels Sec and Tat protein translocases F0F1 ATP synthase NADH dehydrogenase Rieske-cyt b complex Quinol oxidase Septal DNA translocase (FtsK) Two component sensors

with a complex membrane containing many different membrane proteins with specialized tasks, and thus quite a sophisticated organism. In particular, the membrane has channels and pumps for maintaining the ionic balance, transporters for assuring cellular metabolism and the components necessary for a chemiosmotically coupled electron transfer chain and ATP synthesis (Ouzounis et al. 2006). It has been suggested that this coupling may have involved Na+ rather than H+ (Mulkidjanian et al. 2009) though the evidence for this is not particularly strong especially in view of evidence for a proton based Q-cycle in LUCA (Ducluzeau et al. 2009). This inference is based on the presence of cytochrome b-type and Rieske [2Fe-2S] proteins in both bacteria and archaea, with ancient phylogenies preceding the archaeal-bacterial dichotomy. It is interesting to note that the hydrophobic thickness of the membrane is very well conserved through different kingdoms with transmembrane a-helices that have very similar lengths independent of their origin (Arce et al. 2009), and with the hydrophobic parts of membrane lipids that are typically about 16 carbons long. This provides further evidence that LUCA had a well-developed cellular membrane even if the nature of the lipids is unclear. Thus in marked contrast to the situation with the lipid component of the membrane, the proteins appear to have been relatively well characterized and specialized in LUCA. Taken together, these observations strongly suggest that LUCA had a complex membrane containing many different transmembrane proteins and probably a complex-membrane lipid mixture. At the same time, it is not entirely clear whether the genetic material of LUCA was RNA or DNA. In view of the multiplicity of membrane proteins expected in LUCA and the complex metabolism and exchanges with the environment, this gives an image of a protein-rich heterogeneous membrane much like that of many modern cells. The complexity of this membrane, and the variety of the proteins contained in the membrane suggests that the origins of membranes, and membrane proteins considerably ante-date LUCA. It is perhaps important here to note that LUCA was not the first living organism, but rather, one organism among many existing at that distant time that has given rise to all organisms extant today. No doubt this position is due to its invention of some

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universal component of biological systems today that gave it a considerable advantage over its competitors. However, at that epoch there was another pre-LUCA ancestor to all the competing organisms then present and still, in all probability, a complex living organism with genetics, metabolism and membrane. Unfortunately, we do not have access to the properties of this and other earlier pre-LUCA’s by phylogenetic analysis. However, the complexity of the membranes of LUCA suggests that biological membranes already had a long history.

8.3

Origin and Evolution of Membranes

The information presented above gives an outline of the possible components and organization of membranes some time after the transition between the protobiological and biological stages of evolution (Pereto 2005). I will now consider, using a bottom-up approach, the origin of membranes and protocells. This depends on our understanding of prebiotic chemistry and metabolism and as such is strongly influenced by opinions on the heterotrophic or autotrophic origin of cellular life as we know it and the order of appearance of different metabolites and complex molecules. Various divergent and mutually exclusive views have been expressed in the literature for the origin of membranes and cells. W€achtersh€auser (2007) suggests an origin on catalytic mineral surfaces, Pohorille and Deamer (2009) on the other hand build unstable vesicles and soap bubbles, while Egel (2009) has proposed a peptide dominated origin. Other models based on mineral bubbles (Martin and Russell 2003) seem so distant from modern membranes and incompatible with membrane protein evolution; it is hard to envisage an evolutionary scenario that will bring them toward the membranes of LUCA. In this section, I attempt to examine the roles of biological and potentially pre-biological membranes, the importance of these different roles in the development of protoorganisms and cells, and the potential of these roles to participate in the development of the first living organisms. Finally, I will suggest a structure for the first membranes.

8.3.1

Behavior and Nature of Prebiotic Amphiphiles

The various products of prebiotic chemistry are hard to assert with certainty; however, it seems reasonable to assume that smaller products preceded more elaborate chemical structures. This is borne out by the products of various prebiotic synthetic experiments. Among the various chemicals that might have been present as a result of abiotic syntheses, various hydrophobic products could have been formed. This has been argued at length by Pohorille and Deamer (2009) and is necessary to support their

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suggestion of the presence of early membranes formed from mono- and di-glycerides and fatty acyl chains. Undoubtedly, prebiotic syntheses could result in amino-acids and peptides. Early peptides were probably rather short and using a limited vocabulary of amino acids. These conclusions can be reached from various lines of evidence. First, the size of polypeptides was probably limited by the low concentration of amino-acids and high concentration of water making hydrolysis much more thermodynamically favorable than condensation. Second, the original amino acid vocabulary was probably rather limited to a handful of prebiotically synthesized amino-acids. Interestingly, Miller type synthesis experiments (van der Gulik et al. 2009), and analysis of the genetic code (Trifonov 2009) both suggest a similar repertoire of early amino acids containing: glycine (G), alanine (A), aspartate (D), valine (V) and serine (S). Furthermore, it is suggested that this repertoire could have preceded the development of a translation machinery (Shepherd 1981; Johnson and Wang 2010). Small peptides can act as amphiphiles even if they contain only a few amino acids. Perhaps the best known case of this is the bee venom peptide melittin. This 26 residue peptide is able to integrate into membranes and is the principle active component of bee venom. In common with many short peptides in solution, it is essentially non-structured. However, the peptide develops an amphipathic helical structure in certain solvents or in the presence of membranes (Othon et al. 2009). Importantly, small peptides containing a limited repertoire of residues, such as those cited above, could easily form amphipathic structures as both hydrophobic (A, V) and hydrophilic (D, S) side chains are present. I have suggested above that in the prebiotic soup there are likely to have been various different types of amphiphiles including small amphiphilic molecules, larger lipid-like molecules and small amphiphilic peptides. This is important because amphiphiles unlike other solutes show a very complex phase behavior in aqueous solution. Amphiphiles, or mixtures of amphiphiles, are able to self-assemble into a wide variety of different structures in a concentration dependent manner. At low concentrations, amphiphiles are soluble in aqueous solutions; at higher concentrations, they can form complex aggregate phases such as lamellar membrane phases or micelles (small aggregates of molecules). These phase transitions depend strongly on the co-solutes present in the solution (salt concentration, pH etc) and the nature of the amphiphiles present. The nature of the different aggregate structures formed has been rationalized in terms of the structure of the lipids and the relationship between the volume of the hydrophobic part of the molecule and the surface area of the hydrophilic head group (Israelachvili et al. 1977). A micelle is a small aggregate with a hydrophobic interior and a hydrophilic exterior. This structure is favored by small amphiphilic molecules with relatively large hydrophilic parts. Many peptides and lipid like molecules are able to form micelles. Lamellar bilayer structures in which a hydrophobic membrane barrier is formed are favored by larger amphiphiles with smaller hydrophobic parts. All these different aggregates have several important properties for the following discussion. First, they separate a hydrophobic interior and a hydrophilic

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exterior. Second, they provide a chemically complex interface, where the hydrophilic parts of the amphiphilic molecules are concentrated. The hydrophobic interior is important as it can induce small molecules to partition between chemically distinct environments. While the chemically complex surface can result in a very reactive chemical environment.

8.3.2

Possible Stages in Prebiotic Membrane Development

Amphiphiles, and in particular smaller amphiphiles, tend to form micelles. The presence of micelles in a prebiotic environment will result in the appearance of a hydrophobic environment, in the interior of the micelle. I propose that early in prebiotic evolution, self-organizing micellar systems were formed able to segregate metabolites according to hydrophobicity and possibly create, or aid creation of, an early self-replicating metabolic system, for example during tidal concentration cycles (see Chap. 6). Such micelles would be mixed containing a rich mixture of different organic molecules. In many ways, such micelles containing several amphiphilic peptides could represent the first proteinlike assemblies with a hydrophobic interior and a catalytic hydrophilic surface. Even though, unlike proteins such assemblies would neither be entirely made of peptides nor would the catalytic active site represent a necessarily stable structure. The presence in an aqueous abiotic environment of hydrophobic regions, in the interior of such micelles, would facilitate condensation reactions and thus aid the formation of longer oligo-peptides. Such condensation reactions could of course be further enhanced, and partitioning into micelles encouraged by dehydration cycles (Egel 2009; Deamer and Weber 2010). The presence of micelles might thus result in increased chemical catalysis at the active surface together with increased concentrations of some metabolites. These two effects both lead to an augmented possibility of the catalytic closure of a metabolic system. This type of argument is similar to, and can be coupled with, the role proposed for clays and silt in concentrating small organic molecules (Deamer et al. 2006). Furthermore, such chemically complex micelles are likely to adhere to mineral surfaces. The transition between self-assembly into micelles and self-assembly into lamellar structures in a mixture will depend on relatively subtle aspects of the structure of the components involved. However, in general, larger molecules will have a greater tendency to form larger more stable more lamellar structures. Thus, it is easy to envisage a progression from an abiotic micellar metabolic system to a vesicular system. The appearance of a membrane system brings the possibility of confinement and thus immediately the chance of increasing concentrations on one side of the membrane, the inside, relative to the other side, the outside. In this scenario, the appearance of vesicles will bring the possibility of using transmembrane potentials to drive metabolism via vectorial reactions. The nature of the membrane envisaged is however far from the lipid bilayer model of Pohorille

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and Deamer (2009); rather, it is a peptide-rich mixture built around membrane spanning peptides sealed to provide a barrier by a diverse mixture of smaller amphiphiles. This proteo-lipidic membrane thus does not need to acquire either proteins or lipids, but is created with both and thus the capacity for peptide mediated catalysis and transport. In many ways, such a membrane resembles those that have been observed by AFM of bacterial photosynthetic membranes as shown in Fig. 8.1a with a large number of peptides each containing a single transmembrane helix assembled to form a metabolically active vectorial catalytic system. This scenario begs the question as to what metabolism would such pre- or proto-biotic membranes engage in. Various scenarios seem plausible and have been discussed in the literature, an osmotic-driven metabolism as favored by Egel (2009) or a redox based metabolism as favored by others (W€achtersh€auser 2007). Of course in this scenario, it is not clear whether the first membrane bounded vesicles contained the metabolism that produced them, or were associated with other external components necessary for catalytic closure. However, the capture by such vesicles of the metabolic system necessary for its production would present an enormous step from the prebiotic to protobiotic worlds.

8.3.3

A Scenario for Protobiological Membrane Development

The novel suggestion for pre-biological membrane development discussed above allows a relatively smooth and easy transition from these initial membranes to the more well-developed membranes of LUCA. The scenario also suggests that the bilayer forming lipid component of the membranes can evolve separately from the protein components of the membrane. The scenario presented above is drafted in a context of catalytic closure of the metabolic system and the formation of membranes in the absence of genetics. As such, the major necessity for the origin of life is the incorporation of a genetic system capable of controlling the various peptides formed. However, it is possible to re-write the scenario in a context with early appearance of genetics and a later development of membranes. Nevertheless, these membranes differ in several important ways from those deduced for LUCA. Importantly, they are built around small peptides and a mixture of amphiphiles while the membranes of LUCA contain a much richer mixture of proteins, even if the lipid part of the membrane can remain as ill defined. The major development in the membrane during the proto-biological period is thus envisaged as the development of more and more complex protein components capable of acting as the center of aggregation for the lipid molecules. These proteins need to change from simple trans-membrane peptides to complex multipass proteins containing a variety of metabolically important co-factors. Thus, there needs to be, a presumably genetically based, evolution of the membrane protein structures. Beyond this, there needs to be a development of function.

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The initial transmembrane peptides serve a role of amphiphile, participating in the assembly of the membrane, and have associated chemical reactivity and possibly semi-specific transport properties. These functions need to evolve to include specific catalytic, bio-energetic and transport capacities, including the capacity to insert peptides synthesized outside the membrane into the membrane. The development of structure involves the multiplication of the initial module of a transmembrane helix. This module is typically about 24 amino-acids long (Arce et al. 2009) though there is some variability. In genetically piloted systems, module duplication by gene duplication is relatively common place. Furthermore, it has been suggested peptide evolution has, since the dawn of life, been driven by the multiplication of peptide modules of 20–50 amino-acids (Trifonov and Berezovsky 2003). This size, it was suggested, is derived from the physical properties of polypeptides and nucleic acids. Thus, we can imagine that the length of transmembrane helices, and thus the thickness of biological membranes, is derived from the polymer properties of peptides and nucleic acids. The development of function would be the subject of strong evolutionary pressure during the proto-biological period. The diverse functions of a membrane, found among the proteins of LUCA, are of obvious advantages to this life-form. However, importantly, even the simplest of peptide-rich membranes can contain the beginnings of these activities. Thus, for example, a simple transmembrane peptide can introduce defects in a lipid bilayer resulting in increased transport across the membrane. Equally, peptides in a membrane can provide a chemically heterogeneous system able to provide chemical reactivity. Coupling of these two provides the possibility of vectorial transport driven reactivity. Perhaps, the most difficult process to imagine is how these membranes were able to initiate their bioenergetic role. It is however possible that this specialized role developed in special dedicated membranes allowing a leaky membrane to contain bioenergetic membrane vesicles.

8.4

Conclusions

In the sections above, I have tried to develop a view of the origin of membranes and membrane proteins that is consistent with what is known about the prebiological chemical environment and our inferences about the membranes of LUCA. This vision is somewhat different from previously described scenarios. It is based on the complex phase properties of amphiphiles and the formation of peptide-rich membranes, similar in several respects to the bacterial photosynthetic membranes that have been observed by AFM. This work was supported by the CNRS and a grant from the Agence National de la Recherche (ANR-PNANO-06-0089). Ce´line Brochier, Marie-Luz Cardenas, Athel Cornish-Boden, Je´roˆme He´nin and Simon Scheuring are thanked for many stimulating discussions and help with the figures.

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References Arce J, Sturgis JN, Duneau JP (2009) Dissecting membrane protein architecture: an annotation of structural complexity. Biopolymers 91:815–829 Bywater RP (2009) Membrane-spanning peptides and the origin of life. J Theor Biol 261:407–413 Darwin C (1859) On the origin of species by means of natural selection, or, the preservation of favoured races in the struggle for life. John Murray, London Deamer D, Weber AL (2010) Bioenergetics and life’s origins. Cold Spring Harb Perspect Biol 2: a004929 Deamer D, Singaram S, Rajamani S, Kompanichenko V, Guggenheim S (2006) Self-assembly processes in the prebiotic environment. Phil Trans R Soc B 361:1809–1818 Ducluzeau AL, van Lis R, Duval S, Schoepp-Cothenet B, Russell MJ et al (2009) Was nitric oxide the first deep electron sink? Trends Biochem Sci 34:9–15 Egel R (2009) Peptide-dominated membranes preceding the genetic takeover by RNA: latest thinking on a classic controversy. Bioessays 31:1–10 Eigen M, Schuster P (1977) The hypercycle: a principal of natural self organisation. Part A: emergence of the hypercycle. Naturwissenschaften 64:541–565 Engelman DM (2005) Membranes are more mosaic than fluid. Nature 438:578–580 Glansdorff N, Xu Y, Labedan B (2008) The last universal common ancestor: emergence, constitution and genetic legacy of an elusive forerunner. Biol Direct 3:29 Israelachvili JN, Mitchell DJ, Ninham BW (1977) Theory of self-assembly of lipid bilayers and vesicles. Biochim Biophys Acta 470:185–201 Johnson DBF, Wang L (2010) Imprints of the genetic code in the ribosome. Proc Natl Acad Sci USA 107:8298–8303 Maher KA, Stevenson DJ (1988) Impact frustration of the origin of life. Nature 331:612–614 Martin W, Russell MJ (2003) On the origins of cells: a hypothesis for the evolutionary transitions from abiotic geochemistry to chemoautotrophic prokaryotes, and from prokaryotes to nucleated cells. Phil Trans R Soc B 358:59–83; discussion 83–85 Mitchel P (1961) Coupling of phosphorylation to electron and hydrogen transfer by a chemiosmotic type of mechanism. Nature 191:144–148 Mulkidjanian AY, Galperin MY, Koonin EV (2009) Co-evolution of primordial membranes and membrane proteins. Trends Biochem Sci 34:206–215 Othon CM, Kwon OH, Lin MM, Zewail AH (2009) Solvation in protein (un)folding of melittin tetramer-monomer transition. Proc Natl Acad Sci USA 106:12593–12598 Ouzounis CA, Kunin V, Darzentas N, Goldovsky L (2006) A minimal estimate for the gene content of the last universal common ancestor – exobiology from a terrestrial perspective. Res Microbiol 157:57–68 Pereto J (2005) Controversies on the origin of life. Int Microbiol 8:23–31 Pereto J, Lopez-Garcia P, Moreira D (2004) Ancestral lipid biosynthesis and early membrane evolution. Trends Biochem Sci 29:469–477 Pohorille A, Deamer D (2009) Self-assembly and function of primitive cell membranes. Res Microbiol 160:449–456 Scheuring S, Dufreˆne YF (2010) Atomic force microscopy: probing the spatial organization, interactions and elasticity of microbial cell envelopes at molecular resolution. Mol Microbio l75:1327–1336 Scheuring S, Sturgis JN (2006) Dynamics and diffusion in photosynthetic membranes from Rhodospirillum photometricum. Biophys J 91:3707–3717 Scheuring S, Sturgis JN (2009) Atomic force microscopy of the bacterial photosynthetic apparatus: plain pictures of an elaborate machinery. Photosynth Res 102:197–211 Scheuring S, Francia F, Busselez J, Melandri BA, Rigaud JL et al (2004) Structural role of PufX in the dimerization of the photosynthetic core complex of Rhodobacter sphaeroides. J Biol Chem 279:3620–3626

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Scheuring S, Busselez J, Levy D (2005) Structure of the dimeric PufXcontaining core complex of Rhodobacter blasticus by in situ atomic force microscopy. J Biol Chem 280:1426–1431 Shepherd JCW (1981) Method to determine the reading frame of a protein from the purine/ pyrimidine genome sequence and its possible evolutionary justification. Proc Natl Acad Sci USA 78:1596–1600 Singer SJ, Nicolson GL (1972) The fluid mosaic model of the structure of cell membranes. Science 175:720–731 Trifonov EN (2009) The origin of the genetic code and of the earliest oligopeptides. Res Microbiol 160:481–486 Trifonov EN, Berezovsky IN (2003) Evolutionary aspects of protein structure and folding. Curr Opin Struct Biol 13:110–114 van der Gulik P, Massar S, Gilis D, Buhrman H, Rooman M (2009) The first peptides: the evolutionary transition between prebiotic amino acids and early proteins. J Theor Biol 261:531–539 W€achtersh€auser G (2007) On the chemistry and evolution of the pioneer organism. Chem Biodivers 4:584–602

Part III

RNA Worlds: Ancestral and Contemporary

Chapter 9

Nicotinamide Coenzyme Synthesis: A Case of Ribonucleotide Emergence or a Byproduct of the RNA World? Nadia Raffaelli

Abstract Coenzymes likely represent the oldest metabolic fossils within a cell, as suggested by their presence and essentiality in all kingdoms of life and the autocatalytic nature of their biosynthetic pathways. The presence of a ribonucleotidyl group in the structure of most coenzymes that use it as a “handle” for binding to the protein catalyst means that ribonucleotides must have been present at the time coenzymes emerged. An open question remains whether the ribonucleotidyl group has been co-opted from a preexisting RNA in a primordial “RNA world” before the emergence of proteins, or it represents the evolutionary predecessor of contemporary nucleic acids. The nicotinamide coenzyme NAD (P) is one of the oldest molecules, not only in the history of biochemistry, but also in the evolutionary steps towards the emergence of life. Together with relatively simple organics, such as PRPP (50 -phosphoribosyl 10 -pyrophosphate), PLP (pyridoxal 50 -phosphate) and many others, it may have been a crucial prebiotic agent in organizing a collectively autocatalytic protometabolic ecosystem. Here, the NAD(P) biosynthetic pathway will be described with views on its origin. NAD(P)’s peculiar biochemical features will also be discussed with the aim to offer novel arguments to the debate on the sequence of chemical evolution in the origin of life.

Abbreviations FAD FMN NAD NaMN

flavin adenine dinucleotide flavin mononucleotide nicotinamide adenine dinucleotide nicotinate mononucleotide

N. Raffaelli (*) Section of Biochemistry, Department of Molecular Pathology and Innovative Therapies, Universita` Politecnica delle Marche, Via Ranieri, 60131, Ancona, Italy e-mail: [email protected] R. Egel et al. (eds.), Origins of Life: The Primal Self-Organization, DOI 10.1007/978-3-642-21625-1_9, # Springer-Verlag Berlin Heidelberg 2011

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NMN PLP PR PRPP

9.1

N. Raffaelli

nicotinamide mononucleotide pyridoxal-50 -phosphate phosphoribosyl 50 -phosphoribosyl 10 -pyrophosphate

Introduction

Coenzymes are ubiquitous and small molecules required by many protein enzymes to catalyze reactions that are otherwise inefficient or impossible for typical amino acids. By binding to the catalyst, either through covalent or weak interactions, they function as transient carriers of specific functional groups. In cellular metabolism, the same coenzyme is usually used by enzymes with different substrate specifities; for example, NAD(P) is used as electron carrier by several cellular dehydrogenases catalyzing reduction and oxidation of a wide variety of substrates. Given their essential function and broad use, it is evident that a change in coenzyme structure would inevitably result in a fatal outcome for the organism. Therefore, it is reasonable to assume that, in evolutionary time, once coenzymes had gained the most suitable structure for their function, they should have retained it more stringently than the catalysts (Chen et al. 2007). As a consequence, contemporary coenzymes should not be much different from their ancestral precursors. Indeed, they are considered the oldest metabolic fossils within a cell (White 1976). The autocatalytic nature of coenzymes also speaks in favor of an ancient metabolic history. An autocatalytic metabolite is a compound which is required for its own synthesis and cannot be accessed from the food set. Well known is the autocatalytic nature of ATP: in glycolysis, the nucleotide is essential to kick-start the pathway, but once glycolysis has been launched, ATP is able to support the pathway and thus its own synthesis. A recent research based on the metabolic reconstruction of coenzyme biosynthetic pathways in various eubacterial organisms has provided evidence that also some coenzymes, including NAD, coenzyme A and tetrahydrofolate, behave as autocatalytic molecules, that is they are able to support their own synthesis (Kun et al. 2008). Notably, some coenzymes, including NAD, FAD, coenzyme A and adenosylcobalamin, share a ribonucleotidyl group, even though they have completely different biochemical roles. Indeed, in none of them does the ribonucleotidyl moiety participate directly in the coenzymatic function; it rather acts as a “handle” for binding to the protein catalyst, as revealed by a structural analysis of enzymes in complex with ATP, NAD(P), FAD and coenzyme A (Denessiouk et al. 2001). The analysis shows that different proteins recognize the adenine moiety of coenzymes with a common binding motif. Notably, binding to adenine relies on polar and hydrophobic interactions, which are not dependent on the protein sequence and very closely resemble adenine base-pairing and -stacking in DNA and RNA. The presence of such a motif in ancient proteins, which are present in all living organisms, suggests that it has been exploited very early in biotic evolution.

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Accordingly, a bioinformatic study tracing the protein-binding history of early ligands reveals that the ligands that might have bound their host proteins relatively earlier than others are in the order ATP, NAD and FAD (Ji et al. 2007). The occurrence of a ribonucleotidyl moiety in cofactors means that ribonucleotides must have been present at the time coenzymes, as we know them, emerged. A question is whether the ribonucleotidyl group has been co-opted from an existing RNA or it represents the evolutionary predecessor of contemporary nucleic acids. The first assumption presupposes the existence of a primordial “RNA world”, which is believed to be a time before the emergence of template-encoded proteins, when metabolism was guided entirely by RNA (Pross 2004; Chen et al. 2007). In this scenario, nucleotidyl coenzymes might have been synthesized by RNA enzymes and used by them to support all the different types of reactions that are not feasible with only the four nucleotides . The biochemical properties of RNA are indeed compatible with this scenario, as revealed by the in vitro selection through directed evolution of RNAs able to synthesize ribonucleotide coenzymes and RNAs able to bind coenzymes and use them to perform the chemical reactions typical of modern protein enzymes (Jadhav and Yarus 2002). Moreover, the occurrence of natural small eubacterial RNAs carrying at their 50 termini coenzymes like NAD and coenzyme A (Chen et al. 2009; Kowtoniuk et al. 2009), together with the presence in all living organisms of riboswitches that directly bind to, and hence are regulated by thiamine pyrophosphate, flavin mononucleotide (FMN) and adenosylcobalamin (Mironov et al. 2002; Nahvi et al. 2002; Winkler et al. 2002; Sudarsan et al. 2003) bring important insights into the plausibility of the above hypothesis. Based on the consideration that precursors of some coenzymes, like NAD and coenzyme A, can be synthesized from simple molecules under prebiotic conditions, one line of reasoning is that the early chemical environment contained functionalized molecules similar to modern coenzymes, but without the nucleotidyl moiety (Jadhav and Yarus 2002). These molecules would have been highly reactive and initiated a series of chemical reactions. Later on, catalyst RNAs appeared which adopted them to expand their catalytic repertoire, thus initiating coenzyme-RNA-dependent metabolic pathways (Jadhav and Yarus 2002). In this view, modern nucleotidyl coenzymes might have originated as coribozymes and the adenine ribonucleotide moiety might represent a surviving vestige of primordial RNA. On the other hand, the possibility that coenzymes might have been evolved in other scenarios should also be considered. “Metabolism first” hypotheses imply that life started from the assembly of increasingly complex autocatalytic networks from mixtures of interacting organic molecules, before any genetic coding system emerged (W€achtersh€auser 1988; Kurland 2010). In particular, it is suggested that amino acids and short heterochiral peptides generated abiotically might have played a prominent role in prebiotic evolution by contributing to the formation of primordial membranes and by binding inorganic catalysts (e.g. phosphates and iron-sulfur centers), thus acting as uncoded proto-enzymes (Milner-White and Russell 2005). Indeed, amino acids and peptides can be experimentally produced under prebiotically plausible conditions (Plankensteiner et al. 2005); moreover,

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it has been verified in several experiments that certain amino acids and small peptides can catalyze reactions from simple starting materials, with enzyme-like specificity (Weber and Pizzarello 2006 and references herein). In this scenario, synthesis of both the functional and the ribonucleotidyl group of coenzyme molecules might have been assisted by peptide films and favored at the solid surface of minerals or the liquid surface of oil microspheres (W€achtersh€auser 1988; Sharov 2009). Once first coenzyme-like molecules emerged, their autocatalytic nature would have ensured their propagation, and their catalytic ability might have played integral roles in protometabolism. Among their functions, they might have assisted random peptide formation before an RNA translation apparatus (Egel 2009) and been involved in the emergence of bilayer lipid membranes (Sharov 2009). A transition from this scenario to an “RNA world” has been supposed to occur when they polymerized evolving to the early polymers of ribonucleotides, thus paving the way to template-based synthesis (Sharov 2009). In these scenarios, coenzymes might have been the predecessors of contemporary nucleic acids. Here I will focus on the coenzyme NAD(P) with the aim to review the experimental investigations, as well as the hypothetical suggestions, that can be accommodated into the above hypotheses.

9.2

Nicotinamide Coenzyme Function

NAD and NADP are ubiquitous and essential redox coenzymes contributing to a significant percentage of all cellular metabolic reactions in both catabolism and anabolism. NAD(P)-dependent dehydrogenases catalyze the reversible oxidation of a substrate by transferring a proton and two electrons from the substrate to the C-4 atom of the coenzyme’s pyridine ring (nicotinamide) and a proton from the substrate to the aqueous solvent. As a redox coenzyme, NAD(P) shuttles between the oxidized form (NAD(P)+) and the reduced form (NAD(P)H), and the total concentration remains constant (Fig. 9.1). A peculiar feature of NAD is that, in addition to its coenzymatic function, it is used as a co-substrate by several distinct enzymes that consume the molecule, thus rendering its continuous resynthesis indispensable. Notably, such enzymes catalyze the transfer of the ribonucleotidyl moiety of NAD, either in the form of AMP or ADP-ribose, to different functional groups of proteins and nucleic acids (Fig. 9.1). These enzymes are involved in important cellular processes like DNA replication, DNA repair, RNA ligation, cell differentiation and cellular signal transduction. In particular, the eubacterial enzyme DNA ligase uses NAD instead of ATP as the source of the AMP group to activate the 50 -phosphate of nicked DNA ends to allow DNA ligation. In yeast, an NAD-dependent tRNA 20 -phosphotransferase removes from tRNA the 20 -phosphate group which is generated during tRNA splicing. The enzyme transfers the ADP-ribose group from NAD to the 20 -phosphate of tRNA forming an ADP-ribosyl tRNA intermediate; a subsequent transesterification reaction resolves the intermediate into dephosphorylated tRNA and ADP-ribose

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Fig. 9.1 NAD functions The nicotinamide ring of NAD(P) is responsible for the coenzymatic function in redox reactions (1). In NADP the 20 -hydroxyl group of adenine ribose is esterified with phosphate. The AMP moiety of NAD is transferred by DNA ligase to the 50 -phosphate of nicked DNA ends to allow DNA ligation (2); the ADP-ribose group of NAD is transferred by specific enzymes to the 20 -phosphate group of tRNA (3) or to the acetyl group of proteins (4) to remove them as ADP-ribose 100 ,200 -cyclic phosphate or acetyl ADP-ribose, respectively; the ADP-ribose group can also be transferred and covalently attached to target proteins resulting in their functional modification (5)

100 ,200 -cyclic phosphate (Steiger et al. 2005). Notably, yeast homologs with the same enzymatic activity are found in all domains of life, even in those organisms, like eubacteria, where 20 -phosphate tRNAs are not generated because different splicing mechanisms are utilized (Spinelli et al. 1999). The substrates and the biological functions of the enzyme in these species remain to be determined. The same reaction mechanism is used by NAD-dependent deacetylases that catalyze protein deacetylation by transferring ADP-ribose from NAD to the acetyl group, that is subsequently released as acetyl ADP-ribose (Sauve et al. 2006). A family of enzymes catalyze the covalent attachment of either single ADP-ribose units or ADP-ribose polymers to target proteins modifying their properties (Hassa and

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Hottiger 2008). Finally, free ADP-ribose can be released from NAD by enzymes that catalyze its transfer to water. Both ADP-ribose and its cyclic form have important roles as signal molecules in eubacteria and eukarya (Rodionov et al. 2008; Koch-Nolte et al. 2009). Notably, all organisms whose genomes have been fully sequenced so far contain proteins with evolutionarily highly conserved domains (i.e. the Nudix domain and the macro domain) able to specifically bind ADP-ribose (Karras et al. 2005; Huang et al. 2009). This argues for an ancient origin of these domains and for a physiological significance of ADP-ribose also in primordial cells.

9.3

Nicotinamide Coenzyme Biosynthesis

In contemporary metabolism, NAD(P) can be synthesized both de novo, starting from simple precursors, and through salvage routes that allow NAD synthesis from both the nicotinamide liberated by the NAD-consuming enzymes and the pyridine ring that is available from the food set, in the form of nicotinamide and nicotinic acid (also known as vitamin B3) (Magni et al. 1999). Two alternative de novo routes exist, one starting from aspartate and dihydroxyacetone phosphate, the other from tryptophan. The former is operative in most bacteria, archaea and plants; the latter occurs in animals, including humans, fungi and few bacterial species. Both routes converge to the formation of quinolinate. NAD synthesis from quinolinate is depicted in Fig. 9.2. Quinolinate is first converted to nicotinate mononucleotide (NaMN) by the enzyme quinolinate phosphoribosyltransferase that uses 50 -phosphoribosyl 10 -pyrophosphate (PRPP) as the phosphoribosyl (PR) donor. NaMN is then converted to NAD by NaMN adenylyltransferase and NAD synthetase that catalyze two consecutive reactions, common to both salvage and de novo pathways. ATP-dependent phosphorylation of NAD by NAD kinase leads to NADP formation. Figure 9.2 also shows the most common salvage routes: nicotinamide can be deamidated to nicotinic acid via nicotinamidase, followed by the PRPPdependent phosphoribosylation of nicotinic acid to NaMN by nicotinate phosphoribosyltransferase; alternatively, nicotinamide can be directly converted to NAD via two consecutive reactions catalyzed by nicotinamide phosphoribosyltransferase that uses PRPP to form nicotinamide mononucleotide (NMN), and NMN adenylyltransferase which adenylates NMN to NAD. A salvage pathway also occurs in which NAD is synthesized from the pyridine nucleosides nicotinamide riboside and nicotinate riboside, after their conversion to the corresponding mononucleotides (not shown). A peculiar feature of NAD biosynthesis is that in extant living species different combinations of the metabolic routes described above are operative, depending on the organism (Sorci et al. 2010). This results in a great diversity of the NAD biosynthetic machinery in various species, implying some evolutionary complexity. Phylogenetic and structural studies on the three PR transferases involved in NAD biosynthesis suggest that quinolinate phosphoribosyltransferase (enzyme 1 in

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Fig. 9.2 Schematic overview of major NAD biosynthetic routes starting from the free pyridine ring. The pyridine ring is used in the form of quinolinate (Qa), nicotinate (Na), nicotinamide (Nam). Enzymes catalyzing the reactions are: Quinolinate phosphorybosyltransferase (1); NMN/ NaMN adenylyltransferase (2); NAD synthetase (3); Nicotinate phosphoribosyltransferase (4); nicotinamide deamidase (5); nicotinamide phosphoribosyltransferase (6). Other abbreviations used are: DHP diydroxyacetone phosphate, NaMN nicotinate mononucleotide), NaAD nicotinate adenine dinucleotide, NMN nicotinamide mononucleotide, PRPP 50 -phosphoribosyl 10 -pyrophosphate)

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Fig. 9.2) is the most ancient: it might have given rise to nicotinate phosphoribosyltransferase (enzyme 4 in Fig. 9.2), which in turn evolved to nicotinamide phosphoribosyltransferase (enzyme 6 in Fig. 9.2) (Brenner 2005; Chappie et al. 2005). It is therefore likely that primordial cells synthesized NAD de novo starting from aspartate and only later the salvage routes emerged. A phylogenetic analysis of the two enzymes salvaging nicotinamide, that is nicotinamide deamidase (enzyme 5 in Fig. 9.2) and nicotinamide phosphoribosyltransferase, points to an earlier emergence of the former (Gazzaniga et al. 2009). The gene coding for the deamidase is in fact present in a larger variety of living species and is distributed more deeply in the tree of life while the gene coding for the PR transferase is present only in few eubacterial species, bacteriophages, sponges and vertebrates (Gazzaniga et al. 2009). Accordingly, NAD biosynthesis from nicotinamide through the deamidated pathway might have predated the coenzyme synthesis through the amidated one. Notably, among the few organisms using nicotinamide phosphoribosyltransferase are some a-proteobacteria which are considered relatives of the first mitochondria. Indeed, in vertebrates a mitochondrial homolog of the enzyme is present, which is essential for maintaining physiological levels of mitochondrial NAD under genotoxic stress and nutrient restriction (Yang et al. 2007). Under these conditions, in fact, mitochondrial nicotinamide phosphoribosyltransferase is upregulated and the increased enzymatic activity provides protection against cell death. This suggests that NAD levels might have controlled cell survival in the bacteria that gave rise to mitochondria, and the survival pathway might have been conserved up to the present day in vertebrates (Yang et al. 2007).

9.4

Pyridine Ring Prebiotic Synthesis

While phylogenetic studies based on structural and comparative genomic analyses provide insights into the evolution of the NAD biosynthetic machinery, synthesis of the coenzyme in prebiotically plausible conditions still remains a matter of speculation. Both nicotinamide and nicotinic acid can be successfully produced from inorganic elements in experiments simulating early-Earth conditions. In particular, nicotinonitrile, which hydrolyzes to nicotinamide and nicotinic acid, can be synthesized under the action of an electric discharge on ethylene and ammonia (Friedmann et al. 1971) or from the reaction of cyanoacetaldehyde, propiolaldehyde and ammonia, which are in turn synthesized from a spark discharge (Dowler et al. 1970). An alternative scenario for a prebiotic synthesis of the pyridine ring has been suggested following the discovery of an easy and efficient way to nonenzymatically generate the pyridine ring in the form of quinolinate starting from the amino acid aspartate and ribose or deoxyribose (Cleaves and Miller 2001) (Fig. 9.3a). Studies of the intermediates of the reactions involved in this process suggest that methylglyoxal, which is one of the major acid degradation products of both ribose and deoxyribose, reacts with aspartate in a five-step nonenzymatic sequence of

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Fig. 9.3 Quinolinate synthesis: (a) non-enzymatic synthesis of quinolinate in prebiotic conditions, as proposed by Cleaves and Miller (2001); (b) biosynthesis of quinolinate in contemporary metabolism

reactions, yielding quinolinate and, to a lesser extent, nicotinate. An even higher amount of product is obtained starting from dihydroxyacetone phosphate, which spontaneously dephosphorylates to methylglyoxal. It is interesting to note that the non-enzymatic production of quinolinate reflects the upstream metabolic pathway of the de novo NAD biosynthetic route starting from aspartate and dihydroxyacetone phosphate. Here, quinolinate is formed via two consecutive enzymatic steps (Fig. 9.3b). The first reaction is catalyzed by L-aspartate oxidase, which uses a FAD molecule to oxidize aspartate to iminoaspartate. This enzyme directly couples the amino acid oxidation to reduction of fumarate to succinate to regenerate the oxidized form of FAD, which explains how the pathway may also occur in anaerobic conditions. Iminoaspartate is a rather unstable compound and, once formed, is immediately condensed with dihydroxyacetone phosphate to form quinolinate by the enzyme quinolinate synthase. Thus, an efficient spontaneous synthesis of quinolinate is feasible from simple precursors, likely to have dominated in abiotic environments. The same precursors are used by the metabolic pathway that allows quinolinate synthesis in most of contemporary organisms. This strongly suggests that NAD synthesis, at least in the first steps, might have arisen independently of enzymatic chemistry. It can reasonably be assumed that the extant metabolic pathway resulted from a preexisting pathway, with gradual enzymatic takeover of the various steps (Cleaves

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and Miller 2001). In this view, among all biosynthetic routes, NAD biosynthesis from quinolinate might represent the most ancient one. Routes salvaging nicotinic acid and nicotinamide should have appeared later in evolution, as also supported by phylogenetic analyses. The finding that in some organisms, like methanogenic archaea and the deep-branched bacterium Thermotoga maritima, the first step of quinolinate biosynthesis is catalyzed by an NAD-dependent aspartate dehydrogenase, an enzyme with a completely different primary structure from L-aspartate oxidase (Yang et al. 2003), suggests the possibility that in the ancient metabolic pathway the first step might have been NAD-dependent, that is NAD might have allowed its own biosynthesis.

9.5

Evolution of Nicotinamide Coenzyme in “RNA World” Scenarios

It has been speculated that pyridine coenzymes might be fossils of an “RNA world”, in which they were synthesized by ribozymes that used them as cofactors to perform redox reactions (Orgel 1968; White 1976; Huang et al. 2000). This hypothesis is supported by the results of directed evolution experiments which allowed the isolation of RNAs able to bind NMN and NAD, to catalyze NADdependent redox reactions and to synthesize NAD. In particular, RNA aptamers have been selected that bind the adenosine portion of NAD with high affinity (Burgstaller and Famulok 1994), and highly structured RNAs have been isolated that interact with the nicotinamide ring of NMN and can discriminate between the reduced and the oxidized molecule (Lauhon and Szostak 1995). More strikingly, the plausibility of a ribozyme endowed with oxidoreductase activity is supported by the in vitro selection of a ribozyme that uses NAD to catalyze the oxidation of an alcohol in the presence of Zn+2 with a reaction similar to that of an alcohol dehydrogenase (Tsukiji et al. 2003). The in vitro selection approach also suggests that behind a possible role as a coribozyme in redox reactions, NMN might have been able to activate RNA substrates for the ligation reaction to occur. In fact, an artificial ligase ribozyme has recently been reported that catalyzes RNA–RNA ligation using the 50 -terminal phosphate activated by NMN, which behaves as the leaving group (Fujita et al. 2009). The occurrence of a covalent attachment of pyridine coenzymes to RNA early on in prebiotic evolution is supported by the recent discovery of small endogenous RNAs linked to NAD at their 50 -termini in E. coli and Streptomyces venezuelae cells (Chen et al. 2009). The surprisingly high abundance of these molecules (about 3,000 copies per cell) is indicative of a physiological role that is still unknown. The possibility that in an “RNA world” the formation of NAD-RNA conjugates might have been mediated by ribozymes is supported by two findings: (i) a modern Group-I ribozyme can covalently self-incorporate NAD via the same

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mechanism that is normally used for the insertion of guanosine into RNA during the initiation of splicing (Breaker and Joyce 1995); (ii) an RNA with ATP at its 50 -terminus has been selected that is able to catalyze the synthesis of NAD linked to its own 50 -end. This ribozyme catalyzes NMN attachment to the adenylyl moiety of ATP, with concurrent release of pyrophosphate, in a reaction that mimics the last step of NAD biosynthesis, as catalyzed by the enzyme NMN adenylyltransferase (enzyme 3 in Fig. 9.2) (Huang et al. 2000). Altogether, these findings support the hypothesis that NAD might have been synthesized attached to the 50 -terminus of adenosine-initiating RNAs. In this scenario, newly synthesized NAD molecules might have been released by hydrolysis to be utilized as coenzymes by other RNA catalysts: binding of these RNAs to the coenzyme would have been possible through interactions with the AMP moiety (Huang et al. 2000; Jadhav and Yarus 2002; Chen et al. 2007). In this view, the adenylyl moiety of nucleotide coenzymes was initially part of the 50 -terminus of RNAs. Alternatively, ribozymes might have evolved the ability to synthesize NAD in the free form, since ribozymes can be selected that catalyze the formation of pyrophosphate bonds between free nucleotides (Huang et al. 1998). It is assumed that when protein enzymes gradually replaced ribozymes, they also adopted the coenzymes, together with their AMP handles (Jadhav and Yarus 2002). In other views, it is proposed that the “RNA world” was itself preceded by an era when ribonucleotide-like coenzymes already existed, and later on were incorporated into RNA-proto structures, thus expanding RNA’s catalytic repertoire (Kritsky and Telegina 2005; Yarus 2010). Therefore, the ability of RNA to bind and harness the chemical potential of the ribonucleotidyl coenzymes would be indicative of the existence of such an ancient “Coenzyme world.” In particular, ribonucleotidyl coenzymes have been suggested to represent the modern descendants of the Initial Darwinian Ancestor (IDA), the small first replicator initiating Darwinian evolution on Earth before the emergence of the “RNA world” (Yarus 2010). The hypothesis relies on the assumption that the peculiar structure of these molecules, that are 50 -50 linked dinucleotides (see NAD structure in Fig. 9.1), might have provided them with the ability to replicate, and it is based on the observation that ribonucleotidyl coenzymes are more stable than RNA and can perform reactions that are not possible for ribozymes (i.e. redox reactions in the case of NAD and FAD). In such a scenario, it is proposed that a dinucleotide template might have guided NAD synthesis from its activated precursors ATP and NMN, ensuring the coenzyme replication. Therefore it is assumed that an NAD-like molecule might have appeared before the rise of RNA catalysts and later on, an emergent RNA metabolism might have taken advantage from its incorporation. Unfortunately, as also pointed out by the Author, the critical assumption supporting the proposed scenario remains to be experimentally evaluated; in fact, even though it is reasonable to expect that the NAD molecule might be able to engage in basepair interaction with a complementary dinucleotide (Liu and Orgel 1995), no direct evidence exists so far that single activated nucleotides might polymerize on a dinucleotide template.

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Evolution of Nicotinamide Coenzyme in Other Scenarios W€ achtersh€ auser’s “Surface Metabolism” Theory

While experiments simulating the early conditions of life on Earth have been successful in producing both quinolinate and nicotinamide, no experimental data are available at this time to cast light on how the first pyridine nucleotide could have formed from the pyridine ring. Even if we consider the hypothesis of a ribozyme or dinucleotide template-guided NAD synthesis starting from NMN, it is far from obvious how NMN could have formed from its constituent parts (ribose and nicotinamide). Indeed, so far, a prebiotically plausible synthesis has been attained only for pyrimidine nucleotides and it bypasses free ribose and pyrimidine base as precursors (Powner et al. 2009). An original hypothesis on prebiotic NMN formation has been developed by G€ unter W€achtersh€auser more than 20 years ago (W€achtersh€auser 1988). He suggests a prebiotic chemistry on pyrite surfaces carrying positive partial charges and thus capable of adsorbing and activating acidic molecules carrying negatively charged groups, thereby favoring chemical reactions. Some of the extant coenzymes which are polyanionic might have been synthesized on these surfaces before the appearance of a genetic machinery and an enzyme metabolism. On the surfaces, coenzymes might have performed not only heterocatalysis, modifying a large variety of other surface-bonded molecules, but also ensured their own synthesis, acting as autocatalysts. A “surface metabolism” would have evolved via their selection and inherited variation. In this view, it has been proposed that the carboxylic groups of quinolinate and the phosphate groups of dihydroxyacetone phosphate and PRPP might have acted as surface bonders favoring the formation of the first pyridine coenzyme NaMN. The coenzyme would have been able to perform its catalytic function acting as donor and acceptor of a hydride ion between the surface-bonded constituents. At the same time, it would also have catalyzed the oxidation step required to convert aspartate to quinolinate, closing a novel autocatalytic feedback loop and ensuring its own synthesis. The AMP moiety was suggested to get incorporated into the coenzyme to provide additional surfacebinding capability both directly (via the phosphate group) and indirectly, through interactions with nucleic acids which, in this scenario, were also supposed to be surface-bonded. Later on, with the evolution of a semicellular structure with a cytosolic metabolism, the surface-bonding carboxylate group of nicotinate would have been converted to a carboxamide group with the resulting detachment of the pyridine ring from the surface. This would have facilitated the lifting of NAD off the surface by an enzyme able to compete with the surface for the coenzyme binding. The evolutionary sequence proposed to explain NAD formation reflects all the steps of the coenzyme biosynthetic pathway in extant organisms. In addition, the theory gives an elegant explanation of the reason why the coenzyme appeared first in the deamidated form and why enzymes use it only in the amidated one.

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9.6.2

197

The “Peptide-Cofactor World” before RNA Appearance

It is proposed that in a primary “Peptide world,” a population of stochastic polypeptides was produced by prebiotic chemistry, without the help of RNA (Plankensteiner et al. 2005; Rode et al. 2007; Kurland 2010). Such a population, in the form of biofilm-like colloidal associations covering mineral surfaces (Egel 2009) would have been enriched progressively in functional early quasi-proteins, able to sustain a proto-metabolism. As an example, catalytic polypeptides might have been selected through their interactions with metabolites that not only were transformed, but also contributed to the selection by stabilizing the polypeptides themselves (de Duve 1987, 2007; Egel 2009). These early polypeptides might have contributed to the generation of proto-enzymes, as well as to the membranelike structures of the first cells (Milner-White and Russell 2008; Egel 2009). The “Peptide world” hypothesis is supported by several evidences, including the ability of peptides to self-replicate (Issac and Chmielewski 2002) and to spontaneously self-assemble into nanotubes and nanovesicles (Carny and Gazit 2010). Peptides’ self-assembled structures have been proposed to also catalyze the formation of RNA polymers and to stabilize them, thus paving the way to nucleic acid appearance (Carny and Gazit 2010). It is suggested here that in such a scenario, prebiotic peptides might have served as catalysts for the formation of the first coenzyme-like molecules, giving rise to a “Peptide-Cofactor world,” where the interactions of cofactors with early quasiapoenzymes improved the system catalytic efficiency. Later on, coenzymes themselves might have contributed to the transition to the “RNA world,” which in this scenario still goes before DNA, but no longer implies the “RNA first” distinction.

9.6.2.1

Peptide-Assisted NAD Synthesis and the Role of PRPP

It is assumed that some of the first peptides with functional properties in the “Peptide world” might have been conserved across evolution and traces of them might still exist, in the form of active sites, in present-day proteins (van der Gulik et al. 2009). Therefore, relics of them in modern NAD biosynthetic enzymes might be indicative of a likely peptide assisted synthesis of the coenzyme. As shown in Fig. 9.2, a peculiar feature of all routes leading to NAD formation is the involvement of PRPP as the donor of the PR moiety to the pyridine rings to form the corresponding mononucleotides. Indeed, PRPP is a key metabolite in all nucleotides biosynthetic pathways, with its 50 -phosphoribosyl group contributing to the ribose 50 -phosphate moiety of also purine and pyrimidine nucleotides. In the de novo biosynthesis of both pyridine and pyrimidine nucleotides, specific PR transferases catalyze the transfer of the nitrogen base to the C1 of PRPP ribose, releasing pyrophosphate and yielding the corresponding mononucleotide. In the de novo purine nucleotide synthesis, the entire backbone of the purine ring is assembled piecemeal from small components directly on the PRPP ribose. PRPP is also

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an intermediate in tryptophan and histidine biosynthesis, with the ribose ring contributing several of its carbons to the final structure of the amino acids. In modern metabolism, PRPP is synthesized from ribose 50 -phosphate in an ATPdependent reaction catalyzed by the enzyme ribose phosphate pyrophosphokinase. Given that PRPP is required for the synthesis of all nucleotides, including ATP, it is reasonable to assume that in a primordial world it might have preceded ATP as the prevailing energy carrier. Its simpler structure also speaks in favor of a role as an energy rich-compound before ATP emergence. However, the hypothesis of a prebiotic PRPP synthesis has rarely been considered (Cairns-Smith 1982). Some structural features of the active site of type I family of PR transferase suggest that early nucleotides might have arisen from a primordial peptide-assisted transfer of the PR group from PRPP to nitrogen bases. PR transferase type I family includes enzymes catalyzing the PR transfer reaction both in de novo synthesis and salvage routes of pyrimidine nucleotides, as well as in the salvage routes of purine nucleotides (Sinha and Smith 2001). In these enzymes, the PRPP 50 -phosphate group is hydrogen bonded to the main-chain amides of five consecutive residues, closely resembling the anion binding site of so-called nests (Scapin et al. 1995; Focia et al. 1998). Nests are short (3-6 residues) peptides whose main-chain NH groups bind anionic atoms or groups; they are expected to have been very common at the earliest stage of evolution and to have been used for ligands binding, particularly phosphate groups and iron-sulfur centers (Watson and Milner-White 2002; Milner-White and Russell 2005, 2008). Interestingly, the PR transferase crystallization experiments reveal that anions from the crystallization solution, such as sulfate, phosphate or sulfonate groups, nearly always fill the 50 -phosphate PRPP site of PR transferase structures lacking specific ligands (Sinha and Smith 2001). This supports the hypothesis that the PRPP binding pocket of PR transferase might retain the traces of a nest configuration. It is therefore tempting to speculate that peptides containing a nest and able to interact, at least transiently, with other functional peptides might have been involved in the PR transfer reaction: the 50 -phosphate of PRPP might have primarily served as a handle for these peptides to firmly grip the molecule while the functional part, that is the pyrophosphate moiety, took part in the reaction. It is worth noting here that such a function of the phosphate group can currently be found in another molecule considered to be of very ancient origin, the coenzyme pyridoxal-50 -phosphate (PLP). Both the feasibility of PLP synthesis in conditions resembling a primordial atmosphere (Aylward and Bofinger 2006), and the cofactor reactivity even in the absence of the protein catalyst argue for PLP having arrived on the evolutionary scene before the emergence of protein-assisted pyridoxal catalysis. It is also assumed that the cofactor itself might have played a dominant role in the molecular evolution of the PLP-dependent enzyme families (Vacca et al. 2008). In today’s metabolism, PLP is one of the most versatile coenzymes, due to the electron sink properties of its pyridoxal moiety that endows the holoenzyme with diverse reaction specificities, including transamination, decarboxylation, racemization, aldol cleavage, as well as b- and g-elimination, and replacement

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reactions. Similar to other cofactors, also PLP acts autocatalytically on precursors of its own biosynthesis (Begley 2006). Intriguingly, in most PLP-dependent enzymes, the phosphate group of the cofactor does not participate in catalysis, rather it is “glued” to the protein through up to nine hydrogen bonds in a conserved phosphate-binding cup, thus ensuring anchoring of the cofactor and proper steering of the pyridoxal moiety (Denesyuk et al. 2002). Notably, looking at the cofactor de novo biosynthesis consisting of a multistep ring closure reaction starting from phosphorylated precursors, it is evident that the PLP phosphate is simply inherited from the precursors, without any involvement in the various synthetic reactions (Mittenhuber 2001; Fitzpatrick et al. 2007). It is therefore conceivable that the function of phosphates in metabolites of ancient origin, like PRPP and PLP might indeed represent a very ancient relict from an era when mineral-catalyzed surface metabolism gave way to the progressive take-over by interactive networks of organic catalysts. Differently to type I PR transferases, the PR transferases involved in NAD biosynthesis (enzymes 1, 4 and 6 in Fig. 9.2) form a distinct family, known as type II family. They exhibit a completely different topology from type I PR transferases and therefore they are thought to have evolved independently (Sharma et al. 1998). Accordingly, it is not so easy to find the memory of a typical nest in the PRPP binding site of type II enzymes. Nevertheless, the structural analysis of quinolinate phosphoribosyltransferase (which, as already discussed, is most ancient in the type II family) reveals that interactions of the enzyme with the 50 -phosphate of PRPP are required to hinge the PRPP moiety on the protein during catalysis (Sharma et al. 1998). Moreover, as seen for type I PR transferases, a sulfate ion is tightly bound at the 50 -phosphate PRPP binding site in the apoenzyme (Sharma et al. 1998). An intriguing structural feature of quinolinate phosphoribosyltransferase is the cationic nature of the quinolinate binding site: all residues interacting with quinolinate are positively charged amino acids that form an extensive hydrogenbonded network between their main chain and/or side chain NH groups and the oxygen atoms of the quinolinate carboxylates (Bello and Grubmeyer 2010). They are all involved in catalysis and stabilize the reaction intermediate. It is tempting to speculate that basic residues in primordial peptide films might have bound quinolinate in a similar way and assisted a PR transfer from a neighboring peptide containing a PRPP binding nest, to form NMN. In the proposed scenario, NAD might have been synthesized by early protoenzymes able to interact with NMN and ATP and to catalyze transfer of the ATP adenylyl group to NMN. Indeed, the enzyme NMN/NaMN adenylyltransferase that catalyzes this reaction in modern metabolism (enzyme 2 in Fig. 9.2) belongs to the large family of P-loop nucleotidyltransferases. Members of this family share the typical P-loop motif, a motif found in the majority of the nucleotide-binding enzymes, whose function is to properly position the triphosphate moiety of the bound nucleotide. Notably, the P-loop has a nest incorporated within its active binding (Milner-White and Russell 2008).

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Once formed and ensured its own synthesis through an autocatalytic network, NAD or NAD-like molecules might have contributed directly to the emergence of a protometabolism by functioning together with surrounding peptides. 9.6.2.2

Nicotinamide Coenzyme Role in Early Evolution

It has been proposed that the “RNA world” before DNA was itself preceded by an era when ribonucleotide-like coenzymes and other cofactors already existed and took part in many catalytic roles (Kritsky and Telegina 2005). Yet, as cautiously noted by these Authors, even the demonstrated attachment of coenzymes to RNA “does not explain how their presence could have boosted catalytic activity in the absence of a specific apoenzyme.” It has been suggested here that such rudimentary apoenzyme functions emerged from stochastic prebiotic peptides, in a coevolving “Peptide-Cofactor world,” from where the transition to the “RNA world” proceeded later on. No direct experimental evidence exists so far that supports the hypotheses of a peptide-guided synthesis of NAD and the coenzyme involvement in the evolution of the “Protein world”. Indeed, over the last decade, an impressive body of work has been devoted to prove, by in vitro selection and evolution experiments, that ribozymes are capable of catalyzing a broad range of reactions at a rate high enough to likely have sustained life on early Earth. On the contrary, the catalytic potential of primordial peptide films has received very little attention. At any rate, the few experiments performed on this issue warrant the assumption of a “Peptide world” scenario. As examples, the feasibility of a substrate-directed formation of a catalytic dipeptide has been experimentally verified (Fleminger et al. 2005), and catalytic activities of other small peptides were referred to before (Weber and Pizzarello 2006 and references herein). Also, the frequent occurrence of enzymelike oligopeptides has been observed in libraries that have undergone neither evolutionary selection, nor active site design (Wei and Hecht 2004). It is also noteworthy that short peptides have the ability to bind and recognize nucleotides with high specificity. In particular, b-hairpin peptides have been designed able to bind ATP and FMN (Butterfield and Waters 2003; Butterfield et al. 2004; Butterfield et al. 2005). Of significance is the fact that the binding of FMN affects the flavin redox potential, as seen in flavoproteins. Based on these results, it is reasonable to assume the feasibility of also designing peptides with a recognition cleft for the nicotinamide coenzyme. It would also be interesting to establish whether these peptides might potentiate any cofactor-dependent redox reaction. Indirect evidence of NAD being able to support catalysis even in the absence of large protein-catalysts is provided by certain structural features of the NADH coenzyme (Copley et al. 2007). In particular, the NAD(H) pyrophosphate moiety might chelate the metal ions, that in the enzyme-catalyzed reaction polarize the carbonyl group of the substrate, leading to the transfer of the negatively charged hydride ion from NADH to the carbonyl C atom, thus resulting in substrate reduction. In addition, reduction of the substrate might be facilitated by its covalent

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attachment to the 20 or 30 hydroxyl group of the coenzyme’s adenosine ribose. It can be speculated that, at a later evolutionary stage, more effective catalysts might have arisen from the interaction of the coenzyme with peptides and such interaction might have induced the fold of primordial NAD dependent dehydrogenases (Ji et al. 2007). The possibility that in a primordial “Protein world,” NAD might have performed other roles in addition to serving as a redox coenzyme should also be considered. Indeed, the ability of some extant enzymes to efficiently initiate transcription by incorporating NAD into the first nucleotide position of RNA (Huang 2003) or to covalently attach a chain of ADP-ribose residues to proteins to modify their function, suggests that proto-enzymes might have used the NAD ribotide moiety to prime oligomerization reactions. As an example, it has been proposed that the coenzyme might have been coopted by enzymatically active quasi-peptides to activate amino acids for the formation of stochastic peptides (Egel 2009). It is suggested that transition from the “Protein world” to the “RNA world” might have begun when coenzymes polymerized leading to the later emergence of nucleic acids. Interestingly, both the ADP-ribose and the NMN groups of NAD can be polymerized by extant enzymes. As discussed above, specific polymerases are able to use NAD to synthesize a polymer of ADP-ribose on acceptor proteins, consisting of a linear or multibranched polyanion with the ADP-ribose units linked together via 20 –100 glycosidic bonds (Schreiber et al. 2006). More intriguingly, however, the bacterial enzyme polynucleotide phosphorylase is able to efficiently use nicotinamide riboside diphosphate in primer extension reactions, as well as to catalyze the de novo polymerization of nicotinamide riboside diphosphate in its reduced form, yielding long polymers of NMN(H) (Liu and Orgel 1995) (Fig. 9.4a). Notably, nicotinamide can base-pair with both uracil and cytosine (Liu and Orgel 1995), thus resembling a Watson-Crick configuration and substituting for adenine and guanine in modern RNA (Fig. 9.4b). Looking again at extant nucleic acid metabolism to search for hints about pre-biotic pathways, it can be recalled here that while few enzymatic steps are required to synthesize the pyridine and pyrimidine rings, at least ten reactions are necessary to build up the complete purine base in the form of hypoxanthine. Moreover, while in the former pathway the nitrogen bases are synthesized and then transferred to the PRPP ribose by specific PR transferases, the hypoxanthine ring is assembled attached to the PRPP ribose throughout the process, therefore no PR transferases are involved. Inosine monophosphate is the first intermediate that is subsequently converted to both adenine- and guanine-nucleotides. Even though, of course, a perfect correspondence between primordial and extant pathways cannot be assumed, the described differences might support the hypothesis that, in evolutionary time, nicotinamide, uracil and cytosine nucleotides might have predated the purine nucleotides and started complementarity-guided rounds of copolymerization, as aided by appropriate prebiotic peptides. Once hypoxanthine emerged, it might have replaced nicotinamide, due to the same ability to base-pair with both cytosine and uracil and to ensure larger stacking forces between adjacent base pairs. Later on, both adenine and guanine can have been formed from hypoxanthine, thus substituting it in the

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Fig. 9.4 Towards Watson-Crick base-pairing: (a) NMN polymer, as formed by the bacterial enzyme polynucleotide phosphorylase that uses nicotinamide riboside diphosphate as the substrate; (b) base-pairing pattern in a proposed evolutionary path whereby nicotinamide-pyrimidine evolved to purine-pyrimidine through hypoxanthine-pyrimidine base-pairing

polymers and stabilizing the pairing rules of modern nucleic-acid metabolism. Figure 9.4b shows the described base-pairing patterns to illustrate this tentative sequence of evolutionary events, thus offering a supplementary hypothesis supporting the role of nicotinamide coenzyme in the emergence of early nucleic acids.

9.6.3

Coevolution of Genes and Metabolism

Interesting models propose coevolution of genes and metabolism in a world with a productive coexistence of various classes of abiotically generated organic molecules, where ribozymes and enzyme catalysis might have operated side-byside (Mulkidjanian and Galperin 2007). In one such model, it is hypothesized that proto-metabolism was sustained at the earliest stage by simple molecules, like amino acids, keto acids, nucleotides and cofactors. The later polymerization of amino acids and dinucleotides in peptides and longer oligonucleotides would have enhanced the catalytic capabilities; however, given that only RNA has the ability to support both catalysis and genetic information, the system would have moved towards the emergence of RNA as the dominant macromolecule (Copley et al. 2007). In this scenario, NADH is suggested to have assisted the reductive amination

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of a-keto acids covalently attached to dinucleotides, allowing synthesis of the corresponding a-amino acids (Copley et al. 2005). In a different view, short RNA molecules and short peptides would have naturally given rise to an “RNA-Protein world” when oligonucleotides spontaneously became a primordial template capable of accommodating single amino acids or short peptides, catalyzing peptide-bond formation, thus representing early protoribosomes (Agmon 2009; Davidovich et al. 2009). These simple structures would have enabled the emergence of several stochastic, uncoded peptide chains of progressively increased length. Both the uncoded polypeptides and the protoribosomes would have undergone selection pressure for increased stability and efficiency, resulting in the coevolution of the translation apparatus and the proteins (Agmon 2009; Davidovich et al. 2009). The proposed role of NAD in assisting a primordial random peptides formation (Egel 2009) fits nicely in this scenario. Indeed, in an earlier evolutionary stage, NAD-like molecules might have functioned as precursors of proto-ribosomes when, coopted by enzymatically active quasi-peptides, served to activate amino acids and to act as docking guides for their oligomerization to occur.

9.7

Conclusion

The ancient metabolic history of NAD(P) can be inferred by both the autocatalytic nature of its biosynthetic pathway, and the experimental evidence of a spontaneous production of its precursor quinolinate from simple molecules likely to have dominated in abiotic environments via a sequence of reactions that closely resemble the quinolinate biosynthetic pathway in extant metabolism. These findings strongly suggest that the pyridine ring might have arisen independently of enzymatic chemistry and provide evidence for the presence and involvement of the pyridine coenzymes in the earliest metabolic system. No experimental data exist so far to shed light on the origin of the NAD ribonucleotidyl moiety. Its presence is commonly considered a strong indication of the emergence of the coenzyme in an “RNA world”; indeed, the in vitro selection of ribozymes able to synthesize NAD and to use it to catalyze redox reactions together with the recent discovery of eubacterial small RNAs linked to NAD at their 50 -termini, appear relevant to the hypothesis of considering NAD as a byproduct of the “RNA world”. However, it should be pointed out here that no natural ribozymes capable of catalyzing redox reactions have been discovered so far and catalysis of redox reactions seems to be a prerogative of protein enzymes. In addition, given the occurrence of an enzyme (T7 RNA polymerase) able to efficiently incorporate NAD into RNA at the priming step of transcription, it can be reasonably hypothesized that the small RNAs linked to NAD might be the products of an enzymatic reaction. An E. coli polymerase, not yet identified, might exists which uses NAD as the transcription initiator. Therefore other hypotheses on NAD biogenesis cannot be discarded “a priori”. In particular, the possibility should also be considered that NAD might have arisen before

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or contemporary to RNA emergence, playing a role in the appearance of a protometabolism before a nucleic acid-coded protein synthesis. In particular, coenzyme-like molecules might have been synthesized in a “Peptide world” by stochastic prebiotic peptides, in the presence of simple molecules, like quinolinate and PRPP. Assisted by appropriate catalytic peptides, they might have played key roles in the primordial protometabolism, as suggested by NAD functions in presentday organisms. Indeed, the coenzyme is used not only to catalyze redox reactions, but also to control many cellular processes through the transfer of its nucleotidyl moiety to nucleic acids and proteins. An NAD-like molecule might have performed similar roles in the earliest stage of evolution, bestowing redox activity to prebiotic peptides and priming oligomerization reactions. These conjectures offer some new directions for experimental work. In particular, it might be worthy to ascertain the plausibility of peptides able to specifically bind NAD and to potentiate the nicotinamide-catalyzed reduction of carbonylic compounds. Indeed, initial experiments succeeding in including a stable dihydropyridine group within peptide motifs in an aqueous environment support the feasibility of designing such systems (Imperiali et al. 1999). Nowadays, research can rely on novel techniques, like the messenger RNA display, which allows selection and in vitro directed evolution of novel functional peptides, including enzymes (Seelig and Szostak 2007). It would be interesting to use this technique to select structured peptides that can specifically bind NAD and, even more interesting, to isolate short peptides able to catalyze reactions involved in NAD synthesis, like the transfer of the ATP adenylyl group to NMN. It is likely that NAD functions other than those described here will be discovered in the future, which might help to shed light on its eventual role in RNA emergence. Notably, in several eubacterial species, a set of genes likely involved in ribosome biogenesis form a highly conserved cluster that constitutes an operon, which also contains the gene coding for NaMN adenylyltransferase (enzyme 2 in Fig. 9.2) (Galperin and Koonin 2004). The significance of this association remains unknown, but this finding suggests that a possible functional relationship between NAD and RNA metabolism might still occur in extant organisms.

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Chapter 10

On Alternative Biological Scenarios for the Evolutionary Transitions to DNA and Biological Protein Synthesis Anthony M. Poole

Abstract The RNA world hypothesis has become a central part of current thought on the origin of life. For biologists, this has provided especially fertile ground, since the biological origins of genetically encoded protein synthesis and of deoxyribonucleotide synthesis through ribonucleotide reduction can both be understood within an RNA world framework. However, these are not the only possible routes by which proteins and DNA could have evolved. Some proteins are synthesised by modular non-ribosomal peptide synthetases, and E. coli have recently been engineered to synthesise deoxyribonucleotides via the previously hypothetical reverse deoxyriboaldolase reaction. In this chapter, I consider to what degree these alternative processes impact our understanding of the evolutionary transitions from RNA to proteins and DNA.

10.1

Introduction

The RNA world hypothesis, that there was a stage preceding the origin of genetically encoded proteins and DNA, where RNA was both genetic material and an important catalyst, has become the dominant model for the biological understanding the early evolution of life. The model is based on a large number of observations (Benner et al. 1989; Jeffares et al. 1998; Joyce 2002; Yarus 2002), including the demonstration that RNA functions as an information storage molecule in RNA viruses (Atkins 1993; Fraenkel-Conrat 1956; Gierer and Schramm 1956) and viroids (Diener 2003), the broad repertoire of natural catalytic RNAs

A.M. Poole (*) School of Biological Sciences and Biomolecular Interaction Centre, University of Canterbury, Private Bag 4800, Christchurch 8140, New Zealand Department of Molecular Biology and Functional Genomics, Stockholm University, SE-106 91, Stockholm, Sweden e-mail: [email protected] R. Egel et al. (eds.), Origins of Life: The Primal Self-Organization, DOI 10.1007/978-3-642-21625-1_10, # Springer-Verlag Berlin Heidelberg 2011

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(Guerrier-Takada et al. 1983; Kruger et al. 1982; Talini et al. 2009; Wilson and Lilley 2009), and the observation that coenzymes carry nucleotide moieties which are not directly required for function (Szathmary 1999; White 1976; White 1982). Moreover, the identification of regulatory riboswitches in bacteria, archaea and eukaryotes (Breaker 2010; Sudarsan et al. 2003) as well as numerous in vitro selection studies expanding the known enzymatic potential of RNA (Yarus 2002) (including the capacity for limited templated replication (Cheng and Unrau 2010; Lincoln and Joyce 2009) and even its capacity to influence membrane permeability (Khvorova et al. 1999; Vlassov et al. 2001) all serve to strengthen a now satisfyingly mature model for the emergence of modern biology that began as a set of thought-provoking speculations (Crick 1968; Orgel 1968). The RNA world contrasts with most other scenarios for early evolution because it has drawn from both chemistry and biology. Through the comparative analysis of molecular biological features of cells, an emerging consensus is that the ribosome emerged from an RNA world (Campbell 1991; Fox 2010; Gordon 1995; Noller 2010; Poole et al. 1998) (Fig. 10.1). On the chemical side, this view was strengthened by in vitro selection of peptidyl transferase ribozymes (Welch et al. 1997; Zhang and Cech 1997, 1998) and has become widely accepted following structural studies showing that the peptidyl transferase centre in the large subunit of the ribosome is composed of RNA (Ban et al. 2000; Moore and Steitz 2002; Nissen et al. 2000; Steitz and Moore 2003). DNA on the other hand is thought to postdate the origin of protein synthesis, largely on account of the fact that the sole de novo pathway for synthesis of deoxyribonucleotides involves reduction of ribonucleotides (Forterre 2002; Freeland et al. 1999; Lazcano et al. 1988; Poole et al. 2000, 2002) (Fig. 10.1).

Fig. 10.1 The hypothesised evolutionary transitions from RNA to proteins and DNA. This figure provides an overview of the key events, as seen from a biochemical standpoint. Through some means, an RNA replicase emerges in an RNA world. Next the ribosome evolves, resulting in the emergence of genetically encoded protein synthesis. The advent of DNA follows the evolution of ribonucleotide reductases

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Because ribonucleotide reduction is complex, requiring the generation and control of protein-based radicals (Nordlund and Reichard 2006), it has been argued to have necessarily evolved after the emergence of genetically encoded protein synthesis (Poole et al. 2000, 2002), with a separate transition leading to the replacement of uracil with thymine as fourth base (Forterre 2002; Poole et al. 2001). While the evolutionary transitions embodied in the origin of the ribosome and of ribonucleotide reduction seem to point to an earlier RNA world, the case for an RNA world on chemical grounds is far from established, and major chemical difficulties with the origin of the RNA world, dubbed ‘the prebiotic chemists’ nightmare’ (Joyce and Orgel 1999; Orgel 2004), remain. One clear difficulty is in establishing the prebiotic conditions for the emergence of both ribose and, later, ribonucleotides, where good progress has recently been made (Powner et al. 2009; Ricardo et al. 2004; Springsteen and Joyce 2004; Sutherland 2010). Another is the dual problem of the emergence of an RNA replicase that somehow manages to replicate itself, or cross-relicate (Cheng and Unrau 2010; Lincoln and Joyce 2009), plus is capable of sufficiently accurate and processive replication to maintain some minimal RNA genome (Jeffares et al. 1998; Kun et al. 2005; Poole 2006; Scheuring 2000). These difficulties have led to concerns regarding the feasibility of a prebiotic origin for the RNA world (Anastasi et al. 2007), and some have directly questioned the relative timing of emergence of RNA, proteins and DNA (Kurland 2010). Here, I will briefly comment on the latter.

10.2

RNA ! RNP ! Protein ! DNA?

Regardless of the origin of the RNA world, or whether an RNA world sensu stricto is feasible (Cech 2009a; Sutherland 2010), the logic applied in proposing the direction of the transitions from RNA to proteins and DNA (Fig. 10.1) is simple, but based on two different premises. For RNA to protein, the primary argument is that genetically-encoded protein synthesis postdates simpler RNA-based enzymes because the core of the ribosome is RNA-based (Noller 2010). An intermediate step was the transition to ribonucleoproteins – RNA associated with ‘chaperones’ (Cech 2009b; Poole et al. 1998). By chaperones, what is meant is that early proteins were not in themselves catalytic, but were selected because they were capable of stabilising or improving the function of ribozymes. While it seems plausible such simple peptides would not be sequence specific, instead being selected for general nucleic acid binding properties (Tan and Frankel 1998), the expectation is that reproducible production of these would be extremely limited under prebiotic conditions, unless the preferential condensation of basic amino acids is favoured over other types. Even with a sufficient source of prebiotic peptides, this is still a far cry from the transition to complex catalytic proteins. Consequently, it seems that a transition from RNPs to catalytic proteins must require the advent of the ribosome. Consequently, the

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reasoning goes, RNA must have predated genetically encoded proteins. Of course, this does not help explain how an RNA-based system got started in the first place, if indeed it could. That said, proponents of peptide-first models face the same problem as RNA-first proponents: how does one get from building blocks to a viable self-replicating state? The prevailing consensus for the origin of DNA is that the key event was the advent of deoxyribonucleotide synthesis. From a biological perspective, the only de novo pathway for this is via ribonucleotide reduction. This reaction has been argued on chemical terms to be outside the scope of RNA chemistry, primarily because the only way to reduce a hydroxyl to a hydrogen is via complex radical chemistry (Poole et al. 2000) (Fig. 10.2). That radicals lead to indiscriminate cleavage of the sugar-phosphate backbone (Celander and Cech 1990) is a strong argument against the possibility of an RNA-based ribonucleotide reductase (Poole et al. 2000). Moreover, because even the simplest monomeric ribonucleotide reductases are in themselves complex and still dependent on generation of a protein radical (Sintchak et al. 2002), genetically encoded protein synthesis and a large informational coding capacity seem to be prerequisites for the synthesis of deoxyribonucleotides that enabled the RNA to DNA transition (Poole et al. 2000, 2002).

Fig. 10.2 Ribonucleotide reduction and the transition from RNA to DNA. (a) The transition from RNA to DNA is thought to have occurred in two steps. The first is the emergence of ribonucleotide reduction, which enables the generation of deoxyribonucleotides from ribonucleotides. The second step is the replacement of uracil as fourth base, which required the emergence of at minimum a thymidylate synthase. (b) The major steps in ribonucleotide reduction common to all classes of ribonucleotide reductase. In the first step, a thiyl radical is produced, which is then transferred to the substrate, forming a substrate radical. In the next step, the 20 -hydroxyl is protonated with the release of a water molecule. This requires a reductant, though the nature of this differs depending on the class of ribonucleotide reductase. In the final step, the radical is returned to the thiyl group, before being returned to either the cofactor or to another residue on the protein

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Consequently, if synthesising DNA is so chemically complex that sophisticated protein-based chemistry must predate it, it seems difficult to argue that sufficiently complex peptides produced from non-templated (non-genetic) processes could be generated that permit the emergence of complex proteinaceous enzymes. That said, as with peptides-early scenarios (Egel 2009; Kurland 2010), good arguments have been made for an earlier, non-biological origin for DNA (Burton and Lehman 2009; Dworkin et al. 2003). My aim here is not to examine the relative timing of the emergence of certain macromolecules from a chemical perspective. Rather, my goal here is to ask whether the biological transitions depicted in Fig. 10.1 are the sole options open to us, based on what is currently known from biology. Let’s first separate the perceived complexity of these transitions from the notion of an RNA world. In its most basic form, genetically encoded protein synthesis is significant because it enables complex proteins to be reproduced accurately, whereas the alternative, a non-templated process would seem not to be possible for anything but the most simple, low information peptides (i.e. where general properties are more central to function than specific sequence) (Cech 2009b; Poole et al. 1998). And because we tend to assume that evidence of the past history of life is ascertainable from the patterns we see in the most conserved early processes, it seems natural to conclude that this view is largely correct. However, while there has without doubt been a transition to modern genetically encoded protein synthesis, it is reasonable to accept that we know less about the preceding state. The arguments in favour of the transitions shown in Fig. 10.1 are certainly compelling, but if they do not constitute hard evidence for the existence of these transitions and their preceding states, it does pay to keep an open mind. I will now focus on two biological discoveries that give us pause to rethink whether there could be other plausible routes for the biological origins of encoded peptides and of DNA, regardless of their prebiotic origins.

10.3

Non-Ribosomal Peptide Synthesis (NRPS)

Excitingly, non-ribosomal peptide synthesis has been described or genomically identified in a wide number of bacterial and fungal species (Amoutzias et al. 2008), and has key properties of interest to the origin of life: modularity and specificity in synthesis, but without information being stored in a nucleic acid template. What follows is a short summary; for recent detailed reviews, I refer the interested reader to Marahiel (2009) and Strieker et al. (2010). A recent comprehensive review also examines the similar mechanisms involved in production of polyketide antibiotics (Fischbach and Walsh 2006). Non-ribosomal peptide synthetases (NRPSs) are large ‘megaenzymes’ which synthesise small oligopeptides, including a wide range of antibiotics. NRPSs are able to incorporate the 20 amino acids used in ribosome-dependent protein synthesis, but can also generate molecules with both L- and D-enantiomers, incorporate over several hundred additional substrate monomers, including b-amino

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Fig. 10.3 Schematic of non-ribosomal peptide synthetase architecture. Non-ribosomal peptide synthetases are large enzymes with a modular structure. Each module carries at minimum an A, PCP and C domain, enabling substrate recognition, handling and peptide bond formation. The order of the modules determines the synthetic process; the first module is an initiation module, and does not require a C domain as it serves only to select the first substrate monomer. The elongation process involves successive condensations, with each module determining the synthetic pathway. In addition to carrying specific A groups for substrate selection, Auxiliary domains may make additional modifications to the peptide. Finally, the TE domain is required for product release

acids and a wide range of other carboxylic acids. NRPSs also generate lipopeptides (Roongsawang et al. 2010), which is of particular interest to emerging models which envisage a close association between early peptides and abiotic vesicles (Egel 2009). Each NRPS is formed from an array of modules derived from a relatively small number of domain-types (Fig. 10.3) and each megaenzyme carries the information for specifying the order of addition of monomers plus their condensation (Marahiel 2009; Strieker et al. 2010). The A, PCP, C and TE domains form the core suite of domains in an NRPS. The role of Acyl (A) domains is to select specific substrate monomers. Consequently, through their order within a NRPS megaenzyme, A domains act to confer specificity in a code-like manner (Stachelhaus et al. 1999). Adjacent to each A domain is a peptidyl carrier protein (PCP) domain. The growing chain is covalently attached to the PCP domain, which transports both growing chain and substrate monomers between active sites via a ‘swinging arm’. The condensation (C) domain is responsible for the peptidyl transfer reaction.

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In addition to this bare-bones synthetic machinery, a diverse array of auxiliary (Aux) domains are known which may modify the nascent peptide in a number of different ways, including epimerisation, formylation, methylation, cyclisation, oxidation and reduction (Marahiel 2009). Finally, the Thioesterase (TE) domain terminates synthesis in that it is responsible for release of either a linear product, or may cyclise the product prior to release. As depicted in Fig. 10.3, a NRPS megaenzyme may consist of multiple modules. Consequently, specific NRPSs may be very large. For example, cyclosporin synthetase is a 1.4MDa protein, consisting of ~15000 amino acid residues (Lawen and Traber 1993; Lawen and Zocher 1990; Schmidt et al. 1992), yet its product, cyclosporine, is a cyclic undecapeptide (11-mer). The energetic cost associated with producing any oligopeptide via this general route is therefore massive (in excess of 1,000 residues per product monomer). Clearly, the three orders of magnitude discrepancy here is not a minor issue for any peptide-world scenario, especially when weighed up against an in vitro selection study which yielded a 160-mer ribozyme capable of catalysing the formation of 30 different dipeptides (Sun et al. 2002). For the types of syntheses performed by NRPSs to be of relevance to the emergence or chemical study of self-replicating systems, or even the suggested antiquity of secondary metabolites (Davies 1990), for any given level of informational input the system must generate an approximately equivalent informational output. Furthermore, in terms of a historical evolutionary trace, it seems likely that this system is derived, not because it is genomically encoded, but because there are clear similarities between the core modules and other known protein families (Amoutzias et al. 2008; von Dohren et al. 1999). While this does not in itself establish directionality, the question of genetic takeover, in the transition to a nucleic acidcoded world, cannot be ignored. In lieu of an obvious way to back-translate peptideencoded information into a nucleic acid sequence, it would seem that such early peptide synthetase machinery, if it did exist, would have had to have been replaced by a non-orthologous synthetase that generated an equivalent product. The cost of NRPS-based syntheses in modern systems relative to ribosomal peptide syntheses could point to a selective rationale for such a replacement. Consequently, this system cannot be directly linked to a pre-ribosomal stage in the origin of life and its existence does not in itself vindicate arguments that disparage the RNA world model (Kurland 2010). However, that is not what is important here. What the characterisation of non-ribosomal peptide synthesis does is it opens up the possibility that simple peptide-based systems can be explored for self-replicative properties. In the same way that in vitro selection techniques enabled experimental studies seeking to screen for the propensity for self-replication by RNA (Lincoln and Joyce 2009), it may well be possible to assess the minimal information required for NRPS-like modules that can both assemble and specify the information for reproduction of that set of modules. In this regard, it is interesting that the process of substrate selection by some A domains may be determined by as few as 3 residues (Husi et al. 1997). Moreover, there are already some positive indications that NRPSs can be manipulated to generate novel peptides (Mootz et al. 2000; Stachelhaus et al. 1995).

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The next step would be to design minimal NRPSs – there has been direct value for origin of life studies (Kun et al. 2005) in establishing the structure and function of minimal ribozymes, and taking the same approach would directly address the energetic and informational cost problem so obvious in natural NRPSs. The very existence of non-ribosomal peptide synthesis indicates that protein can in principle make protein, even if assessing the self-replicative potential of peptides will be non-trivial. Massive size indicates they are clearly far from a simple selfreplicating system, so for a peptide-based system to have emerged prior to a nucleic acid-based one, what is needed is a demonstration that a minimal NRPS can generate peptides with similar informational content. However, the discovery of this process, together with demonstrations that this system can be manipulated to generate custom peptides, has the potential to move models for a peptide-first origin of life from a largely speculative field to an area of intense experimental endeavour open to study using molecular biological tools.

10.4

Deoxyribonucleotides via the Reverse Deoxyriboaldolase Reaction

In all cellular systems, synthesis of deoxyribonucleotides occurs exclusively through the process of ribonucleotide reduction. Ribonucleotide reductases (RNRs) produce deoxyribonucleotides from ribonucleotides in a complex radicalbased reaction (Fig. 10.2). The fact that ribonucleotide reduction is the only known de novo mechanism for deoxyribonucleotide synthesis, coupled with the fact that genomic screens indicate it is effectively ubiquitous (to date only five cellular genomes are known which lack RNRs, all of which are intracellular parasites reliant on salvage of deoxyribonucleotides from their host – (Lundin et al. 2009)) strongly implies that DNA utilisation evolved directly from an earlier system dependent on RNA (Poole et al. 2000, 2002). As shown in Fig. 10.2, the expectation is that this was a two-step transition. Ribonucleotide reduction requires generation and control of protein-based radicals. This has been argued to place the evolution of ribonucleotide reductases well after the advent of genetically encoded protein synthesis, since the chemistry required to perform ribonucleotide reduction appears to be well outside the catalytic capability of RNA (Poole et al. 2000). Under this view, protein-based catalysts would have been a prerequisite for the origin of deoxyribonucleotides, and hence, we would have definite order to the transitions – DNA would have to postdate protein-based enzymes (Fig. 10.1). However, some workers have suggested that DNA could have had much earlier origins, based on possible prebiotic routes to deoxyribonucleotides combined with the broadening chemical repertoire available to DNAzymes (Burton and Lehman 2009; Dworkin et al. 2003). The other main pathway for deoxyribonucleotide metabolism is salvage. As well as providing a mechanism for recycling or acquiring external sources

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Fig. 10.4 Overview of the reverse deoxyriboaldolase reaction. Glyceraldehyde-3-phosphate from glycolysis and acetaldehyde can be converted to 2-deoxyribose-5-phosphate by the enzyme deoxyriboaldolase. Phosphopentomutase then converts this to 2-deoxyribose-1-phosphate, from which the four deoxyribonucleosides can be produced via specific phosphorylases. Normally, as part of nucleotide salvage, the entire pathway proceeds catabolically, but has been shown to operate in the direction shown (see text)

of deoxyribonucleotides, these can be absorbed into central metabolism: deoxyribose-5-phosphate is broken down into acetaldehyde and glyceraldehyde3-phosphate by the enzyme deoxyriboaldolase; the latter product is a direct intermediate in glycolysis. Of relevance to the current discussion is the fact that the reverse of the deoxyriboaldolase reaction (Fig. 10.4) offers a chemically much simpler alternative to ribonucleotide reduction for deoxyribonucleotide synthesis. Interestingly, prior to the characterisation of ribonucleotide reduction in the 1960s, this energetically favourable reverse reaction was the expected route for deoxyribonucleotide synthesis (Reichard 1989; Reichard and Rutberg 1989). However, biology does not use the reverse deoxyriboaldolase reaction – all de novo deoxyribonucleotide synthesis proceeds from ribonucleotide reduction. Hence, for the biologist there seems little point in discussing the reverse deoxyriboaldolase reaction with respect to the origin of DNA (Poole et al. 2002) – it would seem nothing more than an interesting biochemical vignette. However, interest in utilising this reaction as a means of commercially producing deoxyribonucleosides has yielded a deoxyriboaldolase from Klebsiella which performs the reverse reaction with high efficiency (Horinouchi et al. 2003; Ogawa et al. 2003). While the goal for those authors is the generation of 2’deoxyribonucleosides to meet future commercial demand (much of their work is

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focused on optimising production in a process engineering-type approach (Horinouchi et al. 2006a, b, 2009)), what is interesting for the current discussion is that coaxing E. coli to synthesise deoxyribonucleoside-5-phosphate from intermediates of central metabolism does not require multiple complex changes. One can easily envisage microbial selection criteria that might favour this reaction, and the next obvious step is to develop knockout strains that do not use ribonucleotide reductases at all – this is formally similar to eliminating ribonucleotide reduction under conditions favouring salvage (as is known for some intracellular parasites (Lundin et al. 2009), Fig. 10.4), but with the important difference being that reverse deoxyriboaldolation does not require a source of deoxyribonucleotides in the growth medium. Furthermore, if this reaction can replace ribonucleotide reduction completely in a biological context, it opens the prospect of an alternative evolutionary route to DNA. This is interesting because perhaps the biggest problem for understanding the origin of DNA is that, in its standard form, the simultaneous origin of deoxyribonucleotides (and hence DNA) and ribonucleotide reduction requires a terribly complex reaction to emerge for the de novo synthesis of an unselected product. To date, the only real attempt to address this problem is in Forterre’s host-viral arms race model (Forterre 2002, 2005), wherein viruses drive the evolution of cellular nucleic acid use because modifying the building blocks renders them immune to host defences. As an aside, there is precedent for this, as there are well-documented examples of viruses utilising modified nucleic acids as a means of evading host defences (see discussion in Forterre (2002) and Poole et al. (2001)). If instead deoxyribonucleotides were occasionally generated via the reverse deoxyriboaldolase reaction, it is of course completely possible for these building blocks to be co-opted into a later function, so synthesis of a by-product can subsequently be selected. That this process has moved beyond theoretical plausibility warrants optimism because it makes the problem amenable to experimental investigation – what is the effect of knocking out ribonucleotide reduction in one of the Shimizu lab’s E. coli strains overexpressing deoxyriboaldolase and with a good source of acetaldehyde and glucose? It would be very interesting if it transpires that DNA-based life can use existing biochemistry to generate deoxyribonucleotides solely via an alternative de novo route. From a biologist’s perspective, this would in turn show that there could, in principle, have been viable organisms early in the evolution of life which used much simpler chemistry than ribonucleotide reduction as a means to initially synthesise deoxyribonucleotides.

10.5

Concluding Remarks

In this chapter, I have briefly reconsidered the view from biology that early life underwent an RNA ! protein ! DNA transition series (Fig. 10.1). While this view has been studied in much greater depth when compared to alternative routes to

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both deoxyribonucleotides and peptides (Figs. 10.3 and 10.4), it is significant for biologists interested in early evolution that alternatives are both plausible and known in biology. I have no strong opinion as to the relative chemical feasibility of either the RNA world, or an alternative peptide-dominated world (I am not a chemist), but I do not see a pressing need to dichotomise these scenarios. At any rate, the alternative systems of non-ribosomal peptide synthesis and reverse deoxyriboaldolation highlight the value in reconsidering assumptions from time to time, lest they inadvertently become dogma. Examining the biological plausibility of possible alternatives with an open mind is helpful, especially since even the most ardent RNA world supporter would have to acknowledge that, despite the substantial advances of the last three decades, this theory is far from established fact: as yet, no one has succeeded in generating an RNA-based cell sans protein from plausible prebiotic conditions. I think two important points are worth restating, though neither is new: first, it is essential to distinguish between the biological evidence supporting evolutionary transitions from RNA to proteins and DNA, and the chemical plausibility of an RNA world (or indeed any other scenario for early life emerging from some prebiotic state). Identifying the existence of an evolutionary (i.e. biological) transition does not allow us to extrapolate chemical context. While protein synthesis may be argued to trace vertically back through evolutionary time to some time point preceding the diversification of the three domains, there is no firm evidence that the machinery for the synthesis of deoxyribonucleotides and DNA can be placed this far back in evolutionary time (Forterre 2002; Forterre et al. 2004; Leipe et al. 1999; Lundin et al. 2010; Poole and Logan 2005). Second, as biological systems become more tractable to systems biological analysis, I am sure we will increasingly generalise our questions from historical reconstruction of the earliest stages of life to the nascent science of subsystems biology (infrabiological systems) (Szathma´ry 2006), following on from the seminal work of Ga´nti (2003). I am optimistic that such an approach may soon yield plausible tests of various hypothetical models where a system lacks one or more of the key components we associate with life. Acknowledgements I thank the editors for the invitation to contribute to this book. I thank the Royal Swedish Academy of Sciences for past support via a grant from the Knut and Alice Wallenberg Foundation. Support of the New Zealand Marsden Fund is gratefully acknowledged.

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Chapter 11

Two RNA Worlds: Toward the Origin of Replication, Genes, Recombination, and Repair Dirk-Henner Lankenau

Abstract All modern organisms depend on genomes that encode a diversity of RNA molecules functioning in a plethora of physiological, regulatory, and fundamental functions. Processes like gene transcription into mRNA, ribozyme catalyzed translation in the heart of ribosomes, RNA interference (RNAi), reverse transcription and defense of transposons, retroelements, homing mobility of introns, and many other characteristics of life represent the smoking gun of primordial RNA based, complementary base pairing driven search processes crucial for the origin of life and all successive life. This chapter overviews the necessary steps of RNA dependent evolutionary processes that lead to the emergence of replication, genes, recombination, and repair. The top to bottom journey into the past starts with the concept of Popper’s deductive cycle. It leads us through established knowledge accompanied by the conserved mechanism of homology search engines as a key feature of the modern RNA world to the structure based ribozyme function of the catalytic center of the peptidyltransferase in modern ribosomes. We then jump over to the ancient RNA world in a bottom to top approach, a tactic taken by other chapters in this book as well. We use theoretical evidence for a ~55 nt protoribosome as the endpoint of this bottom to top journey and join again with the current RNA world by pointing out the statistical detection of an ancient RNY code in modern genes.

D.-H. Lankenau (*) Hinterer Rindweg 21, 68526 Ladenburg, Germany e-mail: [email protected] R. Egel et al. (eds.), Origins of Life: The Primal Self-Organization, DOI 10.1007/978-3-642-21625-1_11, # Springer-Verlag Berlin Heidelberg 2011

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Abbreviations BIR dsRNA ESS FRET Ga IDA LTR LUCA(S) Ma NAD ncRNA NHEJ nt ORF QED RdRP RNAi RNP(C) RNY RRR RT SDSA SSA ssRNA

break induced replication (DNA repair) double-stranded RNA evolutionary stable strategy fluorescence resonance energy transfer 1 giga year (anus) ¼ 109 years initial Darwinian ancestor long terminal repeat last universal common ancestor(al) state million years (ani) ¼ 106 years Nicotinamide Adenine Dinucleotide non-coding RNA non-homologous end joining (DNA repair) nucleotide open reading frame quantum electrodynamics RNA dependent RNA polymerase RNA interference ribo nucleo protein (complex) purine – any nucleotide – pyrimidine ¼ hypothetical ancestor of the genetic code replication, repair, recombination reverse transcriptase synthesis-dependent strand annealing (DNA repair) ¼ mechanistic ancestor of RRR single strand annealing (DNA repair) single-stranded RNA

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The origin of life was the chemical event or series of events whereby the vital conditions for natural selection first came about. The major ingredient was heredity.... The origin of life only had to happen once. We therefore can allow it to have been an extremely improbable event many orders of magnitude more improbable than most people realize. . . And the beauty of the anthropic principle1 is that it tells us against all intuition that a chemical model need only predict that life will arise on one planet in a billion to give us a good and entirely satisfying explanation for the presence of life here. (But) I do not for a moment believe the origin of life was anywhere near so improbable in practice. (Dawkins 2006) The word emergence is in a sense the opposite of reductionism where reductionism is the view that any phenomenon can be explained by understanding the parts of that system. (Hazen 2005)

11.1

Introduction: Popper’s Deductive Cycle

The emergence of life is a complex labyrinth, a historical puzzle that cannot be approached directly. However, indirect methodologies are possible but where is Ariadne’s thread? Figure 11.1 shows four interlocked contemporary attempts to crack the origin of life question and to frame a theory of life escorted by a deductive concept that embraces a multidisciplinary, unifying synthesis. One approach (Fig. 11.1b) searches the cosmos for alien or strange traces of recent or fossil life analogs hoping to define constraints that sharpen models for the emergence of terrestrial life (Baross et al. 2007). Another effort (Fig. 11.1d) tries to create artificial Darwinian systems in the laboratory, an approach called synthetic biology (Benner and Sismour 2005; Gibson et al. 2010). Most chapters in this book deal with a third methodology called prebiotic (geo-) chemistry (Chaps. 2, 4–7). They are rooted in Stanley Miller’s famous discharge experiments (Miller and Urey 1959). Miller’s approach addresses the primal self organization of life, starting from abiotic processes, meteorological, astro-geophysical, and geochemical in their nature. Chemistry, is known as the most innovative of the sciences with chemists able to create and design from scratch new organic and inorganic compounds, unique in the entire universe. Many of them serve the well being of humanity. Despite hidden, life threatening dangers such as toxicity, explosiveness, ozone depletion etc. caused by novel chemicals, our civilization would not exist as is, if food chemistry, material grades like plastics or medicals had not been invented and produced by chemistry. Often, the new compounds synthesized by chemists make up tools of all kinds in industry and technology, the colors we paint our homes with and the medicine we so often depend on. Physics, the fundament of science, is likewise responsible for raw materials (steel, silicon chips, semiconductors) as applied in electrical engineering, civil-, and mechanical engineering. Further, physics is known to drive our understanding of nature deeply into the essence of matter itself, discovering fundamental laws, new elementary particles, and presently the existence of dark energy and dark 1 The anthropic principle was named by the mathematician Brandon Carter in 1974 (Barrow and Tipler 1986).

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matter that both only seem to interact with our baryonic matter via gravitation and the weak nuclear force. Murray Gell-Mann once said: You don’t need something more to explain something more.2 What he meant was that all the elementary building blocks and laws that chemistry and physics discovered in history plus a bunch of accidents are sufficient to explain the emergence of something more, i.e., life. Most chapters in this book take the bottom to top approach to experimentally and theoretically explore and reconstruct chemical means and conditions that could have led to the emergence of life (Fig. 11.1). An impressive example of nonDarwinian, purely geo-chemical evolution has been put forward recently by Robert Hazen exploring the evolution of minerals during the formation of our eight planets (Hazen 2010). This analysis shows that the early and present Earth is unique in its mineralogical composition compared to all other planets in this solar system, meaning that the emerging life, as we know it, had a different likelihood to self organize on Earth compared to other extraterrestrial bodies. The chapters of the book try to create a feeling for those geo-chemical processes and settings from which proto-life might have chosen and from which the first entities of life developed that coincide with a most basic definition of life. As of now, we do not know the exact sequence of events life took to emerge. The “golden spike” of Fig. 11.1 has not been bridged yet but a hope evoking concept is on the rise. There is a plethora of chemical compounds from where life could have started. In fact, the Beilstein and Gmelin databases which Stanley Miller assumed as a starting assumption of what he might encounter in the Miller-Urey discharge experiment – include all known organic, metalloorganic and inorganic compounds, and list more than 10 million structures and ten million reactions with 37 million factual datasets. Like in cosmology using quantum physics to elaborate the history of the universe, it is difficult to build up a history of the prebiotic world from bottom-up (Fig. 11.1). The bottomup approach in cosmology is impracticable because in the quantum world there would be a nearly unlimited number of possible histories to follow. Therefore, to understand the origin of the universe, cosmology today uses the top-down approach with a high certainty-amplitude following the anthropic principle as a starting point (Dawkins 2004; Hawking and Mlodinow 2010).3 For this reason, it seems unlikely that we would ever be able to choose the right track toward life based on the Beilstein/Gmelin database alone (Fig. 11.1, bottom wedges B, C, D). Luckily, Stanley Miller found in his spark experiment that some compounds, particularly amino acids, were far more abundant than predicted compared to other compounds such as ribonucleotides (Miller and Urey 1959). Richard Egel points out in the introduction to this book, that some predictable patterns may underlie those chemical reactions relevant for prebiotic synthesis (Egel, this book). Fortunately, the biologist’s anthropic approach strongly assists the equally relevant, Miller-bottom-

2

http://www.youtube.com/watch?v¼ONiWmzrmfuY. Here, I underly a most foundational element of Western philosophy as a firm initial seed of our anthropic analysis: Rene´ Descarte’s matured dictum: “Sum res cogitans” (I am a thinking substance”). 3

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Fig. 11.1 The origin of life and the theory of life escorted by the deductive method – toward a multidisciplinary unifying synthesis. (a) Darwinian evolution and the origin of life conceptionalized by following the Popperian deductive cycle (W€achtersh€auser 1997). It assumes that science moves from the current reality to the past particulars (top to bottom) and back to the general (same track bottom to top) – a circling, reductionistic, holistic process without end. The path backward in time is chosen by cladistic means following the Darwinian tree of life. As described in the text, first, Belozersky, Crick, Orgel, Woese, Britten & Davidson and Gilbert concluded that RNA was a primordial molecule of an RNA world. LUCAS was the Last Universal Common Ancestral State of all organisms but not a real individual entity as horizontal material, i.e., gene exchange, was common. LUCAS defines the ancient key molecules and metabolic processes present ubiquitously in contemporary life. Following these key processes from root level back-up the tree promises insight into the mechanisms of evolution and the historic and coincidental realities. Wedges (b, c, d) show non-Life Science approaches toward a theory of the origin of life. They can be seen as Popperian deductive mini-cycles that have the potential to fuse with any other deductive cycle. Stanley Miller’s discharge experiments (c) represent the initial root of this bottom-up approach. Here, scientists look at chemical reactions that formed primeval, biologically relevant molecules. The goal is to analyze the variables leading to the initial Darwinian ancestor (IDA). The relevance of such reactions for the origin of life on the protoearth is then tested experimentally in the laboratory. Geologic and interstellar findings contribute further data of relevance. Between panel (a) on the one hand, and wedges (b, c, d) on the other hand resides the “golden spike”: The “golden spike” is a paraphrase used by Wills and Bada to describe the bottom-up versus the top-down approach (for explanations see footnote 3). The open pentagon at the center represents the hub of interest for all parties. Benner recently noted that any definition

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up approach (Fig. 11.1c). Biologists use the “backwards-in-time-to-ancient-life” method (Fig. 11.1a) in two ways: first, by simply reconstructing phylogenetic trees, and second, by resurrecting old, currently non-functional sequences and putting them into work in the cell in a Jurassic Park-like scenario after they lost or altered function millions of years ago. The latter approach is called paleogenetics (Dettai and Volff 2006; Eigen et al. 1985; Gaucher et al. 2003; Ivics et al. 1997; Liberles 2007; Noonan et al. 2006; Walisko et al. 2008). This biologist’s approach in fact goes back to Charles Darwin. Since Darwin, biologists used the top to bottom approach to take advantage of the strong anthropic principle. They started their analysis from the most complex levels and varieties of contemporary life, that is the human brain on the one hand and modern species diversity on the other. Then, they observed, catalogued and systemized, and subsequently followed down the Darwinian tree of life using all available morphological traits of living and fossil organisms and from ubiquituously conserved molecular building blocks as a means to grasp the emergence of life and its evolving complexity. To this end, the phylogenetic systematics approach is the major quantitative means to reconstruct ancient beginnings (Dawkins 2004; Hennig 1950; Koonin et al. 2000). Chemists and crystallographers continued this anthropic path and by the same concept reached down and explored the most ancient of the molecular living-fossils. This “Latimeria of the molecular living fossils” is the ribosome, which has now been crystallized and its structure was revealed. The work was honored in 2009 with the Nobel prize in chemistry awarded to Venkatraman Ramakrishnan, Thomas A. Steitz, and Ada E. Yonath. Most important for our RNA world concept, however, was Ada Yonath’s

Fig. 11.1 (continued) of life4 must incorporate a “theory of life” (Benner et al. 2011). Vice versa, any theory of life must address the origin of life. A working-definition (Lankenau 2006), influenced by all wedges shown, that is compatible with Benner’s requirements is taken as a basis. This biologist’s, anthropic definition (for those who prefer a different) should at least help initially to focus efforts at understanding: To comprehend the beginnings of life requires that we explain the origin of replication as well as of metabolism synergistically (Maynard Smith and Szathmary 1997). The genetic aspect of the modern definition of life was first proposed by Muller in 1966: “It is to define as alive any entities that have the properties of multiplication, variation and heredity” (Muller 1966). While metabolism supplies the monomers from which the replicators (i.e. genes) are made, replicators alter the kind of chemical reactions occurring in metabolism. Only then can natural selection, acting on replicators, power the evolution of metabolism. The central idea incorporates the need of “instructive genetic information” that can replicate complements. Panel A further depicts the complex and simple three domains of life as discovered by studies on 16S rRNA (Woese and Fox 1977; Woese et al. 1990); Darwin’s tree of life is rooted in the Last Universal Common ancestor (LUCA) that initially existed not as an entity but as the Last Universal Common Ancestral State (LUCAS) (Koonin 2009). Figure in part based on (Benner et al. 2011). The reticulated tree is based in part on (Doolittle 1999)

4

The distinguished Asperger’s Savant Daniel Tammet notes: “Perhaps the most important logical errors to avoid are those caused by not being clear with definitions that we use. This is because careful and effective reasoning depends on precise definitions . . .” (Tammet 2009, p. 251).

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discovery of the perhaps 4 billion year old ancient catalytic pocket. This is an RNA molecule, i.e., a ribozyme, that carries out peptide bond linkages of tRNA-bound, activated amino acids. Ada Yonath and co-workers called it the protoribosome (Davidovich et al. 2009; Yonath 2009a, b, c). The protoribosome is definitely the most ancient of the ubiquitous living molecular fossils. It is real, not just hot air, and with its crystal structure and its catalytic activity we know a whole lot about it. And, what makes the case even stronger: all ribosomes in all organisms on earth use direct descendents of the same ancient ~55 nucleotide long RNA fragment to synthesize peptide bonds in the typical textbook peptidyltransferase reaction. For many scientists this fossil appears to be the closest we can get to the “golden spike”5 using the top to bottom approach (Fig. 11.1). However, stringent theoretical considerations and experimental simulations allow us to push the top-down lead yet a little bit further beyond the golden spike (Fig. 11.1). Actually, we must conclude that there had to be an even older machine in evolution that secured the existence of the translating protoribosome itself. This was a prototype of the modern replication/repair/recombination (RRR) factories that enabled the protoribosome to either self-duplicate or to co-participate in hereditary processes together with other ribozyme species. Only nucleic acids have the property of forming complementarily paired double-stranded strings, a property immediately recognized in Watson and Crick’s original paper: “It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for genetic material” (Watson and Crick 1953b), and shortly later: “The hypothesis we are suggesting is that the template is the pattern of bases formed by one chain of the deoxyribonucleic acid (in the RNA world it is RNA) and that the gene contains a complementary pair of such templates” (Watson and Crick 1953a). Their hypothesis was confirmed by Meselson and Stahl experimentally showing that DNA is duplicated semi conservatively (Meselson and Stahl 1958a, b). I think, that from these ancient times onward to the present time (Fig. 11.1a right upward arrow), the RRR-factory, by its very nature experienced enormous functional, dynamic changes over 4 billion years of evolutionary history. This RRR-factory in all its facets, i.e., adaptations such as meiotic recombination, DNA-repair, transposition, mating-type

5

“The Golden Spike”: Fig. 11.1 shows the golden spike. While the bottom-up method to the origin of life is focused on the creation of some kind of self-replicating entity in the laboratory, starting with simple substances under some approximation of prebiotic conditions and ending up with a structure that has at least some properties of life, the top-down (anthropic) approach will succeed when it is possible to dissect modern life to its essentials, demonstrating the steps by which the elaborate machinery of living cells first appeared. Wills’ & Bada’s metaphor is based on a North American historical event. The building of the transcontinental railroad, the Union and Central Pacific, was one of the great industrial achievements of the nineteenth century. Workers starting out from Omaha, Nebraska, in the east and Sacramento, California in the west met in triumph at Promontory Summit, northwest of Ogden, Utah, on May 10, 1869. There they drove in a golden spike to mark the railroad’s completion. Like those gangs of railroad workers, science is working in two directions toward the origin of life. But rather starting from the east and the west, they are working from the top downward and the bottom upward (Wills and Bada 2000).

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switching, etc. is the core driving force of life’s diversity and it represents the central interest of evolutionary biology attempting to penetrate the causes and mechanisms of evolutionary change (Lankenau 2006; Mayr 1963). This chapter sketches a reasonable, synthetic story of emerging RNA replicators (whose synonymous nature with transposons, viruses, and genes was recognized only later) synthesized from abiotic geo-metallo-molecular ancestors. Figure 11.1. shows that Popper’s deductive cycle has taught us the advantages of starting out from the general (i.e., our human self, and our human complex brains), then moving to the particulars backward in time and back to the general – a circle without end, an anthropic approach – and hopefully, not just hot air about evolution6 that happily will incorporate astro-geo-physicochemical knowledge whenever it is meaningful.

11.2

The Current RNA World as Hint for an Ancient RNA World

11.2.1 The Modern RNA World The concept of RNA as a primordial molecule preceding DNA was hypothesized first by A. N. Belozersky followed by Francis Crick, Leslie Orgel, and Carl Woese (Belozersky 1957, 1959; Crick 1968; Orgel 1968; Woese 1967) (see also Spirin 2005). In the early 1980s, catalytically active RNAs, called ribozymes were discovered (Guerrier-Takada et al. 1983; Kruger et al. 1982). Early on, it was recognized that RNA molecules may play a fundamental role in regulating eukaryotic gene expression (Britten and Davidson 1969) leading to a current paradigm shift in biology (Jordan and Miller 2008), and hooking RNA up with epigenetics. In search of the evolution of how catalytic RNA gave rise to the intron-exon structure of genes, Walter Gilbert coined the term “RNA world” (Gilbert 1986). First, Herbert J€ackle and colleagues showed experimentally that antisense RNA injection into Drosophila embryos produced mutant effects (i.e., “knockdowns”) of the Kr€uppel gene (Rosenberg et al. 1985). In the mid 1980s, studies on the structure and function of Y-chromosomal lampbrushloops of Drosophila hydei lead to the insight that non-coding RNA (ncRNA) may be a transient recruiting tool for proteins functionally essential for fertility of the male sex (Hennig et al. 1989). The Y-loops also encompassed batteries of proviral retrotransposons that expressed an endogenous antisense RNA complementary to part of reverse transcriptase and the full RNase H (Huijser et al. 1988; Lankenau et al. 1988, 1994) (Fig. 11.2). This finding indicated that not only protein storage for the sake of maintaining fertility played a role but that a balanced parasitic/symbiotic immunity control of proviruses may be achieved by specific, endogenous antisense RNAs – a mechanism now

6 Paraphrase on a paraphrase used for a title by Ramakrishnan: The Ribosome: Some Hard Facts about Its Structure and Hot Air about its Evolution (Ramakrishnan 2011).

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Fig. 11.2 Endogenous expression of the micropia-retrotransposon on Y-chromosome loops in Drosophila hydei. (a) Micropia encoded antisense RNA complementary to reverse transcriptase (RT) and RNase H. The antisense RNA is meiotic-germline specific in primary spermatocytes. (b) Schematic nucleus of primary spermatocyte with giant Y-loop transcripts. Th, giant RNPtranscripts termed Threads. (c) Expression and genomic distribution of micropia retrotransposon clusters on Y-chromosome loops pointed toward a molecular immune system (Huijser et al. 1988; Lankenau 1999; Lankenau et al. 1988, 1994). Transposon silencing pathways were subsequently confirmed with Argonaut subfamily Piwi proteins as the central players of such a molecular immune system controlling endogenous transposable elements of all shades (Aravin et al. 2007; Brennecke et al. 2007; Sarot et al. 2004). Red filling and red arrows indicate location of micropia clusters

known as RNA interference (RNAi) (Joshua-Tor and Hannon 2011). RNAi injection into living organisms as a knockdown tool (i.e., gene-silencing) for studying gene function has been used since then.

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Andy Fire then showed experimentally that antisense RNA injected into the nematode Caenorhabditis interfered quantitatively best with complementary mRNA when injected as double-stranded RNA (Fire 2006; Fire et al. 1998). Similar evidence for a recent RNA world had accumulated from gene silencing phenomena in plants. Here, it was known that infection by viruses was combated by a mechanism that involved destruction of viral RNA. Earlier it had been shown that in plants genes could be switched off by copies of homologous genes in the same cell. These two observations were linked by infecting plant cells with a virus that carried a copy of an endogenous plant gene. This endogenous plant gene was subsequently silenced by the homologous transgene within the virus (reviewed in (Matzke and Matzke 2004). All the phenomena are now known to be mechanistically linked. Most importantly, a genetic screen for RNAi-resistant mutants in Caenorhabditis identified the gene rde-1 (RNAi-defective-1) as being essential for knocking down gene function in response to exogenously introduced-double stranded RNA (Tabara et al. 1999). The rde-1 gene turned out to be a homolog of the gene ago1 in the plant Arabidopsis that had been noticed because of developmental mutant flower phenotypes reminiscent of the pelagic octopus Argonauta argo (Bohmert et al. 1998). Sequence alignments of Argonaut (Ago) proteins revealed a wide spread distribution of this family present in archaea (e.g., Pyrococcus furiosus), bacteria (e.g., Aquifex aeolicus) and eukaryotes, the latter split into three protein-clades (reviewed in Tolia and Joshua-Tor 2007). This actually opened a window into a distinct new world – a modern RNA world with reflections into the past. For their discovery of RNAi and gene silencing by double-stranded RNA, Andrew Fire and Craig Mello received the Nobel Prize in Physiology or Medicine in 2006. Argonaute proteins plus 23 nucleotides long dsRNA fragments called small interfering RNAs (siRNAs) are the signature components of an RNA-induced silencing complex called RISC (Song et al. 2004). RISC itself appears to be a programmable sequence-homology search engine that engages in various genomic targeting activities (Fig. 11.3). How does it work? To explain the collaborating key mechanisms of RNA silencing another key factor must be considered: Dicer. Dicer is an RNaseIII-like enzyme that recognizes and digests long dsRNAs. The genomes of dsRNA viruses are good substrates as they are double stranded from the beginning. Messenger RNAs or transcripts like the 770 nt micropia antisense RNA (Fig. 11.2a) naturally fold into low energy secondary structures giving rise to dsRNA stretches. In some organisms, ssRNAs are made dsRNAs through RNAdependent RNA polymerase (RdRP) before further processing. Such long RNAs are the substrate of Dicer, chopping the RNA into double stranded fragments of 21–26 nt length. These small RNAs (siRNA) are then incorporated into multiprotein silencing-effector complexes. Using the sequence information of the dsRNA fragment, these complexes are then guided by some sort of homology search to complementary nucleic acid targets (Fig. 11.3). As the complex is not necessarily bound to other cell constituents, it is likely that it functions as a diffusible, transacting homology signal. The protein composition of the complex varies and depends on the nature of the target sequence. RISC is just one example of different effector-complexes, with different types of silencing possible (Matzke and Birchler

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Fig. 11.3 The diffusible homology search engine and silencing-effector complex RISC, assembly and function (Homo sapiens). Modified after Gregory et al. (2005). Successive steps indicated by numbers 1 through 8. (1) The microprocessor protein complex comprised of the dsRNA binding domain (dsRBD) protein (Pasha ¼ DGCR8) and the RNAse III protein Drosha recognizes a primary miRNA transcript. Red arrows, microprocessor cleaves specifically the base of stem loop RNAs. A precursor-micro RNA of ~60–70 nt is released with 2 nt single stranded 30 overhang. (2) Transport through nuclear membrane to cytoplasm. Recognition by RISC including Dicer-RNaseIII, Ago-RNase H and double-stranded RNA binding protein TRBP. (3) Dicer cleaves ~22 nt by 2 nt staggered cuts with ~22 nt duplex miRNA remaining bound to the RISC RNP complex. (Note the similarity to the nuclease reactions of retroelement integrases & RNasesH). (4) Guide strand identification in dsRNA for homology search and strand separation. (5) Homology search engine, where the guide strand is kept bound within the RISC. (6) Target recognition. The guide miRNA fragment searches cytoplasmic mRNAs for homology and directs RISC to a complementary sequence. (7) Crooked red arrow, Ago-RNase H endonucleolytically cleaves the mRNA, destroying it partially for posttranscriptional silencing. (8) Cleaved mRNA is released. The RISC homology search engine can engage in a new cycle of mRNA hunting and destruction. According to Gregory et al. (2005), no energy is needed for any of the ssRNA release steps

2005). In the late 1980s and early to mid 1990s, my wife and I worked on the Y-chromosome lampbrush loop of Drosophila hydei. The Y-chromosome is heterochromatic in all tissues and developmental germline stages except during the prophase of meiosis in the male germline. During the primary spermatocyte stage, it forms so-called lampbrush loops (Y-loops) that express giant transcripts of a size of 2,000 kb (Hennig et al. 1989). In addition to the micropia antisense RNA (Fig. 11.2a) mentioned above, we discovered perplexing expression patterns of various transcripts cross hybridizing with single stranded probes detecting micropia sequence-similarity (Lankenau 1996). Figure 11.2b, c shows a graphic summary of the results. Based on early data from prokaryotes and eukaryotes, we

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speculated that the giant transcripts and micropia-embedded transcripts might be subject to RNA degradation representing a kind of molecular immune system against transposable elements (Lankenau 1996). Unfortunately, these Y-loops and the antisense transcript were specific of Drosophila hydei, which is not a good genetic model system, such that we were unable to pursue further functional analysis. Today, we know that RNAi can mediate heterochromatin assembly in the presence of transposon clusters. Volpe and colleagues discovered the involvement of the RNAi machinery in histone methylation and heterochromatin formation at centromeres of fission yeast (Volpe et al. 2002). In yeast, tandem repeats or multiple copies of transposable elements in heterochromatin generate dsRNAs or ssRNAs that can be transcribed to dsRNA through the activity of RdRP. The dsRNA is then cleaved by Dicer producing small interfering RNAs (siRNAs). These siRNAs associate with histone methyltransferases (HMTs) that together represent a silencing-effector complex termed SHREC. In their function to guide the complex to a target RNA or DNA sequence, the siRNAs guide the HMTs to the centromeric chromatin to methylate histone H3 on lysine 9 (H3K9). The modified H3 is then bound by Swi6 or heterochromatin protein HP1, which also associate with methyltransferases, to maintain a silenced state of heterochromatin (for review, see Matzke and Birchler 2005). This RNAi silencing is mediated by SHREC which appears to regulate nucleosome positioning to assemble higherorder chromatin structures critical for heterochromatin functions (Sugiyama et al. 2007). This example demonstrates how deeply the modern RNA world is embedded in the physiology and metabolism (chromatin controls gene expression -> gene expression controls the quantity and content of enzymes etc., -> this controls the making of new metabolites) controlling and penetrating the endogenous integrity of all modern organisms. For example, genome integrity is also modified by RNAimediated pathways. In the ciliate Tetrahymena thermophila during the process of germline-soma differentiation, RNAi mediates DNA elimination of a chromosomal fragment called internal eliminated segment (IES) (Mochizuki and Gorovsky 2004). In other diploid species, RNAi dependent silencing of unpaired genomic regions takes place during meiosis (Shiu et al. 2001). In Neurospora crassa, this mechanism requires RdRP and an Argonaut like protein distinct from “normal” RNAi in this organism (for review, see Matzke and Birchler 2005). During meiosis in the filamentous fungus Ascobolus immersus DNA methylation can be transferred from one allele to another in a pattern typical of gene conversion, indicating the involvement of DNA-DNA pairing (Colot et al. 1996). In summary, RNA plays a crucial role in epigenome regulation and epigenetic inheritance. Today, the prominent role for small RNAs, RNAi, and the multiprotein silencing-effector complexes as diffusible homology search engines in mediating sequence specific silencing at genome levels has been broadly established. The origin of these ubiquitous mechanisms, however, remains to be worked out as it may provide a window into an even more ancient RNA world directly linked to the geochemical origin of replicators, genes, and life itself. As a PhD student, I ran into this modern RNA world by discovering the Drosophila genus-specific micropia retrotransposon family and their perplexing transcripts (Lankenau et al.

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1988). Knowing that the genomes of most eukaryotes consist of significant amounts of transposable elements and other non-coding sequences (Davidson 1986), where the human genome consists of 50% transposable elements and 98% of non-protein-coding DNA (Venter et al. 2001), the idea rose that micropia must be involved in intragenomic molecular immune responses. In addition, the unidirectional tandem repeat organization of micropia (Fig. 11.2c) also pointed to homologous recombination and other DNA repair mechanisms responsible for amplifying a transposon in a way that established the unidirectional tandem patterns (i.e., Synthesis Dependent Strand Annealing, SDSA (Fig. 11.8b) (Lankenau 2006; Paques et al. 1998)). In this context, I am thankful for a note by Matzke and Birchler (Matzke and Birchler 2005) recognizing the intermingled, ancient symbiotic/parasitic correlation of transposons where I cannot better do but to quote: “Consistent with the role of RNAi in defense against invasive sequences (Matzke et al. 2000; Vastenhouw and Plasterk 2004), transposable elements and related repeats are preferred natural targets of the RNAi-mediated silencing pathways in the nucleus. Although often considered solely as molecular parasites, transposon sequences that function as foci for RNAi-based chromatin modifications benefit the host through these mechanisms by contributing to gene regulation and to chromosome structure and function.” Recent, more general theoretical work further underscores this statement (Branciamore et al. 2009) and its spirit leads us directly to address the question of the origin of transposons and to follow the Popperian deduction further top-down (Fig. 11.1). Another modern, prominent example of the RNAi world relates to the human X-chromosome. One of the two human female X chromosomes is transcriptionally silenced in every cell by ncRNAs. X-inactivation is controlled by Xist and Tsix, two non-coding genes of antagonistic function. The Xist gene produces a 17-kb-long non-coding RNA that localizes along one of the two female X chromosomes and triggers chromosome-wide silencing X-inactivation by coating the inactive X. Tsix RNA is transcribed in antisense to Xist, and is critical for the labeling of the active X-chromosome through cis-expression of Xist RNA accumulation. It has been proposed that LINE-1 retrotransposons (L1) serve as DNA signals and mediate the spreading of the X inactivation signal along the chromosome. Apparently, a subset of L1 elements in the X chromosome is enriched that were active less than 100 Ma ago (Bailey et al. 2000; Lyon 2000). Thus, the X-inactivation center (Xic) represents an idiosyncratic RNA world of gene-dosage compensation that evolved about 50–200 Ma ago in Eutherian mammals (Chaumeil et al. 2006; Chow et al. 2005; Lee 2011; Navarro and Avner 2010; Ng et al. 2007). From the human perspective, it appears to be very old. However, in terms of geologic deep time, the X-inactivation center is only a relative recent evolutionary novelty. It may encompass primordial mechanisms but for a first anthropic top-down causality in the Popperian deductive cycle, it would be necessary to find evidence for an ancient RNA world. The X inactivation world is not yet far enough explored to tell us anything. What could a

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relict from the primordial beginnings of an old RNA world within Xic or any other RNAi related process7 look like? What shall we call young and what is old? Let us briefly address the latter key question. Since a long time, we know about one component of the modern RNA world that is actually very old and dates back to an old if not very distant ancient RNA world – a true molecular living fossil that likely is conserved within all life for more than 3.5 Ga. Such a molecular living fossil in fact exists. It is a constituent of all life’s genomes: the ribosome mentioned in the introduction. As long as none of the modern RNA world constituents – and with modern I mean younger than 3.5 Ga (Giga years ¼ billion years) – can be causally connected to the times the very old ribosome emerged (~ 3.5 Ga) this modern transposon/RNAi RNA system world like the Xist/Tsix world, mentioned above, could in theory have evolved de novo from scratch. 4.3 Ga ago, the earth had cooled enough such that water had precipitated. We know this from zircon crystals whose age could be determined using radioisotopic methods. ZrSiO4 only builds crystals in the presence of water. Zircon crystals from the Murchison district in West Australia were dated using the 18O-isotope and provided evidence for liquid water at the Earth’s surface 4.3 Ga ago. Around this time, the astro-geophysical parameters were mild enough such that primeval synthesis of life could have started. We then have to ask, what is ancient from the modern RNA world that could still exist and is detectable in modern cells and genomes in addition to the ribosome? Fortunately, there are numerous links that connect RNAi systems such as Xic (via LINE elements) to the primordial world. First, Argonaut proteins belong to an ancient family! They are related to the RNase H clade of enzymes typical of retroviruses and LTR-retrotransposons. At their core, Argonaut proteins have the typical RNase H fold and the two conserved aspartates invariably present on flanking b-strands of retroelements (Joshua-Tor and Hannon 2011). Above, we learned about RISC as an example of a silencing-effector complex and its role as a homology search engine. Among other functions, the most conserved function of Argonaut proteins is endonucleolytic cleavage of a target sequence (Song et al. 2004) similar to the activity of RNase H during reverse transcription-replication of retrotransposons, retroviruses, and other retroelements (Kohlstaedt et al. 1992). The Argonaut-RNase H activity creates a 50 product 30 OH and a 30 product carrying a 50 phosphate, where the target DNA strand is generally replaced by a guide RNA. The RNase H family is ubiquitously conserved in all domains of the tree of life consisting of well-characterized enzymes such as the integrases and transposases of DNA-transposons and retroelements (Nowotny 2009). While Escherichia coli RNase H1 catalyzes a single reaction resulting in substrate cleavage, integrases, and transposases catalyze two consecutive reactions such as during DNA transposon integration events resulting in strand transfer (Rio 2002). The nucleophile in these reactions is a water molecule or the 30 -OH of a nucleotide. Involved is a two-metal ion catalysis mechanism, with one metal activating the nucleophile and the second

7 Ubiquity of proteins or genes or ncRNAs in all organisms means that they are likely of primordial universality.

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stabilizing the intermediate (Nowotny 2009). Crystal structures of substrate-bound complexes of the transposon Tn5 transposase and of human and Bacillus halodurans RNase H1 revealed the involvement of two metal ions likewise (Lovell et al. 2002; Nowotny et al. 2005, 2007). So far, the participation of one metal ion has been confirmed for Argonaut protein structures as well as the two invariant aspartates and a conserved histidine critical for catalysis (Joshua-Tor and Hannon 2011). Also Piwi clade proteins possess conserved catalytic activity, and are supposedly active, based on their conserved mechanism of transposon recognition and silencing (Faehnle and Joshua-Tor 2007). Argonaut-RNase H active proteins also participate in processing of human immuno deficiency virus (HIV) transcripts (Fig. 11.3). HIV is the most well studied retrovirus and we can learn a lot from its cellular functions and its association with ancient RNA world processes. A silencing-effector complex containing Dicer, Argonaut and HIV-tar-RNA binding protein (TRBP) is able to guide-strand loading and multiple rounds of target cleavage, stimulated by nucleotides (Gregory et al. 2005). This example together with Julius Brenneke’s ping-pong model (Brennecke et al. 2007), where transposon transcript abundance is kept under control involving Ago3 complexes, piwi-interacting RNA (piRNA) cluster transcripts, and transcripts of active transposons, may turn out as the explanation for what is going on with the micropia Y-loop transcripts in primary spermatocytes (Fig. 11.2), and may further give the explanation for why and how these unusual Y-loop structures evolved after all. Probably their existence is a sort of exaptive molecular drive involving the RISC-machine and invasion-control of the micropia family. RNA world derived defense strategies against mobile genetic elements have now also been characterized in prokaryotes and compared to RNAi in eukaryotes (Jore et al. 2011; Shah and Garrett 2011).

11.2.2 Ribonucleases: RNaseH and Integrase Meet Reverse Transcriptase The evidence is overwhelming for a contemporary RNA world being a central element of living cells and cell differentiation. The major signatures that underscore the importance of RNA in modern cells are: (1) transcription into mRNAs from DNA genes; (2) protein translation from mRNAs mediated by tRNAs in a ribosome with 23S or 28S rRNA as catalytic ribozyme; and (3) we now know that gene and chromosome regulatory control mechanisms involve a full panoply of RNA associated activities that even tightly pervade metazoan germline processes (Lankenau 2007, Box 1). Now, why do we think RNA is relevant for the origin of life? Would protein or DNA not suffice? Did DNA or proteins precede RNA in the primal synthesis? The answer seems “no” for the following, selected, rational arguments:

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Anthropic arguments: 1. RNA is both a biocatalyst and an informational molecule. Thus both, genotype and phenotype are a feature of RNA. Protein on the other hand has extremely limited ability to transmit information (as in prions). 2. DNA has an excellent ability to transmit information but it lacks the biocatalyst property in modern cells. 3. RNA is capable of replicating itself and can perform the chemistry needed for RNA replication (Cech 1986). 4. DNA replication in modern cells always needs the 30 OH end of an initial, independently synthesized RNA primer. 5. The ribosome ubiquitously uses the catalytic activity of RNA to perform peptide synthesis. 6. RNA preceded DNA, because in modern organisms the biosynthesis of deoxyribonucleotides is by reduction of previously synthesized ribonucleotides. Two additional enzymes are needed to make DNA from ribonucleotide precursors: ribonucleotide reductase and thymidylate synthase. Bottom up arguments: 7. Prebiotic synthesis of ribose appears simpler by alkaline aldolcondensation than to think of the synthesis of deoxyribose from scratch. 8. The nucleophilicity of the 20 , 30 OH-groups is higher than of the 30 OH group of deoxyribose (Lohrmann and Orgel 1977). 9. It is more parsimonious to think of a single type molecule (i.e., RNA) replicating itself than to imagine that two different molecules (e.g., random peptide plus nucleic acid) were synthesized by random chemical reactions at the same time in the same place – and that repeatedly for only by redoing the trick over and over again a new individual entity would emerge.8 In summation of our current deductive top down analysis, we can so far ask if there is a preliminary unifying theme from the modern RNA world that might unite all three domains of life (i.e., bacteria, archaea, eukaryota) with LUCA at its root? As RNA polymerization is the basic requirement for any cellular RNA world, it is not surprising that two proteins are ubiquitous in all living organisms: DNAdirected RNA polymerase, subunits a, b, b0 responsible for the transcription of genes and transcription antiterminator NusG (Charlebois and Doolittle 2004; Koonin 2003) (not further elaborated here). The second conserved topic is that of two key players of RISC, i.e., two families of RNases (Fig. 11.3). The first is represented by Drosha and Dicer which belong to the RNase III enzyme family. RNase III enzymes specifically bind to and cleave dsRNA. There

8

This statement must not be confused with the assumption that the first RNA replicators emerged in sterile environments. There always must have been organic and anorganic compounds coexisting with any informational RNA molecule. Peptide replicators are possible (Ashkenasy et al. 2004). They possibly have coexisted and facilitated RNA relicators from begin on. For an emergence scenario encompassing systemic properties, see Dyson (1999); Kauffman (1993).

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are three classes of RNaseIII: Class 1 process precursors of ribosomal RNA and in fungi small nuclear RNAs (snRNAs). Class 2 comprises Drosha with rRNA and miRNA processing functions. Class 3 includes the Dicer family involved in RNAi. Especially interesting is the processing aspect of 12S pre-rRNA pointing toward an ancient pathway (Fukuda et al. 2007; Redko et al. 2008). The second ubiquitous RNase enzyme activity in the modern RISC RNA world however is that of the Argonaut proteins, which are related to the RNase H clade. As mentioned above, Argonaut proteins have the typical RNase H fold and the two conserved aspartates invariably present on flanking b-strands like RNase H encoded by LTR retrotransposons (Joshua-Tor and Hannon 2011). Even more prominent is the structural similarity to integrases and DNA transposon transposases, and the Double Holliday Junction processing protein RuvC, the latter providing a link to DNA repair (Yang and Steitz 1995). However, there appears yet another even more crucial link to that stage of LUCA, where RNA started to be transcribed into DNA. Modern retrotransposons and retroviruses represent this transition of an RNA world into the DNA world. Retroviral RNaseH activity in retroviral replication is tightly connected to reverse transcriptase (RT) activity (Kohlstaedt et al. 1992). Let the HIV RT protein serve here as the role model for other retroviruses and retrotransposons. RT is carved out from the pol gene product as a 66 kD polypeptide. A 66 kD peptide includes both RT and RNaseH functions. Two 66 kD monomers aggregate to form a dimer, where both monomers have slightly different conformations. One is susceptible to proteolytic digestion by the retroviral protease such that a RT/RNaseH heterodimer is released with a 66 kD unit containing RT/RNaseH and a 51 kD unit with a second RT subunit (Arnold et al. 1992; Huang et al. 1998). The p66-p51 heterodimer has one RT polymerase active site, one RNaseH active site, and one tRNA binding site. This intricate interrelationship between RT and RNaseH reflects an intriguing replication mechanism that likely dates back to the times of LUCAS. The most interesting part linking retroids9 to RISC are the intramolecular and intermolecular strand exchange reactions during reverse transcription (details see below). As the RNaseH is always an integral part of homology-dependent strand transfer reactions, we may speculate that the RISC homology search machine including Argonaut-RNaseH proteins was evolutionarily derived directly from the RT/RNaseH driven mechanism (Fig. 11.5).

9 All elements and sequences encoding a reverse transcriptase: retroviruses, retrotransposons, group II introns, LINEs, plus-strand RNA viruses, pararetroviruses, retrons.

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11.2.3 Reverse Transcription: An Ancient Mechanism of Replication Retrotransposons and retroviruses are rather autonomous folks that personify idiosyncratic toolboxes of replication. How do retroids replicate? The proviral structure of micropia representing a typical modern RNA world LTR-retrotransposon is shown in Fig. 11.4 (Lankenau et al. 1988). Initially, an RNA polymerase, guided by upstream enhancer bound transcription factors, binds to a promoter such as the classical TATA box. Without the need for the 30 OH end of a primer, it starts transcription about 28 nucleotides upstream. Typically, the retrotransposon transcript terminates after about 5 kb at a AA(G)TAAA termination signal – still inside the boundaries of the proviral DNA sequence entity. As transcription started from a transposon internal promoter, and terminated at the stop signal well before the 50 end of the transposon this transcript by no means represents the full-length boundaries of the transposable element. And yet, this transcript – an incomplete copy of the transposon – is the only source to restore a complete, new copy of the proviral DNA fragment. How is that possible? How can an incomplete RNA fragment restore a complete DNA provirus? The trick is that the mRNA10 encompasses all sequence information necessary to restore the full length proviral DNA entity which sooner or later may integrate at a different site in the “host” genome. It also contains a sequence fragment called R duplicated at its opposite ends. Retroviral replication is complex. Figure 11.5 shows the mechanism of reverse transcription as combined from several sources (for review, see Telesnitsky and Goff (1997), Varmus and Brown (1989), Voytas and Boeke (2002)). Both Figs. 11.4 and 11.5 represent structural and mechanistic details of the replication steps mediated by RT/RNaseH and, because these two proteins can be seen as a homology search engine like the RISC complex, they represent a key to the modern RNA world as well as to an ancient RNA world. Figure 11.5 is simplified. In vitro experiments suggest that an intact virion core is required for efficient elongation and cis or trans strand transfers. There is evidence of limited DNA synthesis in HIV-1 virions prior to the entry of the virion into a target cell. But reverse transcription generally appears to be activated by entry of the viral core into the cytoplasm of the target cell (Telesnitsky and Goff 1997). As endogenous LTR retrotransposons or endogenous retroviruses such as the insect gypsy retrotransposon form virions or virus like particles (VLPs), there are many variations in detail in the timing and location of reverse transcription processes (Adams et al. 1987; Flavell and Ish-Horowicz 1983; Garfinkel et al. 1985; Kikuchi et al. 1986; Mellor et al. 1985; Shiba and Saigo 1983; Song et al. 1994).

10

The long mRNAs of retrotransposons are better called full length RNAs. There may be epigenetic differences between the two.

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Fig. 11.4 Structure and priming model for micropia Dm11 of Drosophila melanogaster. (a) Nucleotide sequence of 30 LTR. The putative order of U3, R, and U5 sequences are colored green, red, and blue respectively. Putative upstream elements relative to TATA box are indicated. (b) Genome organization with encoded genes. LTR regions colored as in A. (c) tRNA priming model, data based on (Lankenau et al. 1988)

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Fig. 11.5 Mechanism of reverse transcription and synthesis of proviral DNA. The processes are drawn as linear cis and trans primer strand-transfer reactions; circular cis-reactions are possible as well. The top and bottom drawings represent the proviral, genomic DNA-integrates of a LTRretrotransposon with gag, protease, RT, RNaseH, and integrase genes – before and after a reverse transcription driven replication cycle. Between the top and bottom proviral schemes there are numerous cell physiological steps not shown, including formation/unpacking of virus like particles (VLPs or virions) and the cytoplasmic processes where replication takes place. (a, b) Two

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Once a full length “mRNA” has been produced,11 a host-endogenous tRNA is binding by complementary base pairing to the 50 tRNA primer binding site (Fig. 11.5a). This initiates the DNA polymerization steps of RT and the RNA degradation steps committed by RNaseH reminding us of the RISC-RNAi shredder mill. The most significant aspect of the replication of a complete copy of a provirus from an incomplete retroid mRNA however is the use of redundancies and unannealing, strand transfer and annealing reactions. In the modern RNAi world, this is performed by the RISC “search engine” (Fig. 11.3), for retroid replication it is performed by RT/RNaseH (Fig. 11.5c, d and g, h). Interestingly, the modern DNA world encompasses a similar genome wide homology search program as part of homologous recombinational DNA repair-reactions using the synthesis dependent strand annealing pathway (Engels et al. 1994; Lankenau 1995). The SDSA DNA repair homology search of modern organisms however faces the far more byzantine world of modern chromatin factors. SDSA will only sporadically be mentioned here (see section “Group I Introns” and Fig. 11.8b) (Lankenau et al. 1996, 2000). The HIV RT/RNaseH search engine serves as a prime model system here as well. Recently, with the aid of smFRET techniques it was possible to understand the fundamental molecular-dynamics of the homology-search at single-molecule resolution (Abbondanzieri et al. 2008; Liu et al. 2008, 2010; Tinoco et al. 2011). This work shows that on a DNA template, RT/RNaseH binds to the template with a DNA or RNA primer in two opposite orientations. Either the DNA polymerase domain or the RNaseH domain are located close to the 30 end of the primer. This is what would be expected from the activity of RT/RNaseH functioning in ä Fig. 11.5 (continued) molecules of full length, poly adenylated mRNA are present within or released from the VLP or virion. The 30 OH end of a host tRNA anneals to one mRNA molecule and primes minus-strand DNA synthesis. RT synthesized until the 50 end of the full length transcript generating a short DNA fragment on the order of 100–150 nucleotides length. This fragment is termed the minus strand strong stop DNA (()ssDNA). (c) Accompanying DNA synthesis the RNaseH activity of RT/RNaseH degrades the mRNA fragment already copied into ssDNA. (c, d) The ssDNA contains the repeated (R) sequence that is occurring at both 50 and 30 ends of the mRNA. The first strand transfer (here hypothetically shown as mediated by RT/ RNaseH working as a homology search engine) leads to the annealing of ()ssDNA with R either on the same mRNA molecule in cis or on the second mRNA molecule in trans. (d, e) RT/RNaseH synthesize the full mRNA and simultaneously degrade all mRNA except a short purine rich fragment (prr) (f). (g) The 30 OH end of the prr RNA primer serves RT/RNaseH to start plus strand DNA synthesis resulting in plus strand strong stop DNA ((+)ssDNA). In a second homology search process the (+)ssDNA fragment translocates and anneals to the opposite DNA end on redundant LTR sequences (h). (i) Plus strand synthesis completes production of a full double-stranded LTR retrotransposon ready to be integrated into the genome with the aid of the self-encoded integrase. Abbreviations: ssDNA, minus- and plus-strand strong stop DNA; prr, purine rich region; cDNA copy DNA; Open arrows and stippled lines: Intra- or intermolecular translocation of complementary cDNA. (Diagram based on: (Peliska and Benkovic 1992; Telesnitsky and Goff 1997; Temin 1993; Varmus and Brown 1989; Voytas and Boeke 2002))

11

The many splice variants and RNA editing is not further elaborated here.

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DNA-directed DNA synthesis (e.g., Fig. 11.5h, i) or DNA directed RNA hydrolysis (Fig. 11.5e, f). On chimeric DNA/RNA primers, RT was observed to occupy both the DNA polymerase competent and RNaseH-competent orientations. The measured rate of primer extension correlated with the fraction of time for which the RT enzyme bound in the polymerase competent orientation. Obviously, RT/ RNaseH distinguishes between different substrates. The HIV genome is ~10 kb long. Therefore, RT, with its low processivity in DNA synthesis (only a few to a few hundred nucleotides on a ~10 kb HIV genome) must accomplish location to its target site very efficiently using a highly capable searching mechanism crucial for RT function. With smFRET, it was demonstrated that RT/RNaseH slides thermally driven between two ends of an experimental substrate even if longer than ~20 nt. Once RT/RNaseH snapped into place at the catalytic target site with atomic precision, it can flip its binding orientation to orient the correct functional domain (DNA polymerization or RNA hydrolysis) close to the target substrate. Thus, the homology search engine combines trembling sliding motions and flipping (Abbondanzieri et al. 2008; Liu et al. 2008, 2010). This key mechanism likely helps to explain the differences of cis- versus trans- genome wide homology searches accompanying recombinational SDSA repair (Engels et al. 1994), but as said before, homology search engine processes and the byzantine organization of chromatin must be viewed on an equal footing (Lankenau 1995; Lankenau et al. 1996).

11.2.4 The Retroelement Ancestor Hypothesis 11.2.4.1

“Linne´’s Revenge” – or the Attempt to Classify Transposable Elements

What is Linne´’s Revenge? Attempting to classify transposable elements of all shades confronts us with similar problems today as Carolus Linnaeus was confronted with when writing his famous Systema Naturae of 1758. Even though Linne´ was not aware of our current understanding and the logics of phylogenetic systematics, his invention of the binomial nomenclature of species nevertheless reflected most aspects of today’s accepted tree of life. While it is the goal of modern systematics to define strictly circumscribed monophyletic groups (Hennig 1950, 1966), Linne´, unknowingly, established polyphyletic and paraphyletic groups that did not necessarily reflect monophyletic common decent. Today, the same holds true for molecular entities. For example, standard classifications of viruses into groups such as Herpes, HepaDNA, Adeno-, Retro-, Onco-, Lentiviridae, etc. do not reflect phylogenetic relationships but rather represent a grouping based on chemical, physical, physiological, or medical similarities. Such classifications are termed paraphyletic and polyphyletic of typological classification systems (Hennig 1966; Lankenau et al. 1988). The problem grows when approaching more and more ancient taxa. Popper’s deductive cycle in Fig. 11.1a circles around the phylogenetic tree of life reflecting many horizontal gene exchanges between different clade-

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branches, especially at the base near LUCA (Doolittle 1999, 2000). With regard to retroelements, it has become clear that between classifications of transposable elements based on the RNaseH or on the RT or on capsid proteins or on the integrase or on protease, there are large inconsistencies (Capy et al. 1998). Here, I have no space to elaborate on the primordial phylogenetic processes and I like to refer to the excellent analyses of Eugene Koonin and colleagues (Jordan et al. 2005; Koonin 2003, 2006a, 2006b, 2007, 2009; Koonin et al. 1980, 2000, 2006; Koonin and Martin 2005; Koonin and Novozhilov 2009; Koonin and Wolf 2008; Leipe et al. 1999). Thus, because the ancient “evolutionary temperature” (Woese 1998), as reflected by frequent horizontal gene exchanges between replicating entities, was very high in the early beginnings, any attempt to root the trunks of the three domains of life, i.e., bacteria, archaea, and eukaryota, appears to be futile. This matter becomes even more tricky if we accept the promiscuity of many proteins acting in historically totally unrelated biological functions and phenomena (Khersonsky et al. 2006). Stephen Jay Gould coined the concise term exaptation, that is, recruitment of an existing structure or function for a new function by unrelated selection forces (Gould 2002). The transposon/virus-like stage in life’s early evolution belongs to the same kind of solutions and might be the most plausible if not the only way to avoid what Koonin termed “irreducible complexity trap” associated with the origin of cellular organization itself and subsequently of primordial entities representing LUCAS (Koonin 2009). To this end, let us get a feeling for the RNA world of retroids and other transposable elements. Many phylogenetic trees have been published on retroid and DNA transposon clades. Neither here can I elaborate fully on this issue and I like to refer to some excellent books for reference (Berg and Howe 1989; Capy et al. 1998; Coffin et al. 1997; Cooper et al. 1995; Craig et al. 2002; Fedoroff and Botstein 1992; Sherratt 1995; Skalka and Goff 1993). Figure 11.6 shows a phylogenetic tree of retroids representing all domains of life based on their RT amino acid sequences. The tree is outlined at two scales. It reflects the pseudo-phylogenetic relationship between RT of the major groups of retroids. It also represents an analysis of the sequence distance of the RT of the primordial Penelope retrotransposon to all major retroids as an outgroup. Penelope represents an average similarity distance to RT of all other retroids of about 14%. The analysis indicates that even though all RTs may share a common ancestor – probably at the time of LUCAS – the evolutionary change of sequences was so significant and sequence stability was compromised by horizontal transfer and xenologous recombination12 (because evolutionary temperature was hot) (McClure 1991, 1993), that we cannot really identify a defined common ancestor among retroid elements. LUCAS therefore is more likely the stem group of the three domains of life with no need to identify an individual ancestor. With this insight, we are getting close to the golden spike of Fig. 11.1 in the top-down approach. But before jumping to the bottom-up path, one of the major retroids

12

Replacement of a resident gene by a homologous foreign gene.

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Fig. 11.6 Sequence relationship of RTs et al. between retroid elements. The pseudo-phylogenetic tree is based on the analysis of Xiong and Eickbush and own data. The tree uses the RT of the

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seems to give us a lead anyway hinting toward mechanisms that circumscribe the molecular conditions at the origin of modern RNA replicators (retroids) and subsequent DNA genes. Penelope already is a very unusual retroid element with links to all retroid groups, but the Group II introns possess ribozyme catalytic properties that strongly help understanding the molecular events at the LUCAS stage.

11.2.4.2

Mobile Introns

For many years, the established term “gene” was dominated by Beadle’s and Tatum’s “one gene-one enzyme” concept (Beadle 1958). The discovery of split genes, i.e., genes that contain introns being removed from primary gene transcripts, then came as a surprise (Sharp 1985). It was even more surprising that RNA transcripts triggered their own excision of introns. Now, the splicing mechanism points toward an ancient origin deep within the realm of LUCAS. Introns are ribozymes that splice by three fundamentally different pathways. Group I introns splice by the guanosine-initiated pathway. Group II introns and the related group III introns splice by the lariat pathway. Introns found in rRNA and tRNA genes of Archaea use the nuclease-ligase pathway (Belfort et al. 2002).

Group I Introns Group I introns are common in eukaryota and bacteria but do not occur in archaea. Intron mobility refers to two mechanistic types of mobility. The first is intron homing that actually is a DNA associated gene conversion process making use of a fundamental recombinational double strand break (DSB) repair reaction called synthesis dependent strand annealing (SDSA) (Gloor and Lankenau 1998; Haber 2008; Lankenau 1995, 2006; Lankenau and Gloor 1998; Nassif et al. 1994). A homing endonuclease is encoded within the group I intron of a host gene. After transcription of that gene with the intron still present in the primary RNA transcript, the 30 -OH group of an exogenous guanosine cofactor triggers a cascade of transesterification reactions splicing out the intron and self-ligating it to a full-length intron circular RNA molecule (Nielsen et al. 2003). Homing endonucleases like the I-CreI homing mega-endonuclease of Chlamydomonas are often used in ä Fig. 11.6 (continued) ancient Penelope retrotransposon of Drosophila virilis as an outgroup for comparison (Evgen’ev et al. 1997). Amino acid sequence identities between the RT of Penelope and other major retroids are shown. Sequence similarities of the putative Penelope RT to all other known categories of RT sequences is, on an average, about 14%. The alignment of RT sequences therefore is based upon groups of conserved amino acid residues that can be identified in published RT-like sequences available (Xiong and Eickbush 1988, 1990). Alignments were carried out with the program MultAlin (Corpet 1988). The groups of conserved amino acid sequences are as defined (Xiong and Eickbush 1990). Sequences of Ulysses, and micropia were taken from published data (Evgen’ev et al. 1992; Huijser et al. 1988; Lankenau et al. 1988)

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Fig. 11.7 Structure of group II intron RNA and the splicing mechanism. (a) Secondary structure of a group II intron (Perlman and Podar 1996; Schmidt et al. 1996). EBS exon binding site, IBS intron binding site; dotted curved lines link complementary binding sites. (b) Transesterification and self splicing mechanism producing lariat RNA. See Fig. 11.8c for group II intron retro homing and SDSA of the lariat molecule

experimental gene targeting systems (Lankenau 2006; Lankenau et al. 2003). The circular RNA molecule can be translated into the mega nuclease (Perriman and Ares 1998). The endonuclease then specifically produces a staggered double strand break within an intronless copy of the host gene. Using the intact, complementary host gene sequences as template, the DSB then is repaired via SDSA gene conversion with donor DNA that still contains the original group I intron DNA (Belfort et al. 2002). The group I introns therefore represent the very dynamic phase of the LUCAS employing recombinational mechanisms that mediate between the ancient RNA world and the modern DNA world.

Group II Introns The second mechanistic type of intron mobility is represented by group II introns. They occur in 25% of the genomes of eubacteria, in mitochondrial and chloroplast genomes of fungi and plants but not in the nuclear genomes of eukaryotes nor in archaea. Like group I introns they are ribozymes as well. However, group II introns do not self-splice without co-factors such as intron-encoded and/or host-encoded splicing factors. These include reverse transcriptase, maturase,13 RNaseH, and endonuclease. The open reading frame (ORF) of these genes are always inserted in domain IV of the group II intron (Fig. 11.7a). Sometimes the ORF extends 50 to

13

Many self-splicing introns code for maturases that help with the splicing process, generally only the splicing of the intron that encodes it.

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form a continuous ORF with the upstream exon. The length of the group II introns are as long as 3,000 nucleotides. The splicing mechanism is shown in Fig. 11.7b. Like group I introns, group II introns possess no conserved sequences at base level. Conserved sequences occur at the intron boundaries, with GUGYG and AY14 as consensus sequence at the 50 and 30 ends respectively. Domain double helix VI contains a buldged adenosine ribonucleotide which defines the branch site of the lariat structure. The free electron pair of the 20 -OH of the buldged A initiates a nucleophilic attack on the 50 splice site, producing a 20 –50 linkage and the lariat RNA structure still remaining attached to the 30 exon. The free pair of electrons of a 30 -OH group at the 30 end of the free 50 exon attacks the 30 splice site resulting in exon ligation and lariat RNA release. The exact recognition of the splice sites is mediated by defined complementary base pairing interactions between intron binding sites (IBSs) and exon binding sites (EBSs) and between d and d0 (Fig. 11.7a). Figure 11.6 shows that group II intron RT is closely related to non-LTR retrotransposons – of which one representative, L1, makes up 5% of the human genome. The mechanism of transposition of LINEs within DNA genomes is related to that of group II intron retro homing (see color plate 47 in Craig et al. 2002; Belfort et al. 2002; Luan et al. 1993). The mechanism of retrohoming involves retrotransposition of the intron lariat RNA into genomic DNA. Details of the mechanism were explored in yeast and in bacteria (Belfort et al. 2002). The protein encoded within the ORF (Fig. 11.7a) is translated after transcription of the premRNA and before splicing sets in. It then binds to the intron and induces RNA splicing with the aid of its maturase activity. The active RNP-lariat complex is in fact a homology search engine like the RISC complex in RNAi mediated pathways (Fig. 11.3) and in the RT/RNaseH strand transfer reactions during retroviral replication (Fig. 11.5). The lariat-RNA contains the EBS1 and EBS2 sequences of 14–16 nucleotide length. These, together with the active RNP complex perform the homology search and recognize the complementary IBS1 and IBS2 sequences in the DNA target site (Guo et al. 1997; Matsuura et al. 1997; Mohr et al. 2000; Saldanha et al. 1999). Other nucleotides in the homing site are recognized by the intron encoded protein in a restriction enzyme like fashion (Yang et al. 1998). The first cut is made by the lariat RNA reverse splicing into the sense DNA strand (Fig. 11.8a). The endonuclease domain of the protein cleaves the complementary DNA strand creating a staggered DSB. Figure 11.8b, c shows a comparison between a fundamental mechanism of recombinational DSB repair called SDSA and intron retrohoming. From Fig. 11.6, it appears that the RT of group II introns is not the most ancient of the RTs of other retroids. However, like the multicellular group of algae, Volvocinae, which serves as model of a line of evolution toward multicellularity but in fact is too young to be the real ancestor of metazoans (Lankenau 2007),

14

Y designates pyrimidine.

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Fig. 11.8 SDSA and group II intron retrohoming. (a) Landed homology search engine RNPC initiating group II intron retrohoming on DNA target. The ovals symbolize the intron-domain IV encoded protein consisting of the following catalytic domains: RT, integrase (¼endonuclease), maturase, DNA-binding. Integrase is an Mg2+ dependent endonuclease. Stippled rays indicate Watson-Crick base pairing interactions between intron and exon binding sites 1 and 2. Interactions

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within the framework of our deductive cycle group II introns serve as a good model for an ancestor of the eukaryotic spliceosomal introns as well as the eukaryote non-LTR retrotransposons (i.e., LINEs) (Lambowitz and Zimmerly 2011). The relationship between group II introns and non-LTR retrotransposons is evident from similarities in DNA sequence of their RTs (Xiong and Eickbush 1988, 1990). The target-primed reverse transcription mechanism (TPRT) used by nonLTR retrotransposons to integrate in DNA is very similar to the retrohoming mechanism of group II introns as well (Fig. 11.8) (Luan et al. 1993). Based on phylogenetic studies, the LTR-retrotransposons and retroviruses then subsequently evolved from non-LTR retroelements. Group II introns are rare in archaea and are probably derived from horizontal events. The assumption that group II introns originated in bacteria of the archaic LUCA age as retroelements is supported by the observation that bacterial group II introns include all known lineages and generally behave as functional retroelements whereas organellar introns belong only to two clades that are frequently eroded. The ancestral eubacterial retroelements might be a descendent of a self-splicing ribozyme with or without RT dating back to the LUCA age. Group IIC introns appear to be the earliest branching clade perhaps dating back into the LUCA age, but as with the tree in Fig. 11.6 statistical significance is weak and in agreement with the stem group character and evolutionary temperature of evolution in the LUCA age (Simon et al. 2009). PostLUCA, when the eukaryote nucleus had evolved, group II introns are thought to have invaded the nucleus and proliferated to many genomic sites as is typical of hybrid dysgenesis causing invasions of other transposable elements (Engels 1997; Evgen’ev et al. 1997; Kidwell et al. 1977). As with transposons, over several generations, the ribozyme structure degenerated producing non autonomous copies that became fragmented into snRNAs that now function in trans in a common splicing gadget, the modern spliceosome (Sharp 1991; Will and L€uhrmann 2011). A recent hypothesis posits that at the root of the three domains of life the introduction of group II introns by bacterial endosymbionts was the trigger for a fundamental step in the evolution of eukaryotes. This step was the formation of the nuclear membrane separating transcription from translation aiding to prevent translation of incompletely spliced RNAs (Martin and Koonin 2006). In any case, the separation of transcription and translation into different cellular compartments prevents direct ä Fig. 11.8 (continued) shown here and in Fig. 11.7 have been confirmed by crystal structure analysis. (b) Mechanism of synthesis dependent strand annealing (SDSA) (Gloor and Lankenau 1998; Lankenau 1995; Lankenau and Gloor 1998; Nassif et al. 1994) compared to (c) group II intron retrohoming. In (c) only one example involving the intron lariat RNA is shown. (c) Step 1, the group II intron is transcribed and the lariat forms as in Fig. 11.7b, the lariat RNA EBS sequences associate with complementary IBS sequences in DNA target. Step 2, RT synthesizes along lariat. Step 3, in order to anneal with target DNA and proceed replicating the entire intron information, the reverse transcript DNA performs strand transfer and annealing reactions as in bitemplate SDSA repair (3a). Step 4, fill in reactions on both DNA strands and removal of lariat RNA by RNaseH. Step 5, fill in and flap removal as in standard SDSA DSB repair (see also color plate 47 in Craig et al. 2002; Belfort et al. 2002; Lambowitz and Zimmerly 2011)

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access of the intron’s domain IV ORF-encoded protein to the intron RNA. This triggered the evolution of splicing factors functioning in trans (Lambowitz and Zimmerly 2011). Eukaryotic genomes contain numerous introns. In an adaptationist setting, they evolved snRNAs derived from group II intron domains into a general RNA-based catalytic machinery replacing that in individual introns. Modern snRNAs still recognize modern introns via conserved 50 - and 30 sequences and a branch-point nucleotide similar to those of group II introns. Splicing then occurs by the same transesterification reactions. This intricate RNA-based spliceosomal machinery of modern eukaryotes seems to be a persisting living molecular fossil like the ribosome. It is actually the strongest evidence that the eukaryotic splicing engine evolved from mechanisms represented by group II introns with probably lots of related molecules replicating in LUCA settings (Lambowitz and Zimmerly 2011). Further, the homology search engines performing genome wide homology searches today also trigger in modern organisms a presumably ancient DNA repair mechanism, i.e., SDSA (Fig. 11.8b).

11.3

The Ancient RNA World

As we have seen above, a thriving modern RNA world is ubiquitously present within all contemporary organisms (see also Wang et al. 2011). It follows that there must have been an ancient RNA world within their ancestors during the LUCA(S) age as well. Indeed, since long it was suspected that an RNA world was historical reality. In 1976, White noted that RNA fragments attached to various cofactors are widely distributed in modern terran life (White 1976). These RNA-cofactors today serve ubiquitous metabolic key functions: adenosine triphosphate (ATP) transfers phosphate transfer energy, S-adenosylmethionine performs one carbon transfers, flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide (NAD) serve in redox electron transfer chains, and coenzyme A (CoA) assists carboncarbon bond formation. Therefore, these cofactors were likely present in the LUCA(S) and indicate that they facilitated the historical RNA replicators from the beginning. Other molecules such as random peptides, lipid molecules, and inorganic atoms must have coexisted and facilitated RNA strand formation and replication as cofactors as well. Entering this primordial RNA world, we now, more often have to rely on reconstructions and simulations in our rhetorical disquisition, but a trajectorial concept is visible. We shall accept that about every strategy available to draw conclusions about the primal synthesis leading toward an ancient RNA world is welcome. Therefore, physico-chemistry based bottom up-approaches as well as geological and astronomical data and experimental approaches likewise are most welcome to nourish ideas about the LUCA(S) age and to feed concepts for better comprehension into Popper’s cycle (Fig. 11.1).

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11.3.1 The Protoribosome, Bridging the Current and the Ancient RNA Worlds Group I and II introns opened a window into the past toward understanding the LUCA world at the root of the three domains of life, i.e., archaea, bacteria, and eukaryotes. Group I and Group II introns have secondary structures conserved within each clade but distinct from each other between the clades. The common feature is that splicing is mediated by intron RNA with ribozyme activity that can proceed in the absence of proteins and both intron groups involve SDSAlike features including complementarity search and strand transfer reactions (Saldanha et al. 1993) (Fig. 11.8c). The sequence structure of mobile introns however is not conserved in evolution and that makes it difficult to move further back in time with mobile introns. Fortunately, the universal sequence conservation of another molecule, i.e., the ribosome, grants moving back even further in deep time. The three essential functions of the ribosome are: (1) decoding of the genetic code, where tRNA anticodons pair with a complementary triplet on a mRNA strand; (2) peptidyl transferase, where two amino acids are covalently linked to a di- or polypeptide; and (3) the translocation reaction, where an aminoacyl tRNA at site A moves into a new sterical position in the peptidyl site P within the ribosome. In contemporary bacteria, the 23S rRNA component of the ribosome contains the peptidyl transferase activity, central to translation, and acts as a ribozyme. Proof that none of the ribosomal protein moieties plays a role in the catalytically active site was revealed by the atomic crystal structure of ribosomes (Nissen et al. 2000). No protein moieties ˚ of the active site. Therefore, peptide bond formation is indeed were found within 17A catalyzed by RNA with no part of any protein component close enough to play a direct (chemical) role in the reaction (Moore and Steitz 2011; Ramakrishnan 2011). Because of its universal distribution in all life, the catalytic center of the ribosome is the most ancient living molecular fossil on earth. The subcomponents of the ribosome are shown in Table 11.1. The 23S rRNA of prokaryotes and the 28S rRNA molecules Table 11.1 Ribosome composition Sedimentation Domain coefficient rRNA 70S Small subunit 30S 16S ¼ 1,500 nt 5S ¼ 120 nt Prokaryotic Large subunit 50S 23 S ¼ 2,900 nt 80S Small subunit 40S 18S ¼ 1,900 nt 5S ¼ 120 nt 5.8S ¼ 156 nt 28S ¼ 3,400Eukaryotic Large subunit 60S 4,700 nt

Proteins

Catalytic activity

21 proteins S1–S21 31 proteins L1–L31

Peptidyltransferase ribozyme

~33 proteins S1–S33

~50 proteins L1–L50

Peptidyltransferase ribozyme

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of eukaryotes are 2,900 nt and 3,400–4,700 nt long respectively. In all modern cells, this translation activity catalyzed by the ribozyme must be facilitated by a large number of co-factor proteins and other rRNAs (Table 11.1) that, figuratively speaking, were added successively in evolutionary history like the shells around the core of an onion (Bokov and Steinberg 2009). The long standing hypothesis, that the first ribosome-like entity, which probably appeared between 3.5 and 4.0 Ga ago, was made entirely of RNA, today seems more likely than ever. Nevertheless, it would be foolish to assume that the biological world in which that first ribosome appeared was one in which RNA was the only relevant polymer (random or informational) (Moore and Steitz 2011). It likely needed co-factors from start on (this book). Everything we know about the ribozymal catalytic site in the ribosome is based on modern ribosome crystal structures, but we cannot go back in time 4 Ga to check out the primordial ribosome directly. However, based on structural data, Ada Yonath and colleagues suggest that there is evidence for a ~55 nt long RNA molecule which they termed the protoribosome (Davidovich et al. 2009; Yonath 2009c). It would be a member of the ancient RNA world from which our present RNA world evolved by Darwinian evolution. According to their work, the ancient translation apparatus may have survived selection pressures, and its vestiges may indeed be embedded within all modern ribosomes (Davidovich et al. 2009). The secondary structure of the protoribosome comprises an RNA-homodimer of stem-elbow-stem elements. From the basic ~55 nt RNA, the ribosome evolved successively over billions of years into the modern ribosome, which includes many protein components and other rRNAs (Bokov and Steinberg 2009). The protoribosome originated from gene (!) fusion and gene (!) duplication – a process that reminds us of the SDSA-like processes mentioned in Fig. 11.8 (Yonath 2009a). Independently from the sequence (RNA and protein) the three-dimensional structure of the two halves of the ribosomes that carry out the peptidyl transferase reaction, has been conserved stereo chemically until today. According to this, the ancestral ribosome possessed a central pocket built of two chains of the same ~55 nt RNA forming a dimer. These two RNA molecules were sufficient, according to Yonath’s hypothesis, to carry out peptide synthesis. The ~55 nt short protoribosomal RNA is an extrapolation from atomic ribosome structures (Davidovich et al. 2009; Yonath 2009c). Nevertheless, it is hypothetical, and we have now reached the other side, down under the golden spike, and outside Popper’s cycle (Fig. 11.1). At this point, there is only one theoretical extrapolation from the preceding RNA world possible where we are still on firm ground: The very first protoribosome-like entity that existed must have done so in multiple copies and not just in one. If it had been only one molecule, it would not have existed as an entity. Reproduction was mandatory. Any further, reproduction needed to be reproducibly stable reproducing similar molecules over many generations. Thus, because there had to be a multi-copy population of protoribosome molecules in order to be called an entity, something (mechanistic or structural) must have been responsible for making copies of the protoribosome. This something would be called “template.” The protoribosome already encompassed a distinct secondary structure (stem-elbow-stem) that was enforced by some sort of primary RNA sequence and

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base-pairing constraints. Thus, the protoribosome likely was in state to self-prime fragments of its own sequence and possibly could have served as template and ribozyme in mobile intron-like priming and retro-replication activities (Fig. 11.8c). If we would proceed further from here, the amount of speculation likely became too much for the good. Therefore, we will now briefly leave the RNA world and make a jump to a new, bottom up hypothesis based on the simultaneous consideration of many bioenergetic, physical, and geological constraints. This hypothesis was recently developed by Armen Mulkidjanian (Mulkidjanian 2009; Mulkidjanian and Galperin 2009). It leads us back into a distant, post-hadean age, bare of life and filled with hardening minerals, consolidating igneous rocks, and sunlight triggered photo polymerization resulting in the emergence of the first replicators. The hypothesis is so attractive because it unifies seemingly opposing ideas such as “metabolism first” versus “replication first” scenarios. According to the Zn world hypothesis, life emerged on earth as a “proto metabolism-driven replication.” From a sun light penetrated, primordial hydrothermal field environment, RNA molecules started to replicate at some point, facilitated by many coexisting and interacting protometabolites (Chap. 9) possibly already involving simple protopeptide replicators (Ashkenasy et al. 2004). From there, we shall arrive at the protoribosome again as the earliest trace record of a replicator bridging the ancient and modern RNA worlds.

11.3.2 The Zn World Hypothesis and First RNA Replicators The Zn world hypothesis represents a perfectly reasonable deviation from a beaten track15 of alternative hypotheses. The accompanying chapter by Mulkidjanian and Belozersky (this book) unfolds the details and handles the matter with utmost expertise. Therefore, let me only give a brief, overlapping account in order to link the two RNA worlds above and below this section. In two seminal papers, Mulkidjanian proposes that the Zn world involved sub-aerial, sun exposed ZnS (sphalerite) precipitates supporting a mechanism of continuous abiogenic photosynthesis of prebiotic metabolites and their further conversion by ZnS-confined replicating entities (Mulkidjanian 2009; Mulkidjanian and Galperin 2009) (see Fig. 1 in Mulkidjanian & Belozersky, this book) Two basic assumptions are critical for the hypothesis: 1. Availability of enough UV-irradiation from the Sun 2. The requirement of high atmospheric pressure According to the “faint young Sun puzzle” 4–3.5 Ga ago, the Sun was about 30% less bright than today (Nisbet and Sleep 2001). If such a sun shone on the current

15

In paraphrase to Richard Feynman’s letters collected in the book: “Perfectly Reasonable Deviations from the Beaten Track.”

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Earth, there would be global glaciation. However, in contrast to visible light, X-ray and UV luminosity emitted from the early Sun was likely higher by analogy with other Sun-like stars that generally show decrease in X-ray and UV luminosity with age (Cnossen et al. 2007). Therefore, the Zn world hypothesis starts from the premise that without the ozone shield solar UV-radiation reaching Earth’s surface land or water was 10–1,000 times higher than today. Further, the atmosphere was dominated by CO2, with smaller amounts of CH4, N2, and condensing H2O vapor. The surface pressure was 10–100 times higher than today changing the critical points for phase transitions of precipitating minerals according to their specific phase diagram. Currently, it is thought that CO2 was the main material from which primal organic polymers were formed. CO2 reduction occurred close to sub-aeral hydrothermal vents that were common in costal and shallow waters as well as on land like nowadays in Yellowstone, or in the Afar Danakil depression. The energy for forming organic compounds came from UV light and CO2 embodied the building blocks for photo polymerization. The ZnS hypothesis is based on W€achersh€auser’s insight that an abundant mineral on earth could have provided the energy for carbon fixation in a redox process converting ferrous ions and hydrogen sulfide of basaltic origin into pyrite (FeS2) (W€achtersh€auser 1988). The detailed reasons for why ZnS or MnS are better candidates for this process is explored in the chapter of Mulkidjanian and Belozersky (this book). Indeed, the ZnS and MnS precipitates close to sub-aerial or coastal hydrothermal vents appear ideal as a primal model system to focus efforts at understanding the emergence of life from abiotic settings. Pumice-like textured ZnS precipitates formed honeycomb micro compartments of <1–100 mm in diameter, such as in fossil and modern vents (Branciamore et al. 2009; Kelley et al. 2005; Koonin and Martin 2005; Martin and Russell 2003). We now can imagine that many, if not all, requirements existed within ZnS compartments for the production of many organic, premetabolic molecules that accompanied or even facilitated the polymerization of the first RNA polymers (see Chap. 1; Mulkidjanian 2009; Mulkidjanian and Galperin 2009). Next to a number of pre-metabolic compounds, the most significant coexisting polymers were abiotic, random peptides or proteinoids, lipids, and RNA precursors – all in a heterochiral mixture. Polymerization of monomers (both nucleotides and amino acids) in aqueous solution under experimental conditions produced only short, let it be, heterochiral oligomers (10 mer). High photostability

Fig. 11.9 Simple oligomerization of tetra nucleotides involving a homology search and primordial, pre-SDSA-like annealing; cp ¼ 20 , 30 cyclophosphate; (Szathmary 1997)

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of the monomers and polymers was a side-effect as the ZnS compartments catalyzed photopolymerization under UV illuminated exposure exerting high selection pressures on photo-instable monomers (Chap. 1). In Chap. 1, Mulkidjanian and Belozersky point out the UV-protection mechanism for the evolution of primordial RNA-like polymers. According to this mechanism, it is likely that small double stranded snippets of RNA, composed of single stranded fragments that were dynamically synthesized and degraded and synthesized again, may have been annealed to random complementary sequences, forming Watson-Crick base pairs, that provided an enhanced photostability and a selective advantage for survival. It was a process very similar to and predating the universal SDSA-mechanism (Lankenau 2006). Surface-bound template polymerization at ZnS surfaces was possible as well. It has been shown that presence of montmorillonite surfaces induces formation of oligomers up to ~55 monomers long, as is the size of the predicted protoribosome (Davidovich et al. 2009; Ferris et al. 1996). Simple ribozyme activity was therefore feasible on mineral surfaces as experiments by the Bartel lab have shown (Bartel and Szostak 1993; Ekland and Bartel 1995, 1996; Ekland et al. 1995; Johnston et al. 2001). These experiments demonstrate the practicability of autocatalytic simple ligations up to multiple, successive ligations of several nucleotides. Multiple ligations in fact represent nothing else but the origin of continuous replication. However, not every sequence is replicated yet in these experiments. An important feature in the evolution of RNA polymers was probably the involvement of complementary annealing. Tetranucleotide-20 , 30 - cyclophosphates were shown to assemble into oligomers of up to 36 nucleotides in dilute solution, with oligomerization proceeding highly chiroselectively (Bolli et al. 1997) (Fig. 11.9). Homochiral templates catalyze the formation of the correct phosphodiester junction between homochiral tetramers that have the same chirality as the template. The origin of homochirality in biomolecules is a significant issue for the evolution of replicators and here is one facet of this subject. Using purine ribonucleoside 5’phosphoimidazolides as activated monomers and oligomeric or polymeric ribonucleotides as templates allows for no enzymatic, template-directed syntheses of oligonucleotides (Naylor and Gilham 1966). Even though it is only a historically unrealistic simulation, von Kiedrowski showed that these imidazolides in vitro grow as artificial replicators without replication enzymes or ribozymes (Sievers and von Kiedrowski 1994; von Kiedrowski 1986). Thus, it appears imaginable that in ZnS micro compartments, homochiral templates of D- and L-enantiomers evolved with the degree of oligomerization growing in time. Each chiral template then gives rise to its own kind. When photochemically synthesized raw-monomer supplies became limiting, the replicator sequences started to compete for raw materials and an imbalance of chiral kinds built up. Once the symmetry of the molecular population was broken only one kind of chirality survived. Of course, there were many hydrothermal vents all over the earth and feeding only one particular kind of chirality into LUCA(S) was likely a long-term Darwinian process. Random peptide replicators could have emerged as well (Ashkenasy et al. 2004; Paul and Joyce 2004). In coexistence with RNA replicators, they might have been selected for coexistence with RNA replicators from the beginning. The most stable and most

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abundant peptides may have had developed binding affinities stabilizing replicators and further photo protecting RNA from destruction. Thus, the first RNP replicator network could have been emerged and continued the Darwinian path toward the protoribosome (Cech 2009). Is all this still to be called pre-Darwinian evolution? At which point of replicators does Darwinian evolution start?

11.3.3 Quasispecies and the Error Threshold 11.3.3.1

The Initial Darwinian Ancestor (IDA)

In a bottom-up journey, we cannot explore LUCA(S) as this is the stemgroup of the three domains of life caught in Koonin’s “irreducible complexity trap” (Koonin 2009) (to be published elsewhere). LUCA(S) remains causally attached to present life forms as viewed from the anthropic perspective (Fig. 11.1). Based on Bayes’ Theorem, i.e., a quantitative version of the anthropic argument, Michael Yarus suggests that there was a substantially older IDA where nicotinamide adenine dinucleotide (NAD) or its congeners were the earliest cofactors to enter the biochemical inventory of all living cells (Yarus 2011) (Chap. 9). According to this hypothesis, in step 1, activated nucleotides and coexisting, interacting molecules oligomerized arbitrarily. In step 2, initially random replicators such as AMPcontaining cofactors became abundant with simple templating and minimal catalysis. In step 3, dinucleotide 50 –50 replicators such as NAD participated and were selected for “usefulness” (increasing fitness) in metabolism. Then, in step 4, the ancestral RNA world emerged (or RNP world, as random peptides may have coexisted in the ZnS/MnS compartments (Cech 2009)), where 50 –50 cofactor initiation and reactivity may or may not have participated. Yarus states that the 50 –30 RNA replicase replicator created the first RNA world at 4 Ga ago. If it took place in the ZnS world, the window would be rather between 3.5 and 3.9 Ga according to the Zn world hypothesis (Mulkidjanian 2009; Mulkidjanian and Galperin 2009). In step 5, translation and coded peptides were invented employing Ada Yonath’s et al. protoribosome (Davidovich et al. 2009). 50 –50 cofactors like NAD, FAD, NADP were adopted by peptide catalysts. They were readily available in spark experiments and several synthetic routes employing plausible prebiotic chemicals (Cleaves and Miller 2001; Friedmann et al. 1971). The stability of the ribose moiety was increased substantially in presence of borate minerals where thermal springs and hydrothermal fluids are an important source of B to continental evaporites (Grew et al. 2011). This points to a sub-aerial relevance as with ZnS. Within sphalerite (ZnS) compartments, photo-excited electrons could further be transferred from the surface of photo-excited ZnS particles to NAD+/NADH and co-electron acceptors in order to evolve energy-managing redox systems (Mulkidjanian and Galperin 2009; Mulkidjanian et al. 2009). It has not been shown yet that single activated nucleotides polymerize on dinucleotide templates, especially on 50 –50 dinucleotides – perhaps a challenge to show for photo excited ZnS

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compartments? Indeed, it would be a great step forward if it could be shown that a random replicator such as a dinucleotide- IDA evolved into longer chain replicators under any conditions. In 1975, Sumper and Luce demonstrated that a mixture containing no RNA chain at all but only activated RNA base monomers plus the enzyme Qb-replicase spontaneously generated long, self-replicating RNA chains (Sumper and Luce 1975). Such a simulation does not prove that random prebiotic peptides could do the same job, but it appears just a matter of time for the initial evolution, e.g., in ZnS compartments to imagine the evolution of effective replicators. As a next step, only a population of RNA sequences representing a single consensus sequence stands for such an effective replicator and can be called an individual genetic entity from where Darwinian evolution can start (Robertson and Joyce 2011). A population of such sequences operating at the boundary between chaos and order was called quasispecies (Eigen 1987, 1992) which we will learn about next. 11.3.3.2

In Vitro Evolution and the Spiegelman Monster

Sol Spiegelman is known for his recognition that for a certain fragment of DNA, only one of the two DNA strands (the Watson or the Crick strand) encodes the genetic information. However, for our purpose of the ancient RNA world, it is Spiegelman’s evolution experiments that are of relevance. The Spiegelman Monster is a specific RNA molecule of ~218 nt length that is able to reproduce and evolve in vitro in the presence of an RNA polymerase, i.e., the Qb replicase (Kacian et al. 1972). In the 1960s, Spiegelman and colleagues employed the plus-strand RNA virus, Qb, which infects the bacterium Escherichia coli. The RNA genome of Qb has a length of 4,500 nt and encodes its own replicase and capsid proteins for packaging the virion. Infection starts with the docking of a Qb encoded adsorption protein to the sex-pili on the outside of a cell. After infection, the plus-strand RNA serves simultaneously as genomic and as mRNA inside the infected E. coli cell. When Qb encoded replicase is translated, it rapidly catalyzes replication. Qb-replicase consists of four subunits and recognizes both, plus strand RNA as well as minus strand RNA. It binds to the 30 ends of both RNA strands and both strands act as templates reciprocally to one-another (Domingo et al. 1976). This sets up a replication cycle similar to what we expect might have happened in ZnS compartments or other locations on the primordial Earth. A stable internal secondary structure prevents the RNA from forming an RNA helix during replication. After about ten to one hundred thousand Qb molecules have been produced, the plus strand RNA molecules are packaged into capsid protein again, and a Qb encoded lysis factor ministers lysis of the host cell and release of free Qb virions. A population of 100,000 Qb virions resembles populations of some animal and plant species. However, higher organisms, and even E. coli cells possess the proofreading activity of modern DNA polymerases, making replication of their genomes highly reliable and safeguarded against too many mutations (Kornberg 1989; Kornberg and Baker 1992). The Qb replicase does not possess proofreading

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and the quantity of errors is high, i.e., 3/10,000 ¼ 3 104. Because of this high mutation rate, only a fraction of the released Qb virions are infectious. The whole population of virions - or better the population of RNA genomes - including its mutant spectrum, was termed quasispecies by Manfred Eigen and colleagues (Biebricher and Eigen 2006; Eigen et al. 1988; Epstein and Eigen 1979). In natural populations of the E. coli bacterium, the Qb virus coexists together with a noninfectious Qb variant (called minivariant) that acts as a superparasite on Qb (Orgel 1979). The length of this superparasite is ~220 nt and it evolved from the 4,500 nt long Qb RNA. Since the minivariant does not encode the Qb replicase anymore, replication and the infection potential completely depend on the intact Qb replication and infection machinery. To overcome the negative effects of its dependence on the original Qb virus, the minivariant RNA evolved a size and a secondary structure that increased replication speed by Qb replicase (Eigen 1992). Such a minivariant of ~220 nt length is the naturally occurring equivalent of the experimental Spiegelman Monster. Superimposed systems of parasites are ubiquitous for life and well known from the transposable element world. For example, SINEs (short interspersed nuclear elements) are a subclass of transposable elements that do not encode any proteins themselves but instead have evolved to parasitize the LINE retrotransposition machinery. Many SINES are also chimeric with a 50 region derived from tRNA and a 30 tail derived from the 30 end of a LINE. Most famous are the 280 nt long Alu elements with 1.5 million copies, i.e., 11% of the haploid human genome. They are sequence related to 7SL RNA.16 Despite their parasitic nature, both LINES and SINES are now known (as are many examples of endogenous retroviruses) for being incorporated into novel genes, so as to evolve new functionality. The Qb minivariant, therefore, serves as an in vitro model system simulating the ancient conditions of mass-effects of small RNA replicators as they likely existed in primordial environments such as ZnS compartments 4 Ga ago. At the same time, this gives us information about the dynamics at the genome level in modern organisms. Thus, the question we pose is: under which conditions did minivariants evolve initially? What were the conditions for gliding from the abiotic, non-Darwinian side with “random replicators” as depicted by Yarus (2011) to the side of informational quasispecies employing Darwinian selection, and driving evolution to be discharged into the worlds of adaptations, exaptations, punctuated equilibria, and molecular and organismic drivers under environmental constraint (Burt and Trivers 2006; Gould 1997, 2002). Based on the mechanisms of Fig. 11.5, a new experimental system was set up termed self-sustained sequence replication (3SR) (Gebinoga and Oehlenschl€ager 1995). In 1997, Eigen and Oehlenschl€ager showed that the Spiegelman Monster

16

The signal recognition particle RNA, also known as 7SL, 6S, or 4.5S RNA, is the RNA component of the signal recognition particle (SRP) ribonucleoprotein complex. SRP is a universally conserved ribonucleoprotein that directs the traffic of proteins in the cell and allows them to be secreted.

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Fig. 11.10 Serial dilution of viral suspension with established titer (Drawing from the author’s Baltimore/ Heidelberg lectures)

eventually becomes even shorter, containing only 48 or 54 nucleotides, which are simply the binding sites for the reproducing RNA polymerase (Oehlenschl€ager and Eigen 1997). This size is effectively the size of the protoribosome as reconstructed by Ada Yonath and colleagues (Davidovich et al. 2009). Crucial to all experimental systems is the serial dilution and transfer principle. It was first applied in 1938, when Max Delbr€ uck started to work with bacterial viruses (Ellis and Delbr€ uck 1939). For example, when a bacterial culture is infected with phage lambda, optical density clears after a couple of hours because all replicating bacteria have been lysed and killed. Plating bacteria on a solid agarose plate and pouring phage solution on top will clear the plate off bacteria as well. However, when the phage suspension is diluted such that only very few phages remain, individual phage particles produced by a single ancestor phage produce singular clear plaques in the bacterial lawn. Delbr€uck realized that when diluting the phage suspension such that, on average, no phage particle per vial was the most common dilution event, he could apply the Poisson formula and calculate future serial transfers (Poisson 1837) as shown in Fig. 11.10. Application of the method initiated molecular biology and experiments on mutant distributions as explicated next. 11.3.3.3

Mutant Distributions of Replicators

Sol Spiegelman, Charles Weissmann, and Manfred Eigen set up experimental systems in which sequence evolution relative to the reproductive speed (fitness) of RNA molecules could be exactly monitored and controlled (for review and references, see Eigen 1983, 1992). Qb-replicase is able to replicate any RNA molecule in the presence of the four NTPs (ATP, GTP, UTP, CTP). An important experimental strategy is to clone each individual sequence mutant from large

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quasispecies populations. Based on a known Qb virus titer, a serial dilution must be carried out (Fig. 11.10). Sequencing from over diluted vials allows for sequencing any number of single molecules. An experiment starts by adding the four NTPs and Qb replicase under defined conditions. The reaction takes place in a so called “evolution reactor” (Eigen 1992). It is initiated and stopped by increasing/decreasing the temperature from 0 C to 37 C within a second. Growth of the increasing number of replicons is monitored by a laser fluorimeter with glass fiber optic and the reaction is stopped in the exponential growth phase. A fraction of the RNA molecules will be reverse transcribed, cloned, and sequenced. Today, next generation sequencing can be applied. The experiment can go on by starting a new round of replication from a single RNA molecule. This can be repeated over many generations under defined experimental conditions. The most spectacular molecules, replicating under presence of increasing amounts of ethidiumbromide or RNaseT1, evolved resistance against such detrimental agents. As mentioned above, the Spiegelman Monster itself evolved from a ~4,500 nt long Qb virus RNA as starting material. Finally, a short ~220 nt long, fast replicating RNA molecule emerged which very much resembled the Qb minivariant superparasite of natural E. coli populations (Orgel 1979). After few generations of independent evolution, individual subpopulations possessed the same consensus sequence. But, after many generations of multiplication, each independently evolved RNA population started to establish its own characteristic mutant spectrum represented by a characteristic consensus sequence.17 A combination of quantitative experimental and theoretical simulation assisted research lead to fundamental insights into the very matter of the origin of life. Let me draw the picture by starting out with a population of say 108 RNA replicators each 50 nt long. This population dwells in one of the micro-compartments of Mulkidjanian’s Zn world.18 Here, it is irrelevant by which mechanism the RNAs or RNPs emerged – e.g., either, via random peptide facilitated short RNA chains that became more abundant by von Kiedrowski/Yarus like processes (Sievers and von Kiedrowski 1994; Terford and Von Kiedrowski 1992; von Kiedrowski 1986; Yarus 2011) or by Qb-type de-novo synthesis facilitated by some random peptide (or an RNP) instead of Qb replicase as reported by Eigen’s group (Eigen 1983). Figure 11.11 summarizes Manfred Eigen’s group’s results. Let me recapitulate the example (Eigen 1988, 1992; Eigen et al. 1988): We start in Fig. 11.11a with a theoretical population of RNA replicators, each 50 nt long, like the protoribosome. Only purines (R) and pyrimidines (Y) are considered. Thus, only two types of mutations are possible: Y - > R and R -> Y. The coordinate system depicts the replication error rates (1 – q; abscissa) versus the relative population number (Xd; ordinate). Initially, we assume that all molecules are identical and the replication

17

Consensus sequence refers to the most common nucleotide at a particular position after multiple sequences are aligned. In case of the mutant spectrum of a quasispecies, the consensus sequence is identical with the master sequence. 18 Other locations such as beaches or brine ice may do the job as well.

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Fig. 11.11 Conditions for a replicating entity of protoribosome size, i.e., a quasispecies of 50 nt chain length (n) at different error rates. For detailed explanations see main text (Combined, redrawn, and modified after Eigen (1992); Eigen et al. (1988)))

error rate (1 – q) is zero. Therefore, the population number Xd is 1.0, that is all molecules (100%) are identical. The index d is the distance (in numbers of nucleotides) of a mutant sequence to the best adapted master sequence, called the Hamming distance. As all sequences are identical and the Hamming distance of all RNA replicators is 0, Xd ¼ X0. Therefore, X0 designates the master sequence of the population. Xd is the ratio between the amounts of individual molecules with the distance d and the total amount of all existing individuals. The X values with indices d ¼ 1 to d ¼ 50, represent the sum of all mutants within each error class 1–50. For example, X1 encompasses all 1-error mutants independently to the position within the 50 nt sequence. The X-values in the ordinate are given as relatives between 0.0 and 1.0 (0–100%), summing up to 1.0. The replication error rate (1 – q) relates to the probability in which a position mutates. An average of each 1/100 base wrongly incorporated gives a mean error rate of 0.01. At an error rate zero, there is a strong “all or nothing” judgment. Either the sequence is identical with the master and lives or it will die after the first mutation (survival of the fittest). With a growing rate of mutations, the population number of the master sequence (X0) decreases rapidly to low numbers. Quickly, there are (in the sum) more X1, X2, X3,. . . error mutants than X0 molecules representing the master. Experimentally, such an individual mastermolecule could not be identified by sequencing, as it will become too rare. It is still mastering the population for its superior fitness through positive selection, but it may become nearly extinct, while its mutants are still symmetrically distributed around the master. Nevertheless, the master sequence can be reconstructed from aligning many mutant sequences such that their consensus sequence identifies the master sequence. In other words, with increasing error rate the master molecules drop in

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copy number to nihility, but the master’s information still determines the mutant distribution. Finally, when the replication error rate increases even further, a threshold value is reached (Fig. 11.11a, b at 1 – q ¼ 0.046) and the information represented by the master sequence instantaneously disintegrates. The population number of the master abruptly collapses to 1515 (Fig. 11.11b). The master sequence then is just one of 250 1015 possible variants (remember, we are dealing with binary R, Y sequences). The formerly selected sequence information has now been lost completely, chaos is ruling sequence space. Eigen and colleagues termed this informational break down a phase transition in analogy to thermodynamics, e.g., when a liquid becomes gas upon heating to the boiling point, resulting in abrupt change in the state of aggregation. The information evaporates. Figure 11.11c depicts details at the phase transition point and the error threshold for two similar fit sequences. Just around the error threshold, Eigen identified the most favorable conditions for evolutionary change. In his example (Eigen 1988, Abb. 3 p.127), he examines a binary R/Y sequence of 50 nt length again (i.e., protoribosome size) and explores the consequences of minute fitness differences. The sequence with the highest selective value (I) (master) is arbitrarily set to 1.0 with I0 (zero errors). Its antipode, a sequence that possesses the complement in all 50 positions (i.e., 50-error mutant) is called I50. This mutant has a similar high selective value of 0.9. Therefore, I0 and I50 represent two similar fitness peaks with only slight differences. All other mutants get a low selective value of 0.1, except the 50 possible one-error-mutants of I50 receiving the selective value 0.5. The latter class of mutants are at the same time the 49-error-mutants of I0 and are, therefore, called I49. In this distribution, the best adapted singular sequence I0 competes with the slightly less selectively favored singular antipode I50, which is surrounded by multiple, better adapted I49 mutants. Figure 11.11c details the results of this competition around the error threshold: Shown are again the logarithmic values of the relative population numbers log Xd for all complete error classes I0 to I50 (sum of all individual sequences within the error class) as a function of the error rate (1 – q). With two “races or breeds” of sequences, we observe two phase transitions; with a relative low error rate, I0 wins, because it is the best adapted individual sequence. This is what would be expected from Darwin’s principle, survival of the fittest. At an error rate of 0.0445, i.e., the phase transition point the information of I0 “evaporates” (Fig. 11.11c red lines: distributions with 0 and 2 mutant molecules are shown). At this point, however, the sequence I50 (a race of its antipode distribution in population biological language) is still stable because it has a better value topography of its neighbors (the I50 mutant with a selection value of 0.9 and the I49 mutant with a selection value of 0.5, whereas I0 with selection value 1.0 is only surrounded by selection values of 0.1). In this way, I50 with its idiosyncratic clan of mutants (fifty possible one-error-mutants I49) now becomes the target of selection (Fig. 11.11c black line distributions). Only at the error threshold with an error rate beyond 0.045, the information finally becomes victim of an error catastrophe. In this example, we see that the target of the selective evaluation is not the individual sequence type (I0 or I50) but the whole mutant spectrum. This is the

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quasispecies, and in my opinion this is a plausible starting-framework of conditions for the IDA as well. Typical of the phase transition are the dramatic changes in population numbers of several orders of magnitude. As mentioned above, a binary sequence with 50 positions has 250 » 1015 possible variants. When the threshold is passed, the population numbers of individual sequences – visible for I0 and I50 (Fig. 11.12c) – fall to statistically insignificant numbers of 1015. The error classes I1 to I49 of course include different individual alternatives. Therefore, the statistically most abundant sequences I25 dominate (Fig. 11.12a). For our analysis of the random RNA molecules in some kind of hydrothermal vent compartment, the experiments and simulations above have tremendous consequences. If we read each plot of Fig. 11.11 from the right to the left, ZnS chambers might have produced RNPs of the type mentioned by Cech (Cech 2009). However, sequence information as in a quasispecies could not have existed a priori. It must have been a chance event when the first single RNP19 molecule arose and produced a replica with a low enough error replication rate and high enough fitness value to overcome the error threshold and make a phase transition into a stable quasispecies. In natural systems, however, the error rate topic can be much more complicated. For example, the Eigen threshold made us believe for long that primitive RNA sequences could not evolve into significantly longer chains exceeding about 50 nt. This created the Eigen’s paradox: “no protein without longer gene, no gene without protein.” A recent paper however argues that replication seizes and slows down in speed at a mismatched point mutation using the complementary sequence as template. In that view, it becomes anticlimactic that master sequences are preferably replicated and longer chains can evolve (Rajamani et al. 2009). An experimental indication that long chains indeed can evolve using imidazolid-replicators was demonstrated for sea ice micro chambers (Trinks et al. 2005). Further, an individual ZnS micro chamber in which a quasispecies had emerged, could then have started to export molecules into the environment infecting other micro chambers, neighboring vents, entire hydrothermal systems but also beaches and ice etc. Vice versa, ZnS chambers could have absorbed solvents from a primordial soup generated elsewhere. Relevant is that at some point, replicators evolved that exchanged RNA fragments interdependently through homology search processes and annealing steps. Synthesis dependent strand annealing (SDSA) as in group II intron retrohoming evolved as a novel mechanism, now ubiquitously present in all life. Simultaneously, the protoribosome-like replicators evolved and learned to translate information into protein. This gave rise to Beadle-Tatum-type genes (one gene-one enzyme concept) (Beadle 1958).

19

With “P” in RNP, I refer to random, non informational, non encoded, abiotic peptides associated with random abiotic ribonucleotides.

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Fig. 11.12 Sterochemical model of a pre-protoribosomal replicator interaction complex based on Woese and Crick (Crick 1968; Crick et al. 1976; Woese 1970). The complex consists of two tRNA anticodon loops in the two possible conformations: FH for the peptidyl tRNA (P-tRNA) shown in green color and hf for the aminoacyl tRNA (A-tRNA) shown in red color. Both tRNA anticodon loops are in antagonistic conformations where the bases 6 through 10 (green) continue the double helical turn from the tRNA stalk of P-tRNA while bases 8 through 12 (red) continue the double helical turn from the tRNA stalk of A-tRNA. Two nucleotides within the anticodon loop are energetically, unfavorably kinked falling out of the double helices (i.e., red bases 6, 7 and green bases 11, 12). This is compensated for by the total base stacking energy of the 10 bp double helix formed between mRNA (gray and black symbols) and the two anticodon loops stabilizing the complex. Solid bars represent two arrays of pentaplet Watson-Crick base pairs between mRNA and tRNA anticodon loop. Graphic symbols and numbering of bases are that used by Woese (Woese 1970). FH and hf designates Fuller& Hodgson and hodgson & fuller conformations of the anticodon loops. This structure is the sterical prerequisite for Crick’s et al. and Schuster & Eigen’s RNY-code hypothesis (Eigen and Schuster 1978b). The energetically unfavorable kinks make sure that the structure is highly unstable and dynamic – switching between pentaplet and triplet Watson-Crick base pairs (for further detail see Woese 1970). The flexibility in addition to the much longer, flexible cloverleaf structure of the tRNA (not shown here) still leaves enough potential for the peptidyl transferase interactions. Most relevant however, the structure allows multiple tRNA like molecules to “creep” along the mRNA strand in a caterpillar like movement (see Fig. 11.13a–d)

11.3.4 Positive Pondering on the Origin of RNY20 The origin of protein synthesis is a notoriously difficult problem. We do not mean by this the formation of random polypeptides but the origin of the synthesis of polypeptides directed,

RNY ¼ purine-any nucleotide-pyrimidine “code.”

20

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however crudely, by a nucleic acid template and of such a nature that it could evolve by steps into the present genetic code, the expression of which now requires the elaborate machinery of activating enzymes, transfer RNAs, ribosomes, factors, etc. (Crick et al. 1976).

In the previous sections, we learned about the central importance of the homology search engines such as RISC, the RT/RNaseH strand transfer mechanism working in trembling, flipping, sliding motions, and the SDSA-step in group II intron homing. SDSA-like templating, annealing, and priming appears to be the universal ingredient of replicating systems taking advantage of perhaps initially random complementarity in RNA sequences, annealing different molecules to each other, serving as informational backups of each other, and continuing in the production of either random RNA chain assemblies (Fig. 11.11a, b right half of abscissa) or of Darwinian, master-governed mutant assemblies, termed quasispecies and called to be alive (Fig. 11.11a, b left half of abscissa). The specific properties of any kind of such RNA(P) molecule assemblies further remain a mystery. However, game theory teaches that some sort of evolutionary stable strategy (ESS) coevally considering Nash-equilibria must have been reached once or at multiple occasions (Maynard Smith and Price 1973; Nash 1994). The RNA(P) assembly of molecules would be but one example of such equilibrated systems. Any system of this kind, replicating and encoding intrinsic genetic information21 that was stable for enough generations to serve as a unit of selection would have been alife and simultaneously fitted the anthropic, deep definition of the term gene.22 Game theory provides a conceptional bridge and is relevant for these initial beginnings of life to the highest complexities, e.g., as in the prisoners’ dilemma or as for the life strategies of social superorganisms such as the honeybee. Thus, game theory represents a well tied Ariadne’s thread throughout life sciences. But finding a concrete, uninterrupted, contiguous path from a first ribozyme-activity of a replicator-quasispecies to the (theoretical) protoribosome (Davidovich et al. 2009; Yonath 2009b, 2009c) calls for bridging substantial leaps of another kind. Only few theoretical and fossil threads remain to conceptualize such a reasonable bridge. The only hint for such a link was motivated by Crick’s et al. recognition that the anticodon loop of present tRNAs encompasses a consensus sequence motive, which reads 3‘NRabmUY, where abm is what we call the modern anticodon (Crick et al. 1976). One possible explanation for the universal

21

i.e., informational replicators as defined (Zachar and Szathmary 2010) with the slight difference that the most basic replicator representing life (i.e., the gene as defined in footnote 22) itself manifests a phenotype (secondary structure) as well. 22 A gene is defined as any portion of chromosomal material that potentially lasts for enough generations to serve as a unit of selection. G.C. Williams cited in Dawkins (1976) – where a unit of selection is any ESS in Nash-equilibrium. This gene definition is more general and it embraces the classic, technical Beadle-Tatum definition of gene (one gene, one enzyme hypothesis) used in everyday laboratory practice. The gene as a physical reality was recognized by physicists only after Muller’s demonstration that radiation triggers mutations in DNA visible as a changed phenotype (Muller 1941).

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conservation of this motive is the stereo chemical theory in contrast to the frozen accident theory (Crick 1968). From the motive, these and other authors deduced an ancient RNY code on what would become mRNA later (see below). Today, these inferences, still provide the best hypothetical link between the ancient and the current RNA world. At present, the most complete study to address the path from random to living replicator-quasispecies (e.g., in ZnS micro compartments) to the protoribosome still is the analysis by Eigen and colleagues (Eigen and Schuster 1977, 1978a, b). As the first replicating RNA(P) entities did certainly not exist alone in the vastness of primordial earth’s geological environments, quasispecies likely exchanged molecular mutants within or between entitary compartments (ZnS/MnS or other analogous ones) and those molecules must have interacted with each other by random complementarities. Therefore, the authors developed the concept of interacting and interdependent hypercycles, representing ESSs as mentioned above. There is no space here to go deep into this matter but the following remark of Friedrich Cramer hits the bull’s-eye best: For the understanding of the mechanism of evolution, the theory of hypercycles is comparably the same as what is the quantum mechanics (and I would rather add quantum electrodynamics (QED)23) for the physics of elementary processes (transl. from Cramer 1989).

As the theoretical protoribosome had to be replicated under the rules of the quasispecies concept, it could have been a part of some kind of hypercycle itself. Several interacting RNA(P) molecules took part as parasites or symbionts in replication cycles in analogy to Spiegelman’s monster or functionally as ribozyme-proto-chaperones analogous to modern peptide chaperones (Karbstein 2010). Thus, molecular drivers (Dover 1986; Dover et al. 1982; Flegr 2002; Strachan et al. 1985) were there from the beginning of life with “transposons” as their modern descendants (Lankenau and Volff 2009). In 1976, Crick et al. published a paper on the origin of protein synthesis (Crick et al. 1976). The authors based their argument only on the assumption, that originally no ribosome at all was necessary and that the ordering of amino acids in protein synthesis was accomplished using only “mRNA” and a few primitive tRNAs. The idea helped to blaze the trail for the modern protoribosome idea (Davidovich et al. 2009). However, the deep key question raised by these classical think tanks was the question how initially do tRNA like molecules ( as emerged from ZnS/quasispecies world origins) anneal to mRNA like molecules and then to move the mRNA further with melting and annealing dynamics of incoming and outgoing proto-tRNAs. May be or may not be that tRNA like molecules such as Noller’s duplicator RNA (dRNA) monomers (Noller 2011) participated in the molecular mechanics of quasispecies RNA(P) replication. Important is that at some point, proto-tRNA anticodon loops did “learn” to anneal with “mRNA” monomers. (The causal reason does not matter primarily.) Protoribosomal

23

Bracketed note, added by D.-H.L related to Feynman (1985).

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ribozymes(P)24, similar but smaller than contemporary 23S and 28S rRNAs, joined into these annealing mechanistics. Aminoacyl tRNAs, i.e., tRNAs that were associated with an amino acid – having some stereochemic stability property – joined as well and linked two different amino acids in a ribozyme-catalytic process for reasons we do not know. In modern ribosomes, two tRNAs bind with their anticodon loop to two adjacent triplets of the mRNA termed aminoacyl (A) and peptidyl (P) site and perform a translocation step. This A- to P-site tRNA translocation comprises two highly correlated motions, i.e., a sideways shift and a ribosomal navigated rotatory motion (Yonath 2009b). According to Crick’s et al. argument early on, there was only a skeletal scaffold without all the proteins added during 4 Ga of evolution. The old pioneers came up with an alternative, much less sophisticated primordial mechanism for translocation along a commafree read-off (Crick 1968; Crick et al. 1976; Eigen and Schuster 1978b; Woese 1970). Their idea was based on perfectly plausible, causal inferences: A primordial code, firmly rooted in an quasispecies-replicator world, must have had a frame structure, analogous to the modern triplet, otherwise any germinating message could not have been read off uninterruptedly. They proposed a particular base sequence to which all codons had to adhere. Crick et al. recognized a consensus sequence regularity found in the anticodon loop of present tRNAs, which reads 3‘NRabmUY, where abm is what we call the modern anticodon. N designates any nucleotide, R and Y stand for purine and pyrimidine. Another requirement of ribosome-free or protoribosome-led translation was the stability of the evolving machine. Until the “complete” message is translated, the peptidyl-t-RNA must not fall off before the translocation of the subsequent aminoacyl-tRNA is accomplished. The stereochemistry with the two t-RNA molecules (peptidyl and aminoacyl tRNAs) each binding to an mRNA via dynamic 7 bp interactions involving the ancient anticodon loop consensus is shown in Figs. 11.12 and 11.13. In this model (Fig. 11.12, slightly altered after Woese (Woese 1970)), the mRNA meanders through two sterically antagonistic anticodon loop conformations (FH and fh) of the two tRNAs binding not to three codon-nucleotides as a triplet but to five complementary nucleotides within each anticodon loop. Note that, according to the current structure of the modern ribosome, tRNA anticodon loops only complement with three nucleotides of the mRNA and the decoding translocation mechanics is highly dependent on higher order ribosomal structures (Ramakrishnan 2011). Because of the crystallography data explaining the movement of the decoding machine, it seems futile to discuss Crick’s et al. RNY code mechanism any further. The 5 nt tRNA/mRNA codon interactions are just hot air (Ramakrishnan 2011). Nevertheless, statistical analysis of hundreds of modern genes revealed that it is

24

P designates the option for the association of a random peptide forming random, primordial RNP chromatin.

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always the coding strand with the open reading frame (ORF) that requires the lowest number of computational back mutations in order to reinstall a comma free RNY pattern. Figure 11.13e, f show two examples of such an RNY rhythm analysis. No matter any criticism that such a pattern might be a statistical artifact or a statistical correlation based on codon bias phenomena, it is stunning that Crick et al. posited their model before the RNY pattern in modern ORFs was discovered (Lankenau 1990; Shepherd 1981a, b). Thus, even with our modern knowledge of a structure based ribosome function, the RNY code hypothesis is still reasonable and would call for being replaced by an alternative better one if that existed. Thus, from our present speculations about the ancient RNA world, the RNY code hypothesis still is the best we can derive in a bottom to top approach. However, what role does the protoribosome (Davidovich et al. 2009; Yonath 2009a, 2009b, 2009c) play in this RNY-code world. Figure 11.13a–d merely explains the translocation by sterical conformation shifts (hf<¼>FH) of tRNA molecules along an mRNA. Thereby, it consents tacitly that the in-brought tRNA-coupled amino acids are “somehow” connected to form a polypeptide chain. Nothing is said about the peptidyl transferase step. At this stage, however, the protoribosome enters. In this hypothesis, the protoribosome is an additional ribozyme quasispecies with polymerization capabilities. In association with the Crick-Brenner- Klug-Pieczenik mechanism (Crick et al. 1976) (Fig. 11.13a–d), the protoribosome was complexed with amino acid-coupled tRNAs (Joyce 2002) and mRNAs (Fig. 11.12) and acted as the primordial translation machine. Only if a combination of: 1. Navigated annealing (as in SDSA) and decoding, 2. Conformation-change based translocation, and 3. Peptidyltransfer was accomplished in the same complex could translation have emerged. The theoretical power of Eigen’s and colleague’s coexistent, interdependent quasispecies and hypercycles, perhaps thriving inside Mulkidjanian-ZnS compartments, truly reflects a fundamental theory similarly important for life sciences as what QED is for the physics of elementary processes. Joyce and Orgel’s note that RNA is a prebiotic chemist’s nightmare (Joyce and Orgel 1999) may be true, but the frameworks concerning two RNA worlds set up to explore the obstacles of the primal synthesis (Eigen 1971; W€achtersh€auser 1997) (Fig. 11.1) stand and thrive firmly.

11.4

From Grassroots Level Back to and Beyond the Golden Spike

The majority of sequences comprising modern genomes express ncRNAs that are now recognized to act as sensors, integrators, catalysts, defenders in a universe of possible metabolic processes (Fig. 11.2) (Wang et al. 2011). The transposon world

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Fig. 11.13 The RNY code hypothesis. (a–d) Decoding and conformation driven, translational progression along a comma free RNY code in mRNA. The pre-protoribosomal mechanism requires annealing of the tRNA anticodon loop in the hf conformation (see Fig. 11.12). Movement along mRNA is driven by conformational flips of the tRNA loop (hf <¼>FH). (e, f) Identification of the RNY code in modern genes – two examples. Results of only three of the six possible frames are shown. (e) RNY pattern analysis of the six reading frames of the micropia retrotransposon. Stop codons indicated as bars. RNY is only associated with the long open reading frame (ORF) encoding RT/RNaseH. (f) Positive correlation of RNY with the gag and pol ORFs of the proviral retrotransposon gypsy. This kind of correlation is ubiquitously conserved in all genes analyzed so far. Method as described in Lankenau (1990) and Shepherd (1981a, b)

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is a driving part of it. This is a reflection of the primal beginnings 4 Ga ago when genetic information emerged. The ancient, short RNA monomers and first short RNA chains certainly interacted with many different prebiotic metabolites where amino acids or even short, random proto-peptides must have interacted with RNA. But only when translation – by whatever geo-environmental, chemical, physical, or mechanistic constraints – was established, the tight interaction of proteins and nucleic acids emerged together as chromatin, transporting the genetic information in an uninterrupted lineage (i.e., line of “clones” as in prokaryotes or germlines as in metazoans) to subsequent generations and protecting it by DNA repair. The phylogenetic tree of life was reconstructed mainly based on ribosomal gene information. Here, the ribosomal 16S rRNA played a major role in assessing phylogenetic relationships (e.g., Woese et al. 1990; Yang et al. 1985). However, the major goal of evolutionary research is not tree-reconstruction but to elucidate the driving mechanisms of evolution. The ribosome is a true molecular living fossil. Charles Darwin did not like living fossils for deciphering evolutionary change, as they do not evolve significantly enough.25 The same holds true for 16S rRNA and other ribosomal components symbolizing the “Latimeria of the molecular livingfossils.” Even though for tree of life reconstructions rRNAs did a great job, they did not evolve speedily enough to enlighten us deeply to understand the major, cutting edge transitions (including promiscuous events) as rooted in particular basic causes and mechanisms of evolutionary change. For example, the evolution of HOX genes gave insight into major evolutionary transitions of metazoans (Akam et al. 1994; Gehring 1998; Holland and Garcia-Fernandez 1996; Valentine et al. 1996). However, mediating both, significant evolutionary changes and evolutionary driving forces throughout all kingdoms of life and spanning the entire deep history of life is the realm of RRR, especially of the homology search engine of SDSA. Therefore, as Ernst Mayr once said, it is these basic causes and mechanisms that are the central themes of evolutionary biology (Mayr 1963). As shown here, (proto)ribosomes were a part of an ancient RNA world where other RRR-factories were as old as the protoribosome or older (Eigen’s quasispecies and hypercycles). Thus, RRR-factory related phenomena were the earliest actors of darwinian evolution. As ingredients of their very own geochemical emergence these reproducing molecule assemblies used homology search processes conceptualized with techniques like FRET (Abd..et al. 2008) to form the anthropic basis the anthropic basis for research in moving the science from the general to the particulars, and back to the general, where moving back means: following Popper’s deductive cycle from bottom to top, beyond the golden spike into the complex worlds of replication, and DNA-repair mechanisms (i.e., NHEJ, SDSA, SSA, BIR) (Lankenau 2006; W€achtersh€auser 1997) Recently, Marcel Weber developed the thesis of “causal specificity” which further strengthens our concept here (Weber 2006).

25 Darwin refers to living fossils as “anomalous forms” or “wrecks of ancient life” saved from competition and extermination (see also Fricke 2010, pp. 81–82).

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To explore modern RNA world interactions with modern, chromatin embedded DNA repair will be a lead for forthcoming research. We now know (and re-experience) that attempts to understand chromatin and DNA repair had a difficult start as Francis Crick remarked: “We totally missed the possible role of . . . repair. I came later to realize that DNA is so precious that probably many distinct repair mechanisms would exist. Nowadays one could hardly discuss mutation without considering repair at the same time” (Crick 1974). Also Roger Kornberg reports on difficulties conceptualizing DNA-repair related processes: “ ‘I’ll bet you a bottle of champagne’ said Buzz Baldwin, a colleague in my department, ‘that the nuclease activity in DNA polymerase is part of the enzyme.’ The preparation of the replicating enzyme we had purified extensively could still degrade DNA chains. In the absence of nucleotide building blocks needed for synthesis, nucleotides were cleaved slowly and serially from DNA. I took Baldwin’s bet because it made no sense to me at the time that DNA polymerase would degrade the very end of the chain it would normally be extending” Kornberg (1989). So the RRR properties of life embedded inside molecular 3D networks of chromatin, membrane enveloped nuclei and cells, and even up to replication phenomena of superorganisms such as ants, termites, and honey bees as spearheads of ecology provide one contiguous trajectory throughout evolution, with enough change at hand for each step and enough depth of conserved traits to represent a current theory of evolutionary synthesis. Acknowledgments I dedicate this chapter to father and son W. Hennig, i.e., Willi Hennig and Wolfgang Hennig – the latter celebrating his 70th birthday this year. I apologize to all colleagues whose key contributions were not cited due to space restriction and focus. I am grateful to Peter Vogt for pointing out to me the original RNY-code concept, to Manfred Eigen for discussing the topic and the quasispecies concept with me a long time ago, and to Carsten Lankenau for his engaged help in writing the RNY pattern analysis software during the 1980s. I also thank Victor Corces and Bill Engels for having me in their labs in Baltimore and Madison where the antisense story and the SDSA connections were established. Many thanks also go to Armen Mulkidjanian for accompanying me while learning about his ZnS theory, and to Susanne Lankenau and Richard Egel for engaged discussions and for comments on the manuscript.

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Epilogue

Chapter 12

Integrative Perspectives: In Quest of a Coherent Framework for Origins of Life on Earth Richard Egel

Abstract By taking an overarching approach, this chapter connects various leads from bottom-up and top-down considerations about origins-of-life research. As no firmly accepted standard model for the emergence and early evolution of life on Earth has yet been established, rethinking of old problems and reevaluation from first principles is given precedence over unreflected repetition of widely held assumptions. In brief, a chain of bottom-up inference focuses on tentative transitions from geochemical pore space reactor conditions in upper sedimentary layers, exposed to sunlight and wet/drying cycles. Photo-active metal sulfides, catalytic minerals, organic heterocyclic cofactors, and short prebiotic peptides are assumed to precede the emergence of oligonucleotides. Oligo-ribonucleotides, in turn, speeded up the generation of more and longer stochastic peptides, later on to be replaced by coded protein synthesis. Up to the level of a peptide/protein-assisted RNA world scenario, a primarily photoautotrophic molecular ecosystem is assumed to develop through a range of sessile precellular stages. Conceptionally, early protocells would resemble certain plasmodial/syncytial organisms, rather than free-living microbial cells. The generation and “escape” of genuine cells as quasiautonomous and propagative entities had to await the assembly of DNA-based integral genomes. Such microbial cells of prokaryotic life style diverged in several rounds. Remnants of the communal precellular systems, however, did not die out completely, but organized themselves as more sluggishly evolving cell-like organisms of a different kind. Specializing in recycling of particulate organic matter, these larger organisms retained and perfected much higher degrees of subcellular compartmentalization and cytoskeletal infrastructure, which directly gave rise to the proto-eukaryotic lineage.

R. Egel (*) Department of Biology, University of Copenhagen Biocenter, Copenhagen, Denmark e-mail: [email protected] R. Egel et al. (eds.), Origins of Life: The Primal Self-Organization, DOI 10.1007/978-3-642-21625-1_12, # Springer-Verlag Berlin Heidelberg 2011

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Abbreviations aaRS ATP DNA LUCA LUCAS mRNA mtDNA NAD(P) PLP PRPP PTC RNA RNP RNPd RNY rRNA rTCA SIPF snoRNA sRNA tRNA UV

12.1

aminoacyl tRNA synthetase adenosine triphosphate deoxy ribonucleic acid last universal common (or cellular) ancestor last universal communal ancestor state messenger RNA mitochondrial DNA nicotinamide adenine dinucleotide (phosphate) pyridoxal phosphate 50 -phosphoribosyl 10 -pyrophosphate peptidyl transferase center ribonucleic acid RNA-protein (complex or particle) RNA-peptide (complex or particle) R: purine (A or G) Y: pyrimidine (U or C), N: any type of nucleic base ribosomal RNA reductive tricarboxylic acid (cycle) Salt-Induced Peptide Formation small nucleolar RNA small RNA non-translated transfer RNA ultraviolet (radiation)

Semantic Preliminaries

Music can be described, but not defined (Isaac Stern). – Perhaps the same is true of life itself. (Lazcano 1994)

The current book addresses the emergence of life on Earth from abiotic sources. This profound transition in our planet’s evolutionary past comprises an intricate cascade of molecular self-organization, which is still far from being understood. For all we know, the primordial Earth began its existence in a sterile and inhospitable state, carrying no life at all to begin with. From a scientific viewpoint, therefore, it is most rational to assume that life as we know it has newly emerged from non-living matter at least once. To be inferred as a series of prehistorical events, however, it is impossible for us ever to know all the details. There are no precedents – no circumstantial evidence other than the existence and universal relatedness of all the living organisms today, and rather spurious fossilized remains from distant past. Hence, scientific approaches to this enigmatic subject are limited to the gathering of plausible arguments from physico-chemical and biological mechanisms or model experiments for a coherent narrative that does not

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violate any widely accepted principles, such as elementary and non-equilibrium thermodynamics. Considerable debate concerns the applicability and range of certain terms to phrase such narratives. What exactly do we mean by “Life” as such? What is so fundamentally different about the functional organization of living organisms, as contrasted with any form of non-living matter? On the one hand, descriptive statements about the essence of life are numerous in the literature (e.g., Popa 2004; Ruiz-Mirazo et al. 2004; Gayon 2010; Forterre 2010; Jagers op Akkerhuis 2010). On the other hand, when it comes to condensing these narratives to a widely accepted – normative – definition of life as such, many authors – this editor included – tend to give up or even question the utility of striving for a normative guideline as a common goal (Hengeveld 2010; Popa 2010). Usually, biologists tend not to pay much attention to defining the essence of life before they investigate various aspects of its manifestation in their particular research subject. They can, in fact, rely on an enormous “outgroup” for referential comparison amongst all forms of “life as we know it” here on Earth. Even when it comes to the ultimate question of how to understand the emergence of organismal life on Earth, the ultimate target is at hand. Yet, the transitional process has likely passed through many stages, with hardly any clearcut boundaries in between. Thus, describing the conceivable paths for the entire range takes precedence over where exactly in such a continual evolution life as such has begun. Exobiologists, however, in considering the occurrence of life-like systems on other planetary worlds, have no actual alternative life-like systems to go by, which gives higher priority to defining the essence of life in decisive terms beforehand (Tsokolov 2009). For biologists in general, “Life means being-alive of discrete entities – organisms” (Penzlin 2009), putting focus on the programming of metabolic reactions, internal homeostasis, and genetic replication. All this is held together by cellular containment, yet allowing controlled exchange with the environment. From a physical chemist’s perspective, however, superior systems properties of preexisting disequilibria, dissipation of potential energy, and the closure of autocatalytic networks appear of paramount importance (e.g. Kauffman 1993; Gladyshev 1999; Bartsev 2004; Ho and Ulanowicz 2005). More generally speaking, the essence of life as such does not only concern the difference between a single living cell and its abiotic surroundings, but also the self-regulating system-wide properties spanning populations, ecosystems, or the entire biosphere. Thus, the emergence of pre-macromolecular collective systems with life-like properties steps in as the critical threshold for the origins of life (Shapiro 2006, 2007). Conceptionally, all life-like entities in the prebiotic transition phase can be subsumed as members of so-called fuzzy sets (sensu Zadeh 1965), applying fuzzy logics of partly overlapping class membership to deal with the enigmatic origins of life itself (Bruylants et al. 2010). A particular operational profile goes as the “NASA Definition of Life” (Luisi 1998): “Life is a selfsustained chemical system capable of undergoing Darwinian evolution” (Horowitz and Miller 1962). – Is this a verifiable criterion at all, and can evolvability as such be subject to rigorous scientific inquiry and elucidation? – Random motions and elastic collisions alone, of ever so many independent atoms or

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molecules, cannot explain the functioning of present life, let alone its emergence in a distant past. In some peculiar manner, it is the self-similar maintenance and propagation of certain patterns of inelastic interactions that distinguishes life from other complex systems. These patterns unfold as contiguous subsets in vast combinatorial spaces of weakly interacting components. This unfolding is characterized by the mutual entanglement of two disparate principles – self-complication on the on hand, and self-simplification on the other (Conrad 1990). As the latter tendency on its own would drive complex unstable systems to disintegration, it must incessantly be counteracted by innumerous adjustments. Self-complication in terms of redundant components and weak interactions are assumed to stabilize the system sufficiently to carry on. For semantic reasons, too, the phrasing of self-organization deserves attention. This catchy term is widely adopted in physical thermodynamics of complex non-equilibrium systems, as pioneered by the concepts of dissipative structures (Prigogine and Nicolis 1967; Prigogine and Lefever 1968) and self-organized criticality (Bak et al. 1988), and followed up in many reviews (Haken 1983, 2006; Lewalle 2008; Barbu 2010). Moreover, a general consensus is building up that such concepts can fruitfully be applied to understand the emergence of life as well (Peacocke 1983; Kauffman 1993; Perry and Kolb 2004; Salthe 2004; Morowitz and Smith 2007; Smith 2008; Pulselli et al. 2009). It is in this tradition that “self-organization” marks the title of the current book. Viewed in historical perspective, about two centuries ago, the self-organizing term was introduced to characterize the peculiar state of living matter. As natural organisms are composed of different organs, all of which are generated in mutually interdependent ways, they are both organized and self-organizing beings (Kant 1790). Later on, as a veritable change of paradigm (Keller 2008, 2009), the concept of self-organization has evolved by generalization into engineering and physicalchemical science, shifting both range and emphasis with time. Thus, adaptive evolutionary processes are by no means reserved for biotic systems, but can unfold in purely physical surroundings as well. Provided the starting set of components is sufficiently complex and appropriate gradients of potential energy prevail, the ambient boundary conditions need not, and will not in general, remain constant over time. This is how compositional and interactive evolution comes about. This shift of paradigm has recently been criticized as “category error” and “illegitimate merging of [two disparate] concepts”, where “self-ordering is confused with self-organizing” (Abel and Trevors 2006; Abel 2009). Yet, this narrow restriction on a generalized and widely accepted descriptive term is bound to cause more confusion than what it is supposed to clarify.1 There is both order and organization in many physical collective systems, living organisms, or entire

1 Intended or not, the underlying insistence on a primacy of deductive logic in deceptive jargon is playing the creationists’ game against scientific modesty. In particular, the various assertions declared as “null hypotheses” evert the scientific usage of this term. As phrased like “Physicodynamics alone cannot organize itself into . . . (functional systems). . .”

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ecosystems. Mere ordering concerns the establishment of static relationships, invariant of time, whereas organizing emphasizes dynamic flow and changing interrelations among the various and numerous components of a complex system. Quite obviously, self-organization of living organisms is more complex than in other collective systems, based on abiotic interactions. To underscore this notion, a novel qualifying term could point out which additional feature decisively distinguishes living organisms from physically simpler organization modes. Codification or encryption of sequential instructions could suite this purpose. The sequence-encoded instruction of protein synthesis by nucleic acids, as commonly observed in all the organisms living today, is indeed the hardest to explain by stochastic fluctuations in the physical environment alone. Within the more general framework of self-organizing trends at various levels, future efforts should specifically aim at narrowing in on this transition, by specifying the most likely conditions for self-codification or self-encryption of marginally biased amino acid sequences, so as to be constrained by and eventually encoded in nucleic acids. Approaching this goal has been a prime objective in conceiving the agenda of the present book. Broadly speaking, two lines are followed by scientists to deal with the tentative origins of life from opposite sides, yet leaving an unbridged gap in between (Penny 2005; Pereto´ 2005; Penzlin 2009). The bottom-up (physical/chemical or forward) approach explores the potential of early-earth conditions and physical/chemical systems of small components to become more complex and gradually develop lifelike properties. The top-down (biological or backwards) approach, on the other hand, takes life as we now know it as a starting point and tries to deconstruct its vast complexity into tentative precursory steps. Whilst bottom-up models preferentially rely on decisive experiments to validate the reliability of pertinent hypotheses, the top-down deduction from the living state is bound to remain more speculative. As yet, a reconstruction of fully functional life-like precursory systems defies experimental verification, and the limited access to experimentally tractable aspects of the top-down approach will always depend on a multitude of tenuous additional assumptions. Nevertheless, the bridging of the conceptual gap between forward and backward analyses of prebiotic evolution remains a valid scientific goal. Over the last 50 years or so, this gap has been narrowed considerably already. The current book is conceived and written in confidence that this trend continues. The main focus is on a number of experimentally tractable model systems to advance and strengthen the bottom-up approach, whilst some tentative connecting lines to cellular life are also laid out in perspective (Sects. 12.12–12.16). Next to the primordial coevolution

(Abel 2009), these statements by no means describe the most trivial explanation from a scientific point of view. If true, that is, they definitely would exclude the emergence of life on earth from any scientifically sound hypothesis in terms of physical/chemical reactions, but should require some extra-scientific or supra-natural input instead. By that token, these so-called null hypotheses take a creationist position for granted, until it be decisively refuted by experimental evidence.

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of metabolism and organic catalysts, nowhere do two disparate aspects of macromolecular evolution appear as intertwined as in the formation of proteins and nucleic acids respectively by polymerization of amino acids and ribonucleotides (Sects. 12.10 and 12.11). Fundamental evolutionary problems are often riddled by conceptual “chicken–egg” alternatives. What came first? – The escape, in fact, can be surprisingly easy. There were certainly eggs before there were any chickens, but those ancestral eggs were no chicken eggs yet. On a more general level, however, the tightly interwoven dependence of two distinct phenomena upon one another can have emerged together from an indistinctive precursory stage, as followed by coevolution in parallel. In terms of complexity theory, this is a critical bifurcation point of symmetry breaking (Sect. 12.3). “Thinking out of the box” to grasp the gist of such a proposition may at times be easier in one language than another. To wit, the concept of metabolism is deeply ingrained in biology as metabolic reactions being programmed by gene-encoded enzymes. Accordingly, the bold idea that a more or less equivalent protometabolism might have preceded any cellular life as we know it may be considered preposterous by observant scholars. In a German scientific background, however, the corresponding term is slightly different – Stoff·wechsel – literally meaning (ex)change of matter, which makes it more readily conceivable that quite profound channelling in the exchange of matter – resembling much of core metabolism – might have preceded the emergence of gene-encoded enzyme synthesis (Sect. 12.9). The semantics of life would be incomplete without mention of information, which is closely related to memory in any form. Evolutionary self-organization always depends on some kind of memory to evade stochastic equilibration in the flow of time. In modern life, the biochemical memory is primarily based on DNA, both as a durable substance and as a propagative agent, preserving self-similar characteristics through many rounds of doubling up. – Was there another life before DNA? – There is indeed consensus about RNA having developed quite similar genetic potential before DNA eventually took over – the “RNA World” scenario (Gilbert 1986; Gesteland et al. 2006; Chaps. 10 and 11). Yet, what was the world like when RNA was about to emerge as a replicating material? Was there some substitute “pre-RNA” (Joyce 2002), or rather no replicator in the genetic sense at all? – The agenda of this book is guided by the concept that any pre-“RNA World” scenario more likely entailed a pre-genetic world without sequential replication, rather than tentative pre-RNA as genetic remedies – more specifically proposing a modified RNA world scenario that from the onset was assisted by prebiotic, stochastic peptides. Even today, each cell has more than a single kind of memory. Each newly replicated molecule of DNA is born into a pre-informed receptacle, comprised of preexisting macromolecules – proteins, RNAs, and so on. Complex membrane assemblies, in particular, often require appropriate seed structures for efficient growth and/or duplication. Their inherent spatial constraints are attributed to memories of structural, positional, or compositional information. Moreover, so-called epigenetic modifications of genetic material can further blur the distinctive gap

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between genetic and non-genetic types of memory. Conceivably, self-organizing repositories of solely compositional and structural information have preceded the emergence of genetic memory. Future progress in comprehending the origins of life will likely focus on this pivotal transition – the genetic takeover from agenetic precursory systems (Sects. 12.9–12.11). It is of vital importance, of course, that the physicochemical continuity across this transition had to be secured in terms of continuous and exploitable energy flows (Chap. 1). To conclude this discourse on conceptual terminology, certain more generally applicable ideas appear too valuable to remain firmly associated with the experimental background in which they first happened to be proposed. Two particular examples are worth mentioning here already, whilst details are discussed further below. For instance, the concept of surface metabolism is so radically different from reaction schemes in free solution that the chemical nature of the supportive surface for which this term was first proposed need not be a defining part of the generic term as such. More colloquially, the underlying difference of scale is epitomized by contrasting primordial soup models in a three-dimensional volume with two-dimensional primordial pizza dynamics in surface-associated conglomerates (Sect. 12.4). Also, the seminal idea that a tentative Peptide world might have been preceded an RNA world, yet free of DNA, need not be exclusively linked to a particular mechanism, by which prebiotic peptides have been proposed to have formed in the absence of RNA (Sect. 12.10). As mentioned before, the emergence of life on early Earth comprised a historical series of transitions, which as such cannot be subject to experimental repetition. By applying scientific reasoning and experience, however, we can try to re-enact in our minds how it most reasonably could have happened, taking as much as possible of current knowledge about physicochemical processes into consideration – relevant model experiments included. Due to the complexity and speculative dimension of this endeavor, split opinion on controversial issues is inevitable (Pereto´ 2005). In particular, the primordial entanglement of mRNA splicing and the origins of eukaryotic cell organization is still subject to one of the deepest schisms on basic evolutionary issues (Koonin 2006a, 2010; Kurland et al. 2006; Penny et al. 2009; Poole 2010). To present a coherent narrative in this chapter, I have deliberately taken sides on these issues beforehand, in favor of introns-first and protoeukaryotesearly scenarios (alternative views are referred to in above citations and Sects. 12.13–12.16). The phylogenetic discourse at the end of this chapter addresses the tentative nature of common ancestry, from where the main subtrees of present life have since diversified. How should the hypothetical organism at the deepest recognizable branching point be referred to? – The currently most popular acronym is LUCA (Last Universal Common Ancestor), but this is not without some disagreement. Its usage traces back to a workshop held in 1996 (Lazcano and Forterre 1999), and it appears to represent a euphonic merger between two preceding terms – Last Universal Ancestor (Koch and Schmidt 1991; Forterre et al. 1992) and Last Universal Cellular Ancestor (Forterre and Philippe 1999; Philippe and Forterre 1999). Whilst the former term should indeed have served its purpose well, it

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apparently fell short of the appealing LUCA acronym. The cellular implication, however, although it allegedly claims priority for the LUCA acronym, is a potentially meaningless overdefinition. It certainly put stress on “cellular” to take exemption of viruses, but it boldly presupposes that the Last Universal Ancestor indeed was organized as cells in a modern sense. More recently, either form has been extended to LUCAS, adding State to the preceding ones (Woese 2002; O’Donoghue et al. 2005; Koonin 2009). As cellularity does not necessarily arise so early (Sects. 112.5 and 12.16) and “common” is rather redundant with “universal” to begin with, LUCAS is used throughout this paper, preferring “Communal” as an alternative qualifier for the collective nature of the precellular state in question. Discussing the likely nature of the tentative LUCAS proto-organism(s) is intrinsically entangled with the Tree of Life concept, which itself has come under intense debate (O‘Malley et al. 2010; Doolittle 2010). The challenge is to match the goal of revealing the organismal phylogeny with the operationally available means, which mainly give insight into trajectories of individual genes. As a matter of fact, more and more genes tell stories that are not the same, and integral views are still evasive. Not unlike real trees, the iconic dendrogram to represent the universal phylogenetic Tree of Life may need to accommodate multiple roots beneath its tentative emergence from a point-like trunk. Moreover, transforming observational data into integral theories is also about sifting through signal-over-noise considerations. Instead of dispairing over the overwhelming nonconformity in 99% of inherently fuzzy data sets, the “trees of one percent” in prokaryotic coherence (Dagan and Martin 2006) are worthy of note all the same.

12.2

Early Earth in Context

An eerie window of fortune facing the Sun

The only life we actually are aware of in the universe is remarkably fit for the Earth we are living on. After all, the living state has uninterruptedly managed to survive on our planet for 3–4 Ga (giga years). The Earth, in turn, is quite fit as well for our kind of life (Barrow et al. 2008) – very much in contrast to our neighbors in the solar system. What is it that actually makes Earth so special? When life happened to appear, the early Earth was certainly quite different from present conditions, and tentative details are still under debate (Lunine 2006). Our planet is rocky and big enough to keep an atmosphere, and there is plenty of liquid water at the surface, which is considered central to the so-called circumstellar habitability zone around the Sun. On either side of this zone, Venus is too hot and Mars too cold to carry a liquid hydrosphere. Although our Moon is likewise in the mid zone, it falls short on these criteria, since volatiles – including water vapor – rapidly escape into open space, due to its weaker field of gravity. Furthermore, these basic conditions are subject to dynamic changes in space and time; so for any

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potentially appropriate planet, the actual window of opportunity may only stay open for a limited period. For Earth in particular, the window of biogenic opportunity opened early, in the aftermath of planet formation and subsequent episodes of violent impact collisions. It is remarkable indeed that Earth’s life-surportive environment has been quite stable over several billion years, and this robustness is directly linked to the existence of the terrestrial hydrosphere (Henderson-Sellers 1981). The prebiotic atmosphere was dominated by CO2 and was at much higher pressure than today, exceeding 20 MPa, before most of the water had condensed in the ocean (Kasting and Ono 2006). With the availability of liquid water, the so-called carbonate– silicate cycle began to adjust atmospheric CO2 concentration to intermediate levels, in balance with calcium weathering from silicate rocks and calcium carbonate precipitation from the relatively acidic water accumulating on the early Earth2 (Morse and Mackenzie 1998). Due to the virtual absence of molecular oxygen, ozone in particular, the influx of ultraviolet (UV) irradiation was considerably higher at the surface of the Earth. Once cellular organisms had become able to spread around the globe, they not only closed the window for the generation of additional independent life forms – due to their effective propagation, adaptive diversification, and competition for limiting resources. They also profoundly changed basic geochemical conditions resulting in the accumulation of molecular oxygen, the virtual elimination of sulfur volatiles from the atmosphere, the precipitation of most iron from the pristine oceans as massive banded-iron deposits, and the sequestration of carbon compounds in biogenic limestone carbonates and hydrocarbon-bearing sediments. Moreover, the Earth is peculiar in other ways, which should not be disregarded in the context of primordial biogenesis. Amongst the rocky planets, only Earth has a large companion at close distance – the Moon. Also, Earth is unique in having dynamic plate tectonics of a relatively thin crust, as opposed to the single-plate, stagnant-lid regime prevailing on all our rocky neighbors. Whilst the formation of the Moon is reasonably explained by a grazing impact with a Mars-sized protoplanet (Cameron 2001; Canup and Asphaug 2001; Canup 2004), the origin of plate tectonics is still considered enigmatic (Condie and Pease 2008; Martin et al. 2008). These phenomena may well be causally related. On a global map (Tarr et al. 2010), the relative motion of crustal plates at subduction zones and spreading ridges is highest on either side of the equator, but tapers off in the polar regions, which somehow links the driving mechanism to the rotation of the Earth. Indeed, rotation and tidal despinning have recently been invoked as critical factors in organizing plate tectonics (Riguzzi et al. 2010). Also, tidal heating has presumably been more pronounced in the distant past,

2 This cycle operates by burrying limestone carbonates in sediments, which in turn are recycled by seafloor subduction and CO2 outgassing in volcanic eruptions. As carbonate precipitation increases with rising pH, the biogenic alcalization of the oceans greatly accelerated CO2 depletion, thus lowering its concentration to modern levels.

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Table 12.1 Time line of landmark events on early Earth ‘Dial’ Gyr Landmark event 00:00 4.6 Solar planets form by accretion from a proto-planetary disc 00:30 4.5 Moon-forming collision 01:00 4.4 Zircons/felsic-granitoid rocks protocrust 03:40 3.9 Late Heavy Bombardment 04:10 3.8 Continental formation started afresh 04:30 >3.7 Isotopic footprint of oxygenic photosynthesis 04:40 3.7 Recycling event of the Hadean crust, subduction-altered slabs in mantle 05:45 3.5 Oldest microfossils 13:35 2.0 Low oxygenation of atmosphere and shallow seas 14:35 1.8 Deep oceans mildly oxygenated 20:20 0.7 2–3 periods of global glaciation 21:25 0.5 Cambrian Explosion of multicellular animals 22:25 0.3 Carboniferous maximum of global oxygenation 23:00 0.2 Dinosaurs 23:30 0.1 Early birds and mammals 24:00 0.0 Modern world

both on the nascent Moon and early Earth (Touma and Wisdom 1994). The Moon, therefore, may have held the key to the origin of plate tectonics, thus starting a massive recycling operation involving essentially all the oceanic crust, and much of the continental crust as well. Perhaps, we even need plate tectonics to have life on Earth at all (Martin et al. 2008). A time line of subsequent events is layed out in Table 12.1. After the cataclysm of the Moon-forming collision had passed, the Earth had consumed most of the proto-Moon’s iron core. Conversely, a significant fraction of the proto-Earth’s mantle was splashed into orbit and accreted on the Moon. Thereupon, the Earth was physically locked to a large companion, constantly dissipating kinetic energy by tidal retardation. The spinning rate has decreased ever since, at the expense of increasing the distance to the Moon. Accordingly, day length was considerably shorter on early Earth, only at ~50–70% its present value (Williams 2000; Denis et al. 2002). Up to today, convection in the disproportionately large iron core of the Earth still drives a strong dynamo and magnetic field, shielding living matter against ionizing cosmic ray particles. Largely protected by the gravitational shield of Jupiter, some 400 Myr are assumed to have been relatively calm on Earth, after impacts from the Moonforming event had subsided. By dating durable zircon minerals, some indirect evidence indicates that proto-continental crust had been differentiated during that time already (Cavosie et al. 2004). Yet, collisional turmoil set in once again. As possibly triggered by an orbital instability of the outer planets (Gomes et al. 2005), the terrestrial planets were subject to a spiking episode of late heavy bombardment, which is best documented for the dating of impact-melt rocks on the Moon. It was generally assumed that the largest impactors were able to sterilize the entire Earth, had any proto-life already emerged by then; but this view has been contested (Chapman et al. 2007). At any rate, both life and modern-style plate tectonics

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appear to have started about that time or shortly after (Rosing and Frei 2004; Kamber 2007). The oldest microfossils hitherto described (Schopf 2006; Javaux et al. 2010) are dated reasonably close to this primordial era, whereafter the slowly increasing oxygenation of the atmosphere and oceans (Holland 2006) began to revolutionize the boundary conditions for further Darwinian evolution, as a necessary precondition for all the macro-organismal “higher” forms of life.

12.3

Dissipative Synergetics

Collective channeling, or How to start a hurricane

Comparing rather diverse kinetic phenomena has uncovered far-reaching similarities, as of tropical cyclonic depressions, orogenic island arcs, continental drainage patterns, or living organisms. All of these dynamic entities couple the dissipation of potential energy to collective flow and concerted redistribution of matter. As such, they are part of superior recycling systems at a global scale, which keep the entire Earth in a gradually evolving state of quasi-equilibrium (Table 12.2). At different levels, the formation of stars and galaxies on the one hand, or socio/ political interactions on the other, can likewise be ascribed to energy-dissipating aggregation. Such entities3 are physically described as dissipative structures (Prigogine 1978). They generally develop in non-linear, far-from-equilibrium conditions, where stochastic fluctuations close to a critical bifurcation point of instability play a crucial role in their emergence. Thus, once started by indeterminate environmental fluctuations and driven by a persistent external energy potential, such systems tend to grow by progressive entrainment, transferring more and more matter through ever widening channels. This focused channeling is not necessarily confined to conspicuous physical structures; in a more figurative sense, it also applies to the concept of biochemical reaction pathways4 and the like. Even beyond the cellular stage of basic life, more complex phenomena at the level of multicellular differentiation or collective behavior of swarming animals, self-organization is a well recognized principle of understanding (Camazine et al. 2001). Notably, the self-organizing channeling effect is often subject to autocatalytic – “winner-takeall” – mechanisms, which progressively alter the external boundary conditions in favor of the maintenance, growth and evolution of the coherent energy-dissipative entity. It is this interactive relationship between a vigorous system and its environment that lies at the heart of self-organization as an emergent property. The hurricane example, in particular, is a physical system dominated by convective flow of gas and vapor, where the temperature/humidity difference between

3

such as ripples on a beach, or a convective supercell creating a tornado. e.g. the regularly oscillating Beluzov-Zhabotinsky reaction

4

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Table 12.2 Dissipative subsystems in global recycling Example Superior system Circumstances Hurricanes Tropical Seasonal north/south convergence excursions, coriolis force

Recycled matter Driving force Water, vapor Solar warming, convective release of latent heat Crustal minerals, Terrestrial heat, Mantle convection, CO2 crust movements, radioactivity, partial melting tidal friction Erosional channeling Water, minerals Gravitation

Orogenic arcs

Plate tectonics, subduction

Watersheds

Precipitation backflow Ecosystems, Metabolism, growth, biosphere, life propagation, inheritance

Live beings

Organic/ inorganic carbon

Atomic electron potential, activated by sunlight or geochemical disequilibrium

tropical ocean waters underneath and cooler air masses of sub-arctic origin in the upper atmosphere has surpassed a critical threshold of unstable layering. The emergence of such coherent entities has been summarized as follows. “There are always small convection currents appearing as fluctuations from the average state; but below a certain critical value of the temperature gradient, these fluctuations are damped and disappear. However, above some critical value certain fluctuations are amplified and give rise to a macroscopic current” (Prigogine 1978). Similar considerations apply to convective cells in the liquid phase as well, such as salt fingers in marine stratification (Inoue et al. 2008) or hotspot mantle plumes, the underlying source of upwelling volcanism (Yoshida and Ogawa 2004). In other examples, convective fluctuations alone can hardly explain how a newly occurring dissipative structure may have started. These have to be considered case by case. As for the emergence of plate tectonics, it is the stiffness of the solid crust that stifles sufficiently large fluctuations, required to initiate subduction of crustal plates down into the mantle. In modern times, at least, full-fledged plate tectonics are primarily driven by buoyant drag of sinking slabs at subduction zones, rather than by actively being pushed apart at spreading rift zones. Accordingly, it is most reasonable to postulate a preceding stage of proto-plate tectonics on top of a magma ocean – well before a solidified, stagnant lid could have covered the entire globe (Stern 2008). The cataclysmic fluctuations accompanying the Moon-forming impact, together with high tidal forces in the aftermath, may have been instrumental in setting primordial proto-plates in motion from the very start. Taken at face value, the remaining examples of Table 12.2 – watershed draining patterns and living organisms – may not have much in common. At any rate, the organizational complexity of life is vastly higher. Yet still, certain commonalities can be noted and might point at comparable mechanisms in their emergence. Both examples are dominated by the restructuring of fractally corrugated surfaces (Sect. 12.4), together with the filling and emptying of transient reservoirs of

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potential energy at various levels. Driven by the energy potential, cascading flow patterns develop and act back on their surroundings. The surfaces in question belong to, respectively, the pristine Earth at the verge of carrying liquid water, and the interface between watery solutions and hydrophobic spaces at various scales. The energies respectively involved are gravitation, directing rain water to the lowest possible destination, and chemical redox potentials to move electrons about in biochemical reactions, together with adsorptive binding forces. The relevant point of which type of geochemical environment could best suit prebiotic selforganization is taken up in the following section.

12.4

The Fractally Corrugated Framework of Being

Rough and kinky at any scale

One of last century’s great mathematical achievements is Benoıˆt Mandelbrot’s realization of fractal objects – a multitude of strangely fascinating entities that populate geometry between the familiar spaces of one, two, or three dimensions. Lying between integral numbers, their dimensionality is fractional.5 Many fractal structures represent the appearance of real objects of the natural world more faithfully than the smoothened regular shapes of classical geometry (Mandelbrot 1977/1983). To add yet another important dimension to published descriptive statements on essential properties of living matter (Sect. 12.1), it is worth noting that living organisms have exaggerated the roughness at the surface of the Earth enormously – starting at the molecular scale of catalytic sites at micelles or membranes, and culminating in the macroscopic appearance of forest communities or coral reefs. A good example of roughness in the inorganic world is a fractal landscape (Wikipedia 2010a) – composed of stochastically distributed sloping angles, ridges, furrows, peaks, and depressions – which is a useful model to facilitate the selforganization of emerging river systems (Rodriguez-Iturbe and Rinaldo 1997). Here, I mainly emphasize the role of surface reservoirs in modulating drainage patterns,6 to be compared with life-maintaining storage facilities later on. Stochastic depressions in a sloping fractal landscape function as natural reservoirs, which retard the flow as soon as rain water begins to collect at the surface. Yet, once they are filled up completely and start to spill over at the sill, the temporarily stored energy is released in backward erosion of the outflow channel. Uphill ponds and lakes, therefore, are geologically unstable. Rain water alone cannot build dams to extend their life time. In the frozen state, however, snow packs and glaciers can likewise retard the flow, and even carve new depressions into the mountain slopes.

5 A common feature of fractal objects is the lack of smoothness in their contures at any scale of zooming in. Thereby they fill much of a 2-D or 3-D enveloping space, but never completely so. 6 Real rivers, of course, are also subject to surface–groundwater exchange.

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On the other hand, balanced series of terraced ponds and ditches can be integrated in natural or man-made life-supporting systems. Most of these, in fact, require the effort of living organisms to be maintained intact. At the borderline of geochemical and biological activities, calcareous terraced ponds tend to grow by CO2 degassing and calcite precipitation at their rims, which is mainly due to physical cooling at travertine hot springs (Chafetz and Guidry 1999), but greatly stimulated by photosynthesis of algal mats and mosses at tufa terraces of karst valleys (Golubic´ et al. 2008). More elaborately, extensive dam/pond systems are organized by beaver colonies, whilst terraced rice paddies, cooperative watermill associations and modern hydroelectric plants have resulted from human industrious activity. Certain macroscopic features of flow, energetics, and evolution in cascading pond and drainage systems formally resemble other processes, which at the molecular nanoscale have led to the organization of living matter. Repeated retardation of flow at intermediate energy potentials, as well as funneling of downward flow into conducting channels can be found again at the biochemical level in diverse reaction pathways. The retardation in reservoirs has an important buffer function, making energy and matter available more evenly than following environmental fluctuations directly. Given appropriate controls, it can assume accumulator function to be discharged upon demand. With proper plumbing, too, the energy difference between adjacent ponds can be extracted for doing work in driving mills or turbines, and sending electricity to users far away. It is the latter aspects of engineering and design that are most difficult to be transferred to formally analogous features of life-maintaining processes. Ultimately, that is, the precursors of composite enzymes and molecular machines have emerged by nanoscale self-organization, the mechanisms of which are yet to be rationalized in physico-chemical terms. For enzymes and molecular machines to work, they rely on interactive surfaces where polar and apolar epitopes are reproducibly arranged in three dimensions, i.e., these surfaces are corrugated or folded in a fractal manner, and they are so in a programmed way. The natural tendency of organic amphiphiles to associate as bilamellar membranes (Sect. 12.6) extends the foldability of biogenic surfaces to protocellular architecture at supra-molecular scales. Inasmuch as the prebiotic evolution towards life on Earth has likely been governed by the structuring of catalytic surfaces in progressively greater detail, the assumption of some kind of primordial surface metabolism (W€achtersh€auser 1988) is quite valuable as a general evolutionary concept. Hence, this term as such should not be solely associated with the particular kind of pyrite-driven chemistry (W€achtersh€auser 1992) for which it was first proposed. In fact, the notion of catalytic surface interactions has earlier roots in clay-driven models of geochemical self-organization (Bernal 1951; Cairns-Smith 1982). Colloquially, the evolutionary potential of surface metabolism models has been referred to as “primordial pizza” dynamics (Maynard Smith and Szathma´ry 1995; Cza´ra´n and Szathma´ry 2000) – in contrast with earlier notions of some “primordial soup”, feeding prebiotic reactions

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in solution, which no longer are considered adequate. The potential importance of pore space in the self-organization of prebiotic matter is likewise well recognized in the related field of exo- or astrobiology (Colgate et al. 2003). In other words, the patchy environment where life can have started may best be conceived as biofilmlike molecular associations in some kind of geochemical reactor with many internal surfaces. As for sustaining modern life on Earth, two primary energy sources are being tapped to activate electrons to higher energy states: Absorption of sunlight and geothermal heatflow. Either source has been considered suitable for charging the transition from a biogenic geochemical reactor to life as we know it, but expressing preference for one over the other has historically shifted from time to time (Chap. 1). Influentual current authors tend to favor deep-sea hydrothermal vents as primordial cradles of life (Russell and Hall 1997; Martin and Russell 2007; Koonin 2007), where sunlight does not prevail and thermo-chemical imbalances are the only potential source of energy. This was not always so, however, and it is not entirely clear why earlier held views that sunlight indeed was the most obvious choice have virtually vanished to oblivion, at least transiently so. Several entries to the current book emphasize various aspects that only prevail at the triple junction where solid minerals are exposed to crucial influence from both hydrosphere and atmosphere – and sunlight irradiation, for that matter – while other entries are readily compatible with either backdrop. Thus, the case for an abyssal origin of life is by no means considered to be closed or conclusively settled yet. Incidentally, the pioneering views of Oparin, Haldane, Miller, and others (for historical perspectives, see Raulin-Cerceau 2004; Lazcano 2010) were also associated with “primordial soup” kinetics, which are no longer considered tenable. Without this unnecessary presupposition, an updated synthesis toward a subsurface pore space reactor scenario in a coastal or river delta setting should be able to integrate effective conversion of solar energy into a primarily photoautotrophic molecular ecosystem (Mulkidjanian 2009; Hagan 2010; Chaps. 3 and 4), from where cellular life eventually can have emerged. In comparing the abyssal-vent and tidal-beach or tidal-mud-flat scenarios, two different modes of reactor dynamics are prevalent. While the porous precipitation chimneys at abyssal vents operate by continuous flow patterns in a permanent thermal gradient field, accompanied by thermodiffusion (Baaske et al. 2007; Koonin 2007), the uppermost layers of sedimentary flats at sheltered embayments of tidal beaches are dominated by reciprocating flows and periodic desiccation. This is accompanied by swelling and shrinking of the hydrated pore space underneath, as well as shifting of chromatographic fronts, where adsorption alternates with desorption. Yet, more importantly, the unending influx of chemically useful sunlight energy, as harvested by ZnS-mediated photo-activation (Mulkidjanian 2009), endows large areas with a biogenic potential. Also, with net degradation of delicate organic molecules prevailing under hot hydrothermal vent conditions (Eschenmoser 2007; Aubrey et al. 2009), the odds for successful biogenesis must have been higher in moderate beach scenarios.

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R. Egel

Selective Chemistry of Living Matter

Biological bulk elements and essential traces

Add water to these fractally corrugated surfaces, and you have the principal ingredients for organizing life – “Life is water dancing to the tune of solids” (Szent-Gy€orgyi 1972, p. 9). The music to this reel dance resounds by means of electrons in resonance – some of them polarized, others not. The solids at the water interface are lipids, proteins, and other carbon-based macromolecules, which locally are semi-fluid, but stick together by numerous noncovalent interactions. Much has been written about the exquisite suitability of water to host the emergence and maintenance of life on Earth, owing to its peculiar and exceptional physicochemical properties (Brack 2002; Pollack et al. 2006). Due to its higher boiling point, it is liquid where ordinary molecules of similar size would be gaseous. Foremost, liquid water is a polar solvent, which is dominated by hydrogen bonds and spontaneously dissociates into H+/OHion pairs, albeit at the low proportion of 107 at 22 C. This moderately reactive medium then engages in a highly productive collaboration with carbon-based chemistry, which progressively diversifies by catalysed reactions at structured water–organic interfaces. This organic kind of chemistry is dominated by rather few elements, which in the periodic table (Wikipedia 2010b) cluster as “other nonmetals” (Fig. 12.1). Four lightweights (C, O, H, N) make up the macromolecular bulk of bio-matter; minor constituents occur as functional groups (S, P, Se). In addition, the autonomous maintenance and growth of macromolecular biosystems requires ubiquitous inorganic ions (Na+, K+, Ca2+, Mg2+, Cl), as well as catalytically important traces of various transition metals (Fe, Zn, Co, Cu, Mn, Ni, Mo). As the list of recognized trace nutrients is still expanding, not all biologically utilized elements are mentioned here. A large variety of organic compounds of potential prebiotic relevance are, in fact, present in carbonaceous meteorites of chondrite type (Chap. 2), so the chemistry for their formation is literally universal. Carbon is uniquely predestined to dominate the versatility of biogenic chemistry. It is the lightest atom with four valency electrons; so the outer shell is exactly half full / half empty, which is equivalent to saying that C is equally prone to accepting or donating electrons to form covalent bonds with other atoms, including single or multiple C–C bonds. Silicon (Si), too, shows similar symmetry, but it is much less reactive and preferentially stores away in crystalline silicate rocks. At the transition stage, however, between sterile geochemistry and primordial biogenesis, the versatile silicate sheets in nascent clay minerals may have cooperated with carbon chain derivatives in keeping biogenic assemblies in close dynamic contact by adsorptive retention and release (Sects. 12.6 and 12.8). Adding C–O or C–H bonds moves carbon chains to opposite sides on a redox scale. The COO carboxyl group is closest to fully oxidized CO2 and provides acidic epitopes to organic surfaces. The less oxidized keto and hydroxy groups of aldehydes and alcohols, as combined in carbohydrates, add surface variety of lesser

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Fig. 12.1 Periodic table of chemical elements (partial). Elements relevant to living matter are shaded grey (Lantanides and the heaviest elements are omitted)

polar character. If many adjacent atoms are saturated with C–H bonds, however, there is no polar interaction with surrounding water throughout an entire surface patch. The formation of aromatic C/H ring systems – with all the p-electrons in planar resonance – adds variance to the hydrophobicity of such patches. Epitopic variation increases further by adding N to the C/H/O organic triad. At hydrophilic patches, essentially all the positive charges depend on ionizable NH3+ amino groups. Moreover, the participation of N in heterocyclic aromatic ring structures can introduce catalytically important polar spots into surrounding hydrophobic pockets. All of these functional groups are to be found in a particular kind of bricks in the repertoire of metabolic chemistry, amino acids. Having both COOH and NH2 in common, they are ampholytes, able to form zwitterions. Not only do amino acids polymerize to form peptides and proteins, they also serve as metabolic intermediates in the biosynthesis of all kinds of other N-containing organic compounds, including heterocyclic coenzymes (Chap. 9) and the basic moieties of nucleotides and nucleic acids (Sects. 12.10 and 12.11). Next in line, still vitally important, sulfur and phosphorus are lesser constituents of living matter. These heavier elements are related to oxygen and nitrogen, respectively, yet add important functional aspects to the chemical toolbox that the major components do not exactly match or share. The biological storage and utilization of chemical energy, in particular, is largely mediated by PO4 phosphate groups (Sect. 12.8; Chap. 3), as well as SH sulfhydryl groups, to a lesser extent. Referring to Lippmann earlier on, Westheimer (1987) ascribed the peculiar suitability of organic phosphate bonds to their “remarkable combination of thermodynamic instability and kinetic stability”. It takes considerable energy to link the phosphate in an organic ester bond; but once made, a lone phosphoester linkage at a hydrocarbon scaffold is remarkably resistant to hydrolysis. To wit, the energy for its

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formation is effectively trapped behind a virtual barrier. The height of this barrier can be lowered by introducing nucleophilic groups in the vicinity, either at intramolecular positions or placed on interacting partners. This opens the door widely for subsequent evolution, selecting for interactive and system-supportive cofactor– catalyst combinations that can stabilize a collectively autocatalytic range of organic products. Closely related to sulfur and phosphorus, selenium and arsenic are potentially of biogenic interest. Of these, selenium is actually used to replace sulfur in the “21st amino acid” selenocysteine, which is specifically incorporated in certain enzymes (Donovan and Copeland 2010). Arsenic, on the other hand, competes with phosphorus and is notoriously poisonous to most modern life forms, due to its higher reactivity, capable of uncoupling oxidative phosphorylation (Shi et al. 2004). Thus phosphorus is considered advantageous, due to the higher stability of its organic bonds. Environmentally, though, arsenic is quite abundant and not ignored entirely by microbial life. The arsenite/arsenate redox system, in particular, is driven either way in numerous organisms. Most commonly, oxidative detoxification leads toward arsenate, but if used as a terminal electron acceptor, “arsenate serves as an anaerobic alternative to oxygen” (Mukhopadhyay et al. 2002). Likewise at the non-metal borderline, boron is not noticeably utilized in modern life, but it may have been important for primordial biogenesis, due to the remarkable stabilization of ribose and ribotides in complex with borate (Ricardo et al. 2004). There are many biochemically relevant redox reactions that are difficult to achieve by organic catalysts alone. This is where the participation of metalcontaining cofactors becomes essential; transition metals are especially suited to this task (Mulkidjanian and Galperin 2009; Morowitz et al. 2010). They have an incomplete inner electron shell, which can be filled or emptied by catching or donating electrons, respectively, so as to ionize and thereby change the redox state. Thus, Szent-Gyo˝rgyi’s “tune of solids” is played not only by organic strings and pipes, but also has a distinctive sound of brass band to it. Notably, the SH group of cysteine often participates in coordinating the active metal ion complex. Comparing the number of thermodynamically feasible reactions in organic chemistry under early-earth conditions with the variety of commonly used reactions in current life, there actually are quite large discrepancies. To understand and rationalize this breach, it is not only important to focus on the more fanciful reactions to be observed, but it is equally worth considering why numerous conceivable reactions are in fact disregarded in real life. Many metabolic intermediates are preferentially observed as one of two stereo-isomeric forms, such as L-amino acids or D-ribose (Sect. 12.9). Why don’t their mirror images occur as well? – Of many different oligosaccharides conceivable (Sect. 12.11), how come just ribose is used as a pivotal hub in both energy management and informational bookkeeping of all living cells? Obviously enough, certain reactions are favored over others, and the evolution of such bias is thought to be comprehended in terms of selective channelling for dissipative energy flow between prevailing reservoirs and sinks in the environment (Morowitz and Smith 2007). To start with, there are no “side reactions” in any sense

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occurring in a pristine word, only various reactions with different rates and other characteristic properties. Yet, if some chemical reactions before others resulted in channels for energy flow of a better conductance than stochastic dissipation, then any catalytic means for coupling and bundling these reactions should be reinforced progressively, thus leading to persistent autocatalytic networks (Sect. 12.9).

12.6

Hydrogels and Membranes

Not solid and not fluid either – or the best of both worlds, really

Modern cells are physically differentiated into membrane-surrounded compartments. The enclosed space inside a cell, however, is not hermetically sealed, but partly permeable to some solutes and perforated by gated channels for other substances. Due to macromolecular crowding, the availability of water molecules is much lower inside a living cell than outside. In fact, it is not a trivial matter to analyze the local interactions with water directly at the surface of a native protein. A recent break-through in this regard is the inclusion of model proteins in nanoscale reverse micelles for NMR analysis (Nucci et al. 2011). When studied in vitro, many soluble enzyme proteins are folded properly and fully active only if they are surrounded by other stabilizing agents, such as gelatine or polyvinylpyrrolidone, so as to resemble the crowded intracellular environment (Schmid 1979). Hence it is reasonable to assume that local hydrogel conditions, in favor of macromolecular crowding, have likely been important in life’s emergence by physico-chemical dynamics and reactions (Trevors and Pollack 2005; Spitzer and Poolman 2009). Gelling agents can consist of organic or inorganic compounds, and a combination of both could have provided a preferential breeding ground supporting primordial biogenesis. Colloidal clay particles indeed flocculate efficiently with carbohydrate polymers, especially their cationic derivatives (Cairns-Smith 1982; J€arnstr€om et al. 1995; Wei et al. 2008). Added phosphate groups, in particular, have become the hallmark of carbohydrate binding and metabolism. Sugary compounds as such belong to the triadic C/H/O class of organic molecules, which could have formed to some extent without involving N as the energetically expensive fourth component. Hence it is not unreasonable to assume that, early on, representatives of this class were more abundant than amino acids and heterocyclic compounds. All modern cells on Earth surround themselves in various kinds of carbohydrates, from slimy sheeths to solid cell walls. Carbohydrate polymers, therefore, have supposedly preceded both proteins and nucleic acids at the earliest stages of geobiochemical evolution (Cairns-Smith 1982; Stern and Jedrzejas 2008), and emerging life may have been embedded in carbohydrates from the very beginning. Likewise belonging to the triadic C/H/O class, carboxylic acids with variable aliphatic tails – the fatty acids – are among the simplest of organic amphiphiles, showing both hydrophilic and hydrophobic properties in water. Together with glycerol and phosphoric acid, they form phospholipids (Chap. 8), as found in

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many bilayered biomembranes.7 Now, full-fledged membranes are quite thick, as compared to the common precursory building block, acetic acid. Typically, it takes nine elongation steps to make C18 tails, as for the stearic acid family of residues. At that length, they are virtually insoluble in water, and complex proteins are required both for their biosynthesis and the subsequent insertion into preexisting membranes. A related class of catenated isoprenoid derivatives shows similar properties, as represented by the isoprenic chains of archaeal membrane lipids. Also, modern membranes are unthinkable without trans-membrane proteins, which provide structural functionality to compartmental boundaries. There are reasons to believe that the Last Universal Communal Ancestor State (LUCAS), from which the three superkingdoms of Bacteria, Archaea, and Eucaria have been derived, already carried a sophisticated load of trans-membrane proteins, even though its capability of forming lipids in the modern sense is unsettled (Chap. 8). Uncertainty as to when proper lipids first emerged is raised by the vastly different modes of lipid biosynthesis in bacterial and archaeal lineages (Pereto´ et al. 2004; Matsumi et al. 2011). Not only are the fatty acid moieties of bacterial lipids linked to glycerol by ester bonds, whilst the archaeal isoprenic chains are ether-linked; yet more disturbing is the different stereo-chemistry of the central glycerol, which is derived, respectively, from sn-glycerol-3-phosphate and sn-glycerol-1-phosphate in bacterial and archaeal lipids. The common achiral precursor of both these stereo-isomeric glycerolphosphates is dihydroxyacetonephosphate, from which different dehydrogenase enzymes generate the bacterial- and archaeal-type glycerolphosphates as enantiomeric lipid precursors (Boucher et al. 2004; Koga and Morii 2007). One way to rationalize this enigma could be that, at the stage of LUCAS, the common precursor dihydroxyacetonephosphate was used directly for lipid biosynthesis, yet only accommodating a single hydrophobic chain. Whilst single-chain lipids, such as dodecyl phosphocholine or lysolecithins, are generally detrimental to bilayered lipid membrane structure (Lee and Chan 1977), they may well be suitable as simple spacefilling units in peptide-dominated membranes, as experimentally utilized in micelle to 2D-crystal transition of integral membrane proteins (Stahlberg et al. 2001). Similar to acidic carbohydrates, also cationic membrane components can productively interact with clay particles, eventually engulfing entire grains (Hanczyc et al. 2003, 2007). Other organic compounds, too, are rapidly adsorbed to sedimenting clay under field conditions (Deamer et al. 2006). Moreover, simple surfactant-like peptides can self-arrange as membrane-like associations (Vauthey et al. 2002). Hence primordial, uncoded peptides could have played versatile roles in early biogenesis (Fishkis 2007; Zhang 2008; Bywater 2009; Egel 2009).

7

Other C/O/H precursors for membrane bulk components are linear terpenoids and more complex steroids, which like fatty acids, are derived from activated acetic acid. The bilayered membrane structure results from arranging all the lipids in parallel on either side, with all the hydrophilic head groups facing the the water outside and the lipophilic tails hiding away inside the membrane layers (Sturgis, Chap. 8).

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The fractally dispersed pore space between sedimenting clay particles appears very appropriate, as a geochemical reactor to have hosted carbohydrate-based hydrogels and proto-membrane forming amphiphiles. Catalytically active epitopes exposed on such surfaces (Sect. 12.7) can have mediated progressive coevolution of all the major components of living matter from feable, non-cellular, yet multi-factorial beginnings. Presumably, hydrogels and membranes shared an extended period of precellular coevolution, where it did not really matter whether the proto-cytoplasmic phase preferentially existed inside or outside membrane-bounded vesicles (Griffiths 2007; Sect. 12.15). Also of note, the fusion and dispersion of reticulate vesicular membrane associations is readily interconvertable in wet/drying cycles (Deamer and Weber 2010).

12.7

Catalytic Minerals

Organics metallized or Metals organized?

Before life there were minerals, and many mineral surfaces show catalytic potential. It is thus commonplace to point out that minerals had decisive roles to play in getting life-like systems under way (Hazen 2005; Hazen and Sverjensky 2010). Bentonites or montmorillonites, in particular, are suited to primordial catalysis (Cairns-Smith 1982; Nikalje et al. 2000; Ferris 2006). Such layered clays arise from aqueous weathering of volcanic ashes and should have been present on the early Earth. The crystalline lamellae consist of aluminium phyllosilicate, with various other metal substitutes. Under anoxic, prebiotic conditions, though, Fe/ Mg-rich smectite clays have resulted from basaltic weathering preferentially (Meunier et al. 2010), instead of the Al-rich montmorillonites formed later on. Layered-clay minerals are also utilized in industrial processes, where interspersed transition metals can catalyse various organic reactions in water-free conditions. This property is preserved in biocatalysts by attaching metal clusters to characteristic nest-type binding sites in peptides (Chap. 7), which in modern proteins often are retracted into narrow pockets in the hydrophobic core. Prominent examples of catalytic metal organics occur in metabolic cofactors, both as coenzymes and prosthetic groups (Theil and Raymond 1994; Mulkidjanian and Galperin 2009). Iron and iron-sulfur clusters, in particular, participate in numerous enzyme activities. A 1960 manuscript (published post-humously, Calvin 2008) already sketched the likely prebiotic evolution of some redox catalysts as follows: 1. Free iron ions (Fe+2 $ Fe+3), in aqueous solution, vibrating amidst six water molecules, display some redox activity8 alone (105).

Catalytic activity is measured as converted matter per time and volume (M L1 s1).

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2. Suspended between four organic N groups in a planar porphyrin ring, and two water molecules outside, the central iron enhances its catalytic activity considerably (102). 3. If the porphyrin-bound iron, in a catalase protein, is further coordinated by two histidine residues instead of water, activity increases very much again (105). Cyclic tetrapyrroles are widespread in nature, forming porphyrin in hemoglobin, cytochromes and various enzymes, as well as related rings in chlorophyll or the essential vitamin cobalamin. Their chelating potential at the center can accommodate various metal ions (Fe, Mg, Cu, Ni, Zn, Co). The pyrrole subunits are synthesized by side reactions from the citric acid cycle (Wriston et al. 1955; Kikuchi et al. 1958), the central hub of the universal metabolic network, which likely has preceded cellular biogenesis (Sects. 12.9 and 12.10). Tetrapyrroles may have been preceded by nest-forming tetra-glycine complexes (Chap. 7). To precede membrane-based containment, alternative concepts of surface metabolism have been suggested (W€achtersh€auser 1992, 2007; Mulkidjanian 2009; Mulkidjanian and Galperin 2009), which are based on metal-catalysed reactions and adhesive coherence among organic product molecules. Bridging the conceptional gap between single metal ions suspended in delicate organic frames, as described above, and crystalline surfaces in prebiotic worlds of pyrite (FeS2) or zinc sulfide (W€achtersh€auser 1992; Mulkidjanian 2009), covalently linked clusters of several metal atoms can still be found in key components of living cells today. In fact, the prototype ferredoxin family of iron-sulfur proteins has long been considered to represent some of the most ancient proteins on Earth (Eck and Dayhoff 1966). These proteins contain Fe2S2, Fe3S4 or Fe4S4 clusters, where the iron atoms, in cubic symmetry, are linked to additional sulfur atoms belonging to cystein residues in the supporting protein chain. Typically, these and other FeS proteins participate in electron transfer chains. Most fundamentally, these are involved in CO2 assimilation, and conversely, in respiration. In bacterial nitrogen fixation, too, electrons and/or protons are relayed via serial FeS clusters to the nitrogenase reaction center (Seefeldt et al. 2009; Dance 2010), which itself consists of a formidable Fe7S9XMo cluster.9 More commonly than not, discussions on primordial membranes have focused on a vesicular role for containment (Deamer et al. 2002), but membrane involvement in energy conversion likewise is important (Deamer and Weber 2010). Photosynthetic membranes, in particular, are quite protein-rich (Scheuring and Sturgis 2006; Jones 2009), and may be dominated more by inter-peptide and peptide–pigment interactions than by the lipid environment as such. Inner mitochondrial membranes, too, have very high concentrations of protein complexes (Wittig et al. 2006; Boekema and Braun 2007), comprising the electron transport chain of respiration. More recently, a number of less frequently studied redox systems have also been localized in protein-rich rafts, caveolae, and tiny vesicles

9

The precise identity of the central X atom (probably N) has not yet been established.

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(Patel and Insel 2009). Not inconceivably, therefore, such electron transfer processes have emerged from tight interactions between mineral metal sulfide clusters, redox-responsive heterocyclics, and amphiphilic peptide patches. Hence, electronic insulation by membrane-like ensheathment of photo-active mineral grains may have been a primal function of organic amphiphiles, together with lipophilic, electron-accepting, organic pigments (Chap. 4).

12.8

High-Energy Bonds

Phospho-, thio- and other esters

Life cannot possibly exist without a metabolic network of biochemical reactions. Many of these proceed downhill on a potential energy gradient, thus tending to approach thermodynamic equilibrium. Yet, reaching this equilibrium would mean the end of life. To minimize this risk, living matter is capable of storing potential energy in semi-stable compounds, and the drainage of such reservoirs is effectively coupled to key reactions that are essential for self-maintenance and/or propagation, yet would not proceed uphill on their own. How this coupling has become readily repeatable by natural processes from randomized coincidences, is one of the major challenges to be solved by origins-of-life research (Chap. 1). The cash flow of energetic coupling in endergonic metabolic reactions is based on relatively few types of chemical bonds and compounds, as characterized by quantized energy release on the one hand and mechanical handles or anchor points on the other. The most readily convertable currency of metabolic energy coupling is represented by ATP and other pyrophosphate carriers. In the top-down ranking of universal metabolism, a close second is the prototype coenzyme, CoA, where thioester linkage activates carboxylic acid moieties for various transfer reactions. In bottom-up considerations, however – as to how a connected protometabolism might have emerged from geochemical reactions – the primal ranking may well have been reversed, proposing thioesters as the primordial energy source of life (de Duve 1998; W€achtersh€auser 2006). The bonding of phosphate and phosphorylated compounds to pyrite (Bebie´ and Schoonen 1999), may even suggest that stably phosphoester-bonded organics started to accumulate due to their handle function (Sect. 12.9), whereafter reactive phospho- and thioester compounds have followed suite in mutual coevolution ever since. These linkages, in turn, give rise to other activated intermediates, such as the activation of amino acids by ester bonding to ribose of tRNA (Sect. 12.11). The tentative origin of phosphorylated biomolecules is discussed by Pasek and Kee (Chap. 3). Ultimately, the energy flow of life must be connected to nuclear fusion inside the Sun or nuclear fission and residual heat in the interior of the Earth. A combination of both these sources is not excluded and could, in fact, be advantageous – involving complementary means of physical chemistry. In particular, certain high-energy states can more readily accumulate under the anhydrous conditions

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of volcanic ash plumes, than at submarine eruptions, and solar radiation can only reach surface-near environments. Of prebiotic relevance, anorganic ZnS surfaces and solar UV radiation can have driven primordial CO2 fixation in the rTCA cycle (Mulkidjanian 2009; Chap. 4), and volcanic ash plumes can have fixed much of the nitrogen needed for amino acids, together with producing energized compounds to activate amino acids for peptide bond formation and other reactions (Chap. 6). The photo-active ZnS surfaces could also have extracted phosphorus from moderately soluble hypophosphite and phosphite in primordial ocean water. If photo-oxidized by ZnS (Mulkidjanian 2009), the resulting phosphate would remain tightly bound to ZnS or other sulfides (W€achtersh€auser 1988; Bebie´ and Schoonen 1999). This would result in a substantial upconcentration effect. Also, the universal role of phosphate handles in small metabolites, such as sugary organics, could have emerged from there, together with the generation of high-energy pyrophosphate bonds. – Could the generation of pyrophosphate, and other polyphosphates, be favored by subsequent wet/drying cycles (recurring at tidal beaches or periodic fresh water ponds in arid terrains)? – Conceivably, an excess of protons from desiccating an acidulous primordial brine should drive out water from the adsorbed and concentrated phosphates, in favor of the formation of polyphosphates. This store of energy could then be mobilized again in the next hydration phase of the cycle. Notably, a potential photochemical driver has been discovered in the uracilcatalyzed synthesis of acetyl phosphate from thioacetate, as mediated by ultraviolet light absorption (Hagan 2010). From acetyl phosphate, in turn, other energy-rich compounds, such as phosphate esters or pyrophosphate, can be readily derived (Chap. 3).

12.9

From Autocatalytic Feedback to Protometabolic Ecosystems

Looping the loops

Living organisms are selective not only in the range of chemical elements assimilated (Sect. 12.5), but also in the number of organic compounds and reactions used by the intermediate metabolism. The sheer variety of organic chemistry is enormous, as documented in the Beilstein database (Reaxys 2010), but not without some underlying patterns. A minority of tightly connected nodal compounds in an inner network core give rise to most other known derivatives and reactions (Bishop et al. 2006; Grzybowski et al. 2009). Reducing this richness to the limited subset of biologically relevant compounds and reactions requires a number of motivated pruning rules (Morowitz et al. 2000), where concepts of autocatalytic reactions and collectively autocatalytic networks play central roles (Kauffman 1993; Benk€o et al. 2009; Hordijk et al. 2010). The efficient channelling into an organized network of metabolic pathways by modern life, of course, is linked to the long-term evolution of effective biocatalysts.

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It is a major challenge, therefore, to comprehend how these or equivalent catalysts can have emerged from thermodynamically acceptable geochemical roots, and serious doubts persist as to whether the geochemical closure of complex prebiotic reaction cycles is chemically plausible (Orgel 2008). By and large, however, it is not unreasonable to assume a certain congruence between metabolic networks in modern life and a prebiotic protometabolism (de Duve 2003). In particular, the shell-like organization of metabolism can be rationalized in evolutionary terms, assuming that the inner shells assembled first in prebiotic, geochemical evolution (Cairns-Smith 1982; Morowitz 1999). The reductive tricarboxylic acid (rTCA) cycle10 comprises the innermost core of this concept, driving the assimilation of carbon from CO2 in a series of autocatalytic steps, where each intermediate compound – by way of cyclic closure – intensifies its own production (Chap. 4). The energetics of this cyclic pathway can readily be associated with prebiotic origins in a tentative iron-sulfur world scenario (W€achtersh€auser 1992), or ZnS-driven photosynthetic CO2 fixation in carboxylic acids (Mulkidjanian 2009; Guzman and Martin 2009; Chap. 4). It is from the rTCA cycle and some related shunts that direct branches lead to the synthesis of fatty acids and terpenoids on the one hand, and carbohydrates on the other – two major classes of the C/H/O organic triad. At the next level, ammonia (NH3) is taken in by various keto acids related to rTCA components, so as to form the most common amino acids (Morowitz 1999), which in turn pave the way to essentially all the other N-containing metabolites, not the least, the various heterocyclic aromatics that dominate many catalytic reactions of molecular biology today. Many heterocyclic cofactors or coenzymes are, in fact, decidedly autocatalytic in their own biosynthesis (Begley 2006). Also, carbamoyl phosphate is considered important as an intermediate of prebiotic nitrogen assimilation (Martin and Russell 2007). Another cyclic network revolves around various sugary compounds, as represented by the reductive pentose phosphate or Calvin cycle in modern photoautotrophic organisms. These reactions are but rarely considered relevant in a prebiotic setting (Olson and Blankenship 2004; Mele´ndez-Hevia et al. 2008). Yet, this is about to change since a promising system of autocatalytic syntheses, advocated as the sugar model (Weber 2001, 2007), has been extended to also include pentose sugars (Pizzarello and Weber 2010). The rTCA cycle and sugar interconversion are interconnected via pyruvate as the central node. A fairly sophisticated sugar interconversion network, of course, is desirable for the emergence of modern nucleotides and nucleic acids (Sect. 12.11). It has been noted that the current enzymology of the complex cycles mentioned is vastly different (Orgel 2008). While both rely on acidic moieties at virtually all reactions, in the rTCA cycle these groups are provided by substrate carboxylic groups exclusively, and thioesters are engaged at exergonic steps. In the Calvin cycle and related reactions, on the other hand, both acidity and energetic needs are provided by phosphoric

10

This is equivalent to the familiar oxidative Krebs cycle of animal respiration, run in reverse.

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bonds, which are not involved at all in the rTCA cycle. This indicates that protometabolic precursory networks of either kind have likely emerged in locally different geographic contexts. Many small uncharged substrate molecules are metabolized as stable phosphoester derivatives, where the phosphate group has no catalytic role in the enzyme reaction, but provides a solid handle or anchoring hinge through several steps in a serial pathway. This applies to alcohols, aldehydes, and sugars,11 as contrasted with carboxylic acids. At the crossroads of carbohydrates with keto- and amino acids, a particular set of phosphate-anchored metabolites leads to the synthesis of pyridoxal phosphate (PLP) (Mittenhuber 2001; Fitzpatrick et al. 2007). PLP is relatively small, yet one of the most remarkable and versatile catalytic cofactors, which participates in many different group transfer reactions and has central roles in the biosynthesis and interconversion of virtually all the amino acids and other metabolites. It has retained the phosphate handle, which fits to highly conserved binding sites, such as glycine/threonine-rich loops (e.g., GTGGT) (Schnell et al. 2007), where the phosphate itself does not directly participate in the catalysed reactions. The first catalysed organic syntheses likely occurred on minerals – at flat surfaces, as well as irregular faults. If organics adsorb to crystalline faces, they are more likely to be extended in planar arrangements than by bushy growth in three dimensions. This might explain why a number of metabolic cofactors are characterized by carrying planar multi-ring systems, such as riboflavine, porphyrin, or corrin moieties. All of these “constitutionally more complex biomolecules [are] generationally intrinsically simple” (Eschenmoser 2007). Although modern biocatalysts are wonderful in many ways, they cannot actually do miracles. Without proper substrates, all their marvelous capabilities would have no effect. What really matters is which potential substrates are present at a given moment in time, and which reactions can possibly lead to other compounds. Primordial catalysts can increase the rate of particular reactions over others and thereby influence the availability of substrates for other catalysts nearby. Inasmuch as entire ecosystems are capable of self-organization (Stewart 2003; Hoelzer et al. 2006), it is the tenet of metabolism-first scenarios that self-organizing molecular ecosystems preceded the emergence and natural selection of cellular/genetic organisms. In terms of network analysis, emerging biocatalysts are both substrates and mediating agents. Acting collectively in a communicative system, this ultimately channelizes a flow of matter, as coupled to the conversion of environmental energy for metabolic work. The effective coupling of energy-rich compounds to endergonic reactions by emerging organic catalysts is arguably the most far-reaching accomplishment in prebiotic evolution, only surpassed by the emergence of

11

This common charge rule has traditionally been rationalized as a precaution to keep such metabolites inside the lipophilic membrane enclosure of the cell (Davis 1958; Westheimer 1987), wheras here the handle function for stable anchorage at a chelating and catalytic surface site is given more weighting and attention.

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sequence-encoded programming later on. In short, “The cascade of metabolism thus functions overall as a relaxation channel for two major sources of geochemical energy: electron transfer from reductants and the hydrolysis of phosphates” (Morowitz and Smith 2007). A relatively simple organic residue engaged in charge transfer is nicotinamide, the active residue in NAD(P) coenzyme. To be useful in various catalysed reactions, it has to be linked to a structural handle that fits to modular universal binding sites (Chap. 9).

12.10

From Mixed Organic Multimers to Interactive Homopolymers

Survival of the fittest complementors

In the course of geochemical evolution, the variety of organics supposedly increased in number and in size. As more and more compounds became available, an evolutionary leap was taken when incremental small-group transfer gave way to modular assembly of moderate-sized reactive subunits. More or less randomly assembled in the beginning, such multimers (de Duve 1991) can contain two or more different types of monomers, also termed chimeromers (Morowitz et al. 2008). The active pantetheine moiety of coenzyme A belongs to this category (Miller and Schlesinger 1993), as well as many other vitamins and cofactors. Some of these multimers will inadvertantly express one or the other catalytic action to increase the availability of small building blocks, thus starting autocatalytic feedback on the entire system. It is widely recognized that such systems may have originated in close association with transition metal sulfides at mineral surfaces (Cody 2005; Sect. 12.7), from where single or complex metal ions are chelated and coordinated by organic multimers. The multimers of collectively autocatalytic systems are not likely to expand as such beyond rather moderate limits in molecular size. At certain length or bushiness of individual compounds, further accretion will less often result in novel, “useful” – network-supportive – properties. At the next level of complexity, however, some channelizing of possibilities can be in favor of copolymers of structurally related subunits, such as oligosaccharides or peptides, by repetitive linking of equivalent subunits, which may vary in peripheral epitopes. A novel emergent property of such copolymers is the potential of forming tightly packed secondary structures, by repetitive interactions between partly equivalent neighboring or juxtaposed subunits,12 whereas mixed multimers are more irregular at any scale. Intriguingly, such copolymer formation can lead to self-organizing evolution under quite reasonable assumptions. As first proposed in a prescient model (de

Of this kind, the formation of a-helix and b-sheets in proteins, and double helix in nucleic acids, have become most successful in biological evolution.

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Duve 1987), selective evolution can result not only from differential replication but likewise from differential breakdown rates. Under general growth conditions, providing that the overall rate of covalent bond formation exceeds the rate of spontaneous degradation, the variety of different multimers is rising. Formally, this entails a random synthesizer function. Further feedback can then result from differential breakdown; if unstructured, flexible, idling multimers are purged before others that are tightly folded, substrate-bound, and stabilized. Complementary systems properties have been proposed by Dyson (1985) and Kauffman (1993), assuming that, in a large set of different multimers, certain members will catalyse the formation of others. In a process of autocatalytic network closure, this provision will select for certain subsets where every member is catalysed in its formation by one or more members of the same set. Collectively, therefore, “selection at the chemical level can operate by the preferential survival of useful molecules” (de Duve 1987). Peptides/proteins and oligosaccharid-related polymers are prominent types of life-associated copolymers, where peripherally variant subunits are joined in chains, repeating the same kind of linker reaction over and over again. While oligosaccharides as such do not substantially extend the repertoire of catalytic capabilities, at a high enough concentration they can change the physico-chemical properties of the watery medium profoundly, and some of them can also serve as a platform for immobilizing catalytic multimers. The propensity to form phosphatelinked derivatives has contributed to their evolutionary success by leading to nucleotide coenzymes and nucleic acids (Sect. 12.11). On the other hand, many peptide assemblies are efficient and versatile catalysts, due to the variety of reactive side chains and consistent folding patterns of the backbone scaffold. Also, peptides can form directly from amino acids, which are expected to arise abundantly as close derivatives from the rTCA cycle (Chap. 4). Yet, forming oligo- or polypeptides under mild to harsh conditions on the prebiotic Earth is not a trivial pursuit. The formation of peptide bonds is a condensation reaction, where a water molecule is released for each amide linkage between two amino acids. Accordingly, this reaction is readily reversible in watery solution. There are two principal means of driving the reaction towards polymerization – withdrawing water in wet/drying cycles and/or activating the free amino acids by chemical modification (Brack 2007). Two different suggestions for peptide formation in a prebiotic setting are represented here in special chapters. In the Salt-Induced Peptide Formation (SIPF) reaction (Chap. 5), two amino acids are coordinated in a Cl-distorted CuII complex and joined by a peptide bond upon water limitation (Rode 1999). The critical point in this hypothesis concerns the availability of CuII, which only could have come from oxidation in a locally energized atmosphere. Notably, this metal complex is inherently chiral to such an extent that the joining of chiral amino acids is slightly biased in favor of L- over D-enantiomers, which may have seeded the subsequent evolution toward L-amino acids, as universally found in the proteins of modern life (Fitz et al. 2007). In the presence of clay particles, the SIPF reaction can yield up to hexamers of glycine. This proves a valid point, but is still too short in product length for the SIPF reaction to stand alone in early evolution.

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What sets copper apart from other transition metals in this context is its unique position in the so-called Irving-Williams series of forming stable complexes, irrespective of the nature of the co-ordinated ligand (Irving and Williams 1948). In the order MnII < FeII < CoII < NiII < CuII > ZnII ; the divalent copper ion clearly provides for maximum complex stability. To evaluate the potential of all these transition metals more realistically, as well as adsorbed-state polymerization, it is highly desirable to analyze a large variety of materials, especially Fe-rich and other minerals that actually may have been present on the early Earth (Lambert 2008; Meunier et al. 2010). As for activating free amino acids before their polymerization under early-earth conditions by other means, a conceivable mechanism is by reaction with isocyanic acid (HNCO), which supposedly entered the primordial atmosphere from volcanic sources. In a bold and ingenious revolving scheme, termed primary pump (Chap. 6), this activation could have facilitated the stepwise elongation of prebiotic peptides, while cycling between tidal wetting at a flooded beach and intermittent desiccation. Owing to the peculiar chemistry, this mechanism would only work during a narrow window in time, when there was some, albeit very little oxygen in the primordial atmosphere, together with some nitrogen oxide (NO), as mainly formed by lightning discharge in volcanic ash plumes. Later on, the rising oxygen of biogenic origin would effectively shut down the reaction by converting NO to nitric acid. The thing primary pump and the SIPF reaction have in common is that they rely on local energizing in a generally anoxic atmosphere, as well as on the regular repetition of wet/drying cycles. Also, they probably would not have worked if they only had to rely on low average concentrations of amino acids in a primordial soup scenario, comprising the bulk of the entire ocean. Yet, in a patchy environment and in close association with a primordial geochemical reactor that already was generating a local stock pile of carboxylic and amino acids, the SIPF and/or primary pump reactions could have provided the critical link to kick-start the tentative biogenic reactor to proceed to the next level. Autonomous peptide formation should then take over, as energized by a system-internal means of amino acid activation and scaffold-guided polymerization of more and longer, yet still stochastic peptides (Sect. 12.11). Neither the SIPF reaction nor the primary pump, however, have left directly traceable relics in modern metabolism. So their potential impact in kick-starting prebiotic peptide formation remains a matter of informed speculation. Notably, though, the primary pump scenario also proposes a link to sugar-phosphate condensation (Chap. 6). What really counts next, from the viewpoint of molecular biology, is how activation mechanisms that are actually used in modern life may have emerged from a geochemical reactor setting. One of these is based on aminoacyl thioester compounds, as used in various types of non-ribosomal peptide synthesis (Zuber 1991), which may link back to cofactor synthesis in a prebiotic thioester world (de Duve 1998).

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Alternatively, the metabolically most important mechanism, as now universally utilized in ribosomal protein synthesis, would prebiotically require that phosphate is readily available in the reactor, in addition to a basic set of amino acids, some carbohydrates, and continual or recurrent influx of energy to generate high-energy phosphorous compounds of various kinds. From such general premises, it should be theoretically possible to derive peptides as well as RNA, and a direct course toward peptide bond formation should, in fact, be most straightforward in chemical terms. Inasmuch as the universal precursor of ribosomal peptide formation belongs to the general class of aminoacyl phosphate monoesters (Kluger et al. 1997), which is then used to acylate one of two vicinal hydroxyl groups (Minajigi and Francklyn 2008), the same kind of mechanism should be chemically feasible before the realization of macromolecular RNA. In fact, the nucleic bases contained in the particating RNA components still have no active role to play in the contemporary activation and transpeptidation reactions as such (Sect. 12.11). At any rate, the recurring wet/drying cycles would augment an initial georeactor’s complexity by providing small random peptides from the uppermost surface and transferring them downward by percolation after chromatographic principles. In the narrow, yet widely ramified pore space between the clay-rich upper sediment layers, these primary peptides were subject to multiple interactions and mutual processing in an internal network. Their non-genetic evolution would be facilitated by preferential degradation of random coil configurations vs. retention of inflexible domains, which had been stabilized by various structural aids (de Duve 1987; Kurland 2010). Lipophilic interactions, in particular, would effectively hide the sensitive peptide bonds in the center of secondary structures (Egel 2009), preferentially arranging lipophilic a-helixes in membrane-like films and amphipolar b-hairpin peptides on top, straddling the lipoid–water interface. At the turns of such peptides, the DGD (Asp Gly Asp) submotif appears to contribute to active sites of various basic enzymes (Van der Gulik et al. 2009). Metal chelation, such as zinc in Zn-fingers (Gamsjaeger et al. 2007) or calcium in EF-folds, as well as phosphate binding in cups or nests (Milner-White and Russell 2005; Chap. 7) can constrain relatively small peptides into rigid conformations, presenting quite specific binding epitopes for secondary interactions. In this pregenetic phase of uncoded peptide evolution, selective fitness had a quite literal dimension, dependent on physically fitting together complementary configurations and eliminating other peptides that did not fit in with any binding partner. Together with phosphorylated metabolites and cofactors (Kritsky and Telegina 2005; Sharov 2009; Chap. 9), this colloidal community of peptides would further diversify by transpeptidation-like splicing reactions and various kinds of crosslinking, resulting in system-wide catalytic closure (Dyson 1985; Kauffman 1993; Morowitz and Smith 2007). To denote the relevance of this important evolutionary stage, the guiding concepts of a peptide world (Plankensteiner et al. 2005) and a cofactor world (Sharov 2009) can blend together in a tentative peptide–coenzyme world (Chap. 9). At this dreamtime stage of prebiosis, regular RNA as a replicative macromolecule did not yet exist (Kurland 2010), and a prebiotic mode of “selection favored communities of molecules that collectively were best able to catalyze

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synthesis of their own constituents” (Copley et al. 2007). By this token, selfsupportive – autotrophic – molecular ecosystems developed before any protocellular organisms, no matter whether the first of those would emerge as autotrophic or heterotrophic entities later on (Lee et al. 1997; Fiscus 2002).

12.11

Ribose at the Crossroads

Reactive agent and scaffolding link

This section is about tentative origins of an RNA World (Chaps. 10 and 11) – for the various versions of this highly influential concept, see Robertson and Joyce (2010). At its highest degree of stringency, this popular concept rests on four main pillars: (1) The precursory role of ribonucleotides with respect to DNA in metabolic and evolutionary terms, together with RNA’s capacity of complementary base pairing; (2) The existence of bioactive ribozymes (Sect. 12.13); (3) The relative ease of selecting new ribozymes from large pools of random sequences; and (4) A general disbelief that uncoded peptide formation as such could possibly lead to any evolutionary utility. Yet, removing the latter presupposition from the theoretical framework does not in any way discredit the validity and importance of items (1–3) as sufficient corner stones for a more general – and arguably more realistic – primordial RNA world scenario. In the preceding phase of protometabolic consolidation, ribose was merely one of many sugars, and the standard nucleic bases were not yet set apart from other heterocyclic agents. Phospho-organics, however, had already entered the scene at various levels, and associations of stochastic peptides very likely existed as well. From this vaguely perceptible background, a decisive breakthrough occurred when a molecular symbiosis began to take shape, forging the generation of more elaborate peptides and polynucleotides into ever more sophisticated interlock systems. What makes ribose so special in chemical terms? Two properties of ribose are crucial, albeit not unique: (1) The 5-membered ring is more rigidly constrained than the 6-membered ring in many other sugars; and (2) the neighboring (vicinal) positions of its 20 and 30 OH groups give ribose a relatively high degree of reactivity. In its multi-phosphorylated state, as 50 -phosphoribosyl 10 -pyrophosphate (PRPP), it is not only the universal precursor of all the nucleotides present in modern RNA, but also the anchoring scaffold for many ribonucleotide-like coenzymes (Chap. 9), which conceivably existed before the emergence of polymeric RNA. From the various heterocyclic compounds formed, the canonical nucleic bases were likely set apart, due to their superior complementarity by hydrogen bonding in pairwise combinations of congruent shape. Furthermore, by way of ester bonds at its 20 - or 30 -position, ribose can activate amino acids for peptide bond formation. This mechanism is universally conserved in ribosomal protein synthesis of modern organisms, where the activating ribose occupies the 30 -terminal adenosine of a tRNA, and both vicinal OH groups are

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essential for aminoacylation at either position (Minajigi and Francklyn 2008). In order to get there, the amino acid is preactivated by a 50 -phosphodiester linkage to another ribose, derived from ATP in the aminoacyl tRNA synthetase (aaRS) reaction. Deep inside the working ribosome, a narrow funnel can snugly accommodate the ends of two tRNA molecules simultaneously, the first one holding the growing peptide chain and the second one providing the next amino acid. All it takes to form the next peptide bond is to bring the two ester bonds together, close enough to interact spontaneously. The only catalytic aid, in fact, appears to be the vicinal OH-group of the peptide-bearing ribose (Steitz 2008). This process is now commonly referred to as substrate-assisted catalysis (Weinger et al. 2004) – or termed “positional”, rather than “chemical” catalysis (Agmon et al. 2005), for it maintains the ribosome’s tentative status as a ribozyme. Unlike most other catalysts, the ribosome itself is not directly involved in the chemical transpeptidation reaction as such; it does not form any reactive intermediate with the substrate. To the contrary, for most of its work cycle it actually forms a particularly unreactive intermediate, so as to protect the energy-rich peptide-bearing ester bond from accidental hydrolysis by ambient water molecules (Schmeing et al. 2005). Overall, the ribosome is essentially a reciprocating ratchet feeder for repetitive transpeptidation, from one ribonucleotide carrier to another (Woese 1970; Zhang et al. 2009). Assembled from many parts, it forms an exquisitely refined and complicated molecular machine. The processivity and quality controls that make it tick today must have gone through many evolutionary steps (Sect. 12.12). It is less obvious, though, how it all might have begun from more stochastic interactions. By structural and informational criteria, the large ribosomal subunit can be deconstructed to a central core, inferred to be of ancient origin (Agmon 2009; Bokov and Steinberg 2009; Davidovich et al. 2009). At the heart of this core unit resides a self-folding substructure, the Peptidyl Transferase Center (PTC). It is commonly assumed that noncoded peptide synthesis preceded the emergence of gene-encoded proteins (Orgel 1989; Schimmel and Henderson 1994). Accordingly, it is likewise assumed that the PTC precursor in the tentative protoribosome originally facilitated the synthesis of uncoded peptides (Davidovich et al. 2009; Belousoff et al. 2010). Notably, the contemporary PTC comprises a conspicuous core with two-fold rotational symmetry, whilst the ribosome at large is highly asymmetric. The active site is surrounded by twisted stems and loops of RNA, lining a funneled orifice on top of the peptide exit tunnel. The paired arrangement accommodates the 30 -CCA termini of P-site and A-site tRNAs13 on either side, with the nascent peptide extruding through the central orifice (Agmon et al. 2005, 2006; Belousoff et al. 2010). Conceivably, the complex ribosome evolved from a primitive dimeric docking site for ribotide-activated amino acids. Snugly fitting into this scaffolded mold, the activated substrate molecules themselves could form a transitional intermediate (Gindulyte et al. 2006),

13

carrying the growing peptide (P) and the incoming amino acid (A).

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Although the central cavity of the PTC docking site appears to be lined by RNA surface exclusively, a number of ribosomal proteins quite closely approach the PTC from outside. Remarkably, many ribosomal proteins carry long and slender extensions, extruding from more compact globular domains (Ban et al. 2000). The extensions consist of unstructured single chains of histone-like tails or b-hairpins. Notably, it is such peptide extensions that come closest to the ancient PTC core. By inference, therefore, these extensions may represent the most ancient types of peptides still present in the modern ribosome. Six of the PTC-approaching proteins surround the peptide exit tunnel, like spokes radiating from the hub of a cartwheel, and thus define a structural plane beneath the PTC, reminiscent of some intrinsic membrane interaction platform quite early on in protoribosome emergence (Smith et al. 2008). Thus, an important function of the early protoribosome could have been to build up peptide-rich patches with membranelike properties, by depositing chains of hydrophobic residues at a lipoid–watery interface. Of such quasi-stochastic chains, several hydrophobic residues in a row would preferentially organize as a-helix bundles in membrane-like patches, whereas more amphiphilic chains would rather form b-hairpins and gather at the interface (Vauthey et al. 2002; Tang et al. 2005; Zhang 2008; Egel 2009). Such membrane-associated b-hairpins could, in turn, tether protoribosomes to the membrane surface. Presumably, therefore, even the smallest functional precursors of proto-ribosomal activity interacted with uncoded prebiotic peptides by forming RNA–peptide (RNPd) complexes, which later on developed into genuine RNP machines, containing coded proteins. The posited PTCs of protoribosomes are assumed to consist of two 50–60 nt Stem–Elbow–Stem elements (Davidovich et al. 2009), which resemble twisted boomerangs, partly overlapping at their ends and forming a central orifice. Not unlike tRNA precursors in size and appearance, such elements would require some means of synthesizing relatively long oligonucleotides efficiently (Di Giulio 1995; Ferris 2006). Diversifying stem–loop structures could develop by templated primer extension, most readily by folding back on itself and, more rarely, switching template to another molecule. Emerging replicase activity can have started as template-facilitated ligation of precursory triplet units (Altstein 1987; Poole et al. 1998; Wolf and Koonin 2007), or by repetitive utilization of the same mini-template, similar to the modern mechanism of terminal extension by telomerase (Blackburn 2005; Blackburn and Collins 2010). Certain repetitive tendencies in primordial nucleotide polymerization are indeed reminiscent in recurring patterns of RNY > RNR > YNY > YNR in contempory 5 S rRNA (Eigen et al. 1985; Chap. 11). Mineral surfaces appear suitable for the emergence of RNA polymerization (Ferris et al. 1996; Ferris 2006), and tidal cycling may have assisted in the periodic separation of template and product in the primordial absence of efficient helicase activity (Lathe 2004). Up-concentration in the pore space of freezing seaice is likewise conducive of polymerization from activated monomers (Trinks et al. 2005; Price 2007). With the emerging ability to replicate a given parental sequence and producing self-similar progeny molecules at higher frequencies than unrelated sequences, chemical evolution has passed a pivotal threshold, so as to enter

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competition between individual molecules for better reproducibility (Sect. 12.14). Together with the emergent tendency for self-similar replication, the prolific exploration of sequence space to reach local or global optima should be greatly accelerated by means of molecular recombination quite early on (Lehman and Unrau 2005; Lehman 2008).

12.12

From Protoribosomes to Coded Protein Synthesis

Boosting peptide formation to a systematic overhaul

Under the tenet of the current book, primitive uncoded peptides had substantial roles to play before the emergence of macromolecular RNA, and even the early phase of RNA-assisted amino acid polymerization on protoribosomes could not right away yield sequence-specific proteins on demand. Still, the emergence of genetic coding in ribosomal protein synthesis remains an enigmatic challenge, no matter what scenario actually prevailed before. As yet, most theoretical attempts to derive the modern encoding/decoding system from more simplified precursory stages have concentrated on the genetic triplet code itself (Woese 1973; Wong 1988; Davis 1999; Knight et al. 1999; Koonin and Novozhilov 2009). The most conspicuous pattern pervading the canonical matrix of coding rules concerns the distribution of aliphatic versus polar and neutral amino acids across the genetic triplet code – such as U at the second base coding for aliphatic residues exclusively.14 This pattern indicates a certain hierarchy of coding principles, which in turn may point at a series of ancient bifurcations, as to the differentiation of discriminative decoding tools. Indeed, the canonical code as we know it appears to be exquisitely optimized to preserve the polar characteristics of amino acid residues (Haig and Hurst 1991; Caporaso et al. 2005). The codon–anticodon pairing is monitored at the decoding center in the small subunit of the ribosome, which is considered younger than the Peptidyl Transferase Center (PTC) of the large subunit (Smith et al. 2008). This age difference is mirrored by the composite architecture of tRNAs (Giege´ 2008); their acceptor stems and anticodon loops interacting with the large and small ribosomal subunits, respectively. The familiar cloverleaf of contemporary tRNA is, in fact, composed of two structurally and functionally independent halves, of which the acceptor stem loop may be older than the anticodon dumpbell half (Maizels and Weiner 1994, 1999; Sun and Caetano-Anolle´s 2008). A third class of molecules essential for faithful reading of the genetic code comprises the aminoacyl tRNA synthetases (aaRSs), each of which chooses only one of twenty canonical amino acids to link it

14

This includes methionine, which strictly speaking is not counted among the aliphatic amino acids, but converted to N-formyl methionine, as in bacterial initiation, the polar nature of a peptide’s amino terminus is concealed effectively.

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to an appropriate tRNA (Hausmann and Ibba 2008). To observe this double specificity correctly, these enzymes, too, are composed of two domains, interacting with the tRNA acceptor stem and the anticodon dumpbell, respectively (Schimmel and Henderson 1994). Focusing on the tRNA acceptor stem, a structural code of amino acylation has indeed been recognized (Schimmel et al. 1993), which may have preceded the coding of mRNA-instructed protein sequences. This code reads local cues in experimental oligonucleotides, in the vicinity of the CCA terminus to be aminoacylated, and the decoding is built into the catalytic domain of the corresponding aaRS. One of the discriminator bases comprising this structural code is the single unpaired nucleotide N next to the universal CCA terminus. Notably, as analyzed by 1998, all the aaRSs for Leu, Val, Ile, Phe, and Met/fMet require the discriminator base A73 at that critical position (Giege´ et al. 1998). This indicates that the entire group of aliphatic amino acids connects backward to a common root of structural coding at the level of primordial acceptor stems. Interestingly, the same structural cues are recognized by two complementary classes of nonhomologous aaRSs, the type of which (I or II) is determined by the respective catalytic domain (Delarue 1995; Woese et al. 2000). When Manfred Eigen considered tRNAs as tentative candidates for the earliest genes, this notion was highly influenced by the geneticist’s preconception that “adaptors without messengers make no sense” (Eigen and Winkler-Oswatitsch 1981). Yet, what kind of evolutionary utility could possibly emerge from aminoacylated oligonucleotides that started to diverge at the acceptor sequence, adjacent to the universal CCA terminus? If the primordial docking site at the transpeptidation centers, together with assisting factors, could deliver its cargo where it fit in best, and if special adaptors and guiding aids preferred CCACCA-aa with aliphatic residues,15 this category could preferentially assemble several aliphatic amino acids in a row. Such peptides would be particularly suited to form membrane patches, especially if the first amino acid was formylated at the amino group, as it is the case in N-fMet, which presently initiates protein chain formation in bacteria. Other, less restrictive adaptors and guiding aids – accommodating any NCCA-aa – would instead assemble stochastic peptide sequences with more polar residues.16 Such peptides, however, would not in general be suited to fully integrate into membrane patches, even though many might still be able to attach to the membrane surface.

15

The peculiar CCACCA element still acts as a powerful signal for terminal processing (Maizels and Weiner 1994), and genomic tRNA sequences still ending in CCACCA in many archaea and bacteria preferentially encode nonpolar amino acids (GtRNAdb 2010). 16 To start with, glycine, alanine, aspartate and valine would have been most abundant (Moeller and Janssen 1992), and others would gradually come up, according to the number of reaction steps required in a diversifying protometabolism based on the TCA cycle (Davis 1999).

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The 21 currently known aaRSs divide into two equally frequent classes, type I and II. Except for lysine, there is exactly one aaRS per amino acid in many organisms,17 only lysine has both types, albeit in different organisms (Koonin and Aravind 1998). Within three corresponding subtypes, the catalytic domains of type I and II aaRSs bind the acceptor stem from opposite sides, with virtually no spatial overlap into one another’s hemisphere (Ribas de Pouplana and Schimmel 2001). There is some peculiar Ying–Yang symmetry at various levels about the canonical codon table of the genetic code, which has appealed to the imagination of model builders ever since the coding scheme has been deciphered (Findley et al. 1982; Taylor and Coates 1989; Davis 1999; Rodin and Rodin 2008; Rodin et al. 2009). Also, the universal cloverleaf structure of tRNAs has both symmetrical and selfcomplementary characteristics. Tentatively, the emergence of the cloverleaf has been attributed to two basic mechanisms, insertion of the anticodon dumpbell domain into the aa-acceptor stem loop (Maizels and Weiner 1999) – perhaps multiple events of this kind (Di Giulio 1999) – or self-duplication from a basic stem–loop precursor (Di Giulio 1995; Tanaka and Kikuchi 2001; Widmann et al. 2005). Either way, the evolutionary history of some 20 aaRSs and corresponding tRNAs must have been tightly coupled. Cumulative evidence indeed suggests that all tRNAs are of monophyletic origin (Widmann et al. 2005), deriving from a primordial stem-loop structure that has been doubled up by fold-back polymerization. How then is it possible to rationalize the sharp dichotomy between type I and type II aaRSs and corresponding tRNAs? The familiar cloverleaf structure of tRNA, in fact, folds into an angular L shape, which can attach to a flat surface in two orientations, as represented by mirror-shaped symbols L and G, respectively. In a self-sufficient RNA First scenario, there would only be other RNAs around to interact with, which in the case of primordial tRNAs could present aptamer-bound amino acids to the acceptor stem. These aptameric ribozymes would in turn be replaced by emerging protein aaRSs in sterically complementary configurations (Rodin and Rodin 2008). There is indeed precedence for such clamping of the acceptor stem region between the RNA and protein moieties of RNase P (Reiter et al. 2010), a universally conserved ribozyme involved in RNA processing (Sect. 12.13). Alternatively, the emerging tRNAs could have attached on to peptide-dominated membrane patches, where rudimentary aaRS activity would be encountered among quasi-stochastic peptide epitopes exposed at the surface. In the fully developed coding-decoding scheme, specificity is determined both at the acceptor stem during aminoacylation and at the anticodon to read the corresponding mRNA codons appropriately. In the L-shaped tRNAs, the corresponding sequence cues lie far apart at opposite ends of the molecule. In contemporary aaRSs, too, the specificity cues are distributed over different binding domains at either end. Conceptionally, such long-distance correspondence could

17

Many bacteria have yet fewer than 20 aaRSs, due to recoding of certain amino acid residues that are restructured on charged tRNAs (Sheppard et al. 2008).

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emerge at close range, if the tRNAs dimerized with peptides at a surface head-totail (L/L0 , where L0 is turned 180º, relative to L), as modeled by contemporary tyrosyl-RS dimers (Yaremchuk et al. 2002). Such dimers adsorbing in the L/L0 orientation would give rise to one type of aaRS, whilst binding in the G/G0 orientation would lead to the other type. Such peptide-connected tRNA dimers would structurally resemble the Stem-Elbow-Stem elements of the primordial peptidyl transferase center (PTC), described above (Sect. 12.11; Davidovich et al. 2009). At the level of the protoribosome, the primordial PTC has likely been assisted by accessory RNA peptide (RNPd) complexes, giving rise to the small ribosomal subunit. Originally, this may primarily have improved on the processivity of more or less stochastic amino acid polymerization, by using some unfolded RNA as a railing to hold the tRNAs in place (Fox 2010). Such a system could initially have consisted of a common type of proto-tRNA, which was loaded with various amino acids, using repetitive RNA complementary to the initial anticodon loop as guiding rails. Thereafter, by expanding the repertoire of both anticodon loops in tRNAs and appropriate railing RNAs, the small subunit became the actual decoding center – affecting the local peptide sequence, as guided by informative and decodable mRNAs. The ratchet mechanics driving peptide formation in a processive manner are inherent in the dynamic interactions between the large and the small subunits of the intact ribosome (Zhang et al. 2009), each transiently binding up to three tRNAs at the respective A, P, and E sites and allowing for intermediate, “hybrid” associations.18 In doing so, the processive ribosome hoards a thermodynamic treasure, keeping the nascent peptide in a receptive position and protecting its activated bond from abortive hydrolysis until the next aa-tRNA takes over and extends its length. Only when the growing peptides are long enough to interact with binding partners in an already peptide-rich hydrogel conglomerate, are they released and consigned to a fate of their own making. It is the ribosome that comprises the origin and perpetual spring of life on Earth.

12.13

Processing of RNA

Heydays of ribozymes

Folded RNA chains capable of catalysing chemical reactions came as a surprise when natural examples were first discovered, comprising a self-splicing intron in ribosomal RNA (Kruger et al. 1982) and the catalytic moiety of RNase P (GuerrierTakada et al. 1983). More examples have since been ascertained, as followed by analyses of the mechanisms of ribozymal catalysis (Doherty and Doudna 2000;

18

The three sites concern the activated amino acid (A), the growing peptide (P) and the exiting tRNA (E). At a transient hybrid state, following trans-peptidation, the peptide-receiving tRNA translocates to the P site in the large subunit while it still occupies the A site in the small subunit.

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Talini et al. 2009); for various groups of introns, see Lankenau (Chap. 11). Except for the involvement of tRNAs and ribosomes in amino acid activation and transpeptidation reactions, all other natural ribozymes engage in splitting and joining of phosphodiester bonds during processing and maturation of RNA substrates. Among all the natural ribozymes, which generally are embedded in ribonucleo-protein (RNP) complexes, only RNase P and ribosomes facilitate more than a single reaction cycle (Kazantsev and Pace 2006), whereas others are used up in their first and only reaction. Besides, ribosomes act more as a mechanical shuttle and funneling aid than a genuine catalyst (Sect. 12.11). Regardless of this narrow range of action in current biology, mainstream confidence is often reposed in the potential ability of ribozymes to manage the numerous organic chemical reactions in a fully connected proto-metabolism, so as to sustain a self-sufficient RNA world early on. In fact, many new ribozymes have been selected in vitro from random sequences (Talini et al. 2009), which is one of the corner stones to support an RNA-first scenario. In general, however, the efficiency of ribozyme action is much lower as compared with corresponding protein enzymes (Doudna and Lorsch 2005), and primeval coevolution as synergistic RNPd machines with uncoded peptides is a preferable option (Kurland 2010). The readiness and specificity of RNA-RNA interactions between single-stranded loops and bulges predestines ribozymes to specialize in the various processing steps involved in the maturation of metabolically active RNAs. The universally conserved RNP ribozyme RNase P, in particular, is pivotal in the maturation of tRNA precursors and related substrates by endonucleolytic cleavage at certain stem structures, so as to precisely generate the 50 end of the active product (Kazantsev and Pace 2006; Ellis and Brown 2009). The related RNase MRP complex is widely conserved in eukaryotes (Lee et al. 1996; Nazar 2004; Aspinall et al. 2007; Woodhams et al. 2007), where it is mainly involved in the processing of ribosomal RNA (rRNA) in the nucleolus (Lindahl and Zengel 1996). Besides, RNase MRP was first discovered in mitochondria, where the same RNA moiety19 is reported to generate the RNA primer for mitochondrial DNA replication (Chang et al. 1987), whereas the proteins attached are different in mitochondria and the nucleolus (Lu et al. 2010). Moreover, stable RNAs, such as tRNA and rRNA molecules, often carry bases that are chemically altered post-transcriptionally by specialized modification enzymes. The conversion of certain uridine sites to pseudouridine, in particular, tends to be highly conserved in evolution, foremost within or close to functionally important loops or bulges (Hamma and Ferre´-D’Amare´ 2006). At another series of conserved positions, rRNA is methylated at the 20 OH group of ribose moieties.

19

The assertion that the same RNA indeed occurs inside and outside of mitochondria needs to be scrutinized more critically, since the only P/MRP-like RNA gene (MRP1) hitherto detected in yeast mtDNA is distinctly different from both the nuclear RNase P and MRP genes (RPR1, NME1), and a translocation process of any nuclear RNA into the mitochondrion is hitherto unprecedented.

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Whilst bacterial pseudouridine synthase and rRNA methylase activities solely depend on protein enzymes, eukaryotes and archaea use multifunctional RNP complexes for both reactions. In these complexes, small guide RNAs constrain the target RNA by base pairing to flanking sequences at either side of a larger loop or bulge, so as to expose the central nucleotide at a sharp bend, where it is presented for modification to the active site of the protein moiety (Li and Ye 2006; Reichow et al. 2007; Duan et al. 2009). These and other small guide RNAs are often referred to as sRNAs (small noncoding) or snoRNAs (small nucleolar) in archaea and eukaryotes, respectively (Dennis and Omer 2005; Dieci et al. 2009). Box C/D and Box H/ACA RNAs, in particular, are conserved from archaea to mammals (Clouet Clouet d’Orval et al. 2001; Kiss et al. 2010). The largest – and arguably most complicated – RNP machines are likewise engaged in the processing of RNA. These are the spliceosomes of eukaryotic cells (Nilsen 2003; Jurica and Moore 2003; Ritchie et al. 2009), and there are two ancient kinds with only partly overlapping composition (Will et al. 2004; Will and L€ uhrmann 2005; Russell et al. 2006; Roy and Irimia 2009). Since the overwhelming majority of eukaryotic proteins are encoded by discontinuous bits and pieces in the genome, the corresponding transcripts need to be spliced into functional mRNAs, prior to meaningful translation by the ribosomes. It is the essential job of spliceosomes to remove all those intervening sequences (introns) – forming a branched byproduct (lariat) en route – and to join the adjacent coding parts (exons) together (Moore and Sharp 1993; Wolf et al. 2009). There is a growing suspicion that spliceosomes indeed are ribozymes at heart (Sashital and Butcher 2008; Butcher 2009). Yet, like ribosomes, they foremost are formidable RNP machines at large (Staley and Woolford 2009). In contrast with most other ribozymes, which esoterically engage in single-shot reactions, spliceosomes are rechargeable in an intricate cycle of dissociation and reassembly steps (Staley and Guthrie 1998). Why this elaborate mechanism only prevails in eukaryotes, but is absent in bacteria and archaea, has intriguing implications for how to interpret the rooting of the universal Tree of Life (Sect. 12.16). As tentative relics from an ancient RNA world are disproportionately more frequent in eukaryotic cells than in both bacteria and archaea (Poole et al. 1998; Collins and Penny 2009; Collins et al. 2009), it is not entirely unreasonable to consider that the basic blueprint for eukaryotic cell organization, too, might be of more ancient vintage than commonly believed (Kandler 1994a, b, c; Poole et al. 1999; Kurland et al. 2006; Glansdorff et al. 2008). It has long been noted that the mechanism of spliceosomal RNA splicing closely parallels certain features of group II catalytic introns (Chap. 11), which have some ribozymal self-splicing capacity on their own and likewise form lariats to initiate this reaction (Michel et al. 1989; 2009). Such introns20 are widespread in nature – not only in organelles of eukaryotic cells, where they were first discovered – but

20

Group I self-splicing introns are structurally unrelated. Also, they excise and circularize by a different mechanism (Cech 1990).

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also in prokaryotes (foremost bacteria and some archaea). They usually encode a reverse transcriptase, allowing them to act as transposable elements, as mediated by a multifunctional RNP complex (Toor et al. 2001; Simon et al. 2008; Lambowitz and Zimmerly 2010). As such, they are subversive or “rogue” agents, depending on others to make for a living. Evolutionarily speaking, they may well contain quite ancient constituents, but they combine rather few of such solitary relics in a shortcircuited device for taking a “free ride” on a smart yet borrowed ticket. While proponents of “introns late” models take it for granted that the obvious relationship between group II catalytic introns and spliceosomal ones implies that the former were ancestral to the latter kind (Martin and Koonin 2006; Koonin 2006a), firstprinciple “bottom up” inference rather suggests a relational order in reverse – deriving self-splicing introns by particular sampling choices from a much broader range of primordial components. Not inconceivably, many of those preexisting functional RNA elements can have resembled ordinary introns by depending on external factors to be processed properly (Sects. 12.14–12.16).

12.14

Gathering Genes on Chromosomes

Archival streamlining

The emergence of replicatable genes with particular metabolic functions is one thing; their integration into genomes is yet another. There are several modes of keeping functionally related genes together, all of which are biologically relevant to various extent. Most directly, the nucleic acid sequences of several genes can be connected into chromosomal entities. Also, groups of genes or their concatenates can be anchored at external scaffolds and/or be gathered in closed compartments (Sect. 12.15). At the RNA world stage already – with or without the help of uncoded peptides – the RNA gene products had to be distinguished and separated from the generative templates (the genes themselves), which likewise consisted of RNA in the beginning. Furthermore, this principle had to cooperate with a growing tendency to gather functionally related genes on a common plasmid or chromosomal entity. These same concerns still apply to RNA viruses, which have developed various propagative strategies to deal with functional and genetic aspects of RNA molecules as a common substrate. Indeed, viruses in general, and RNA viruses in particular, can be considered relics of an ancient evolutionary stage preceding cellular evolution (Koonin et al. 2006; Forterre 2006). Seen from a systems perspective, by combining a cluster of functionally interactive genes and a package coat, viruses were the first semi-autonomous subsystems to emancipate themselves (or “escape”) from the precellular substratum that comprised the protobiotic molecular ecosystem. Yet, all these viruses only refer back to an evolutionary stage where coded protein synthesis had been well established.

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Within the precellular substratum, already at a pre-coding stage of RNA and stochastic peptides, all the genomic templates of the protobiotic gene pool must have comprised both complementary strands, only one of which would in general be processed into metabolic products – be it as guiding RNAs or catalytic ribozymes. Commonly, the functional and complementary sequences are referred to as plus (positive or sense) and minus (negative or antisense) strands, respectively. It is the functional plus strand that is particularly vulnerable, since it must serve two lords, utility and archive. If all its copies would be consumed in metabolic work – first processed into a limited size, and worn out in the end –, all memory of its utility would vanish too. For as long as there still are minus strands around, plus copies can be readily regenerated, but some of these must also remain intact long enough to make new minus strands again. Conversely, the minus strand of RNA genes could solely adapt to archival requirements. This means that it was free to lose the reactive properties that contributed to ribozyme function, yet were hazardous to long-term stability of back-up memory. One way to facilitate an operational distinction between plus and minus templates is to select against all sequences prone to ribozyme processing on the archival minus strands. A more advanced possibility could be to incorporate 20 -methylated ribotides (Poole et al. 2000), as presently exist in stable functional RNAs at exposed positions. The more radical solution is to use 20 -deoxy ribotides instead, as materialized in DNA of present life (Forterre 2002). The second change toward DNA, using the methylated base thymine instead of uracil, has more subtle advantages, which relate to the latent instability of cytosine21 (Poole et al. 2001). The full transition to the modern DNA world required several new enzyme activities, primarily ribonucleotide reductase to provide the precursors and reverse transcriptase to incorporate DNA in the minus strand. Eventually, also plus strand equivalents were made as DNA by emerging DNA polymerases (Koonin 2006b), which fully separated the genomic archives from functional RNA products. In addition to being more stable in single-stranded chains, the overall doublestrandedness of DNA provided a basic layer of genetic redundancy, which is of utmost importance for the evolution of effective damage repair pathways. Back at the RNA world stage already, the subpopulation of plus strand molecules destined for metabolic tasks had to be processed to individual ribozymes, especially if several prospective ribozymes were embedded in a common precursor sequence. Ribozymes at large are characterized by consisting of multiple stem–loop structures, and tying their outmost ends together can decisively stabilize overall RNA structure, as demonstrated for circularized constructs (Puttaraju et al. 1993; Wang and Ruffner 1998). Naturally occurring introns are commonly processed by ribozyme action at specific terminal sites, and various means of circularization keep

21

As cytosine spontaneously deaminates to uracil, such a premutational damage could not systematically be reversed, for so long as uracil was a naturally occurring constituent in all the genes. Now that the genes carry T instead of U, newly appearing G:U pairs (from G:C) are detected as being anomalous and subjected to a special repair mechanism.

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their ends together (Sect. 12.13). This has led to proposals that the ribozyme genes of the RNA world stage were actually contained in intron-like segments of larger RNA genomic entities, as specified by the introns-first theory (Jeffares et al. 1998; Penny et al. 2009) or the mighty introns model (Fedorov and Fedorova 2004). These ribozyme gene segments were supposedly connected by unstructured linker sequences, which were discarded as redundant byproducts of intronic ribozyme processing. Coincidentally, the newly formed reactive ends of the spacers flanking a given intron arise in close proximity, which can have favored their joining in a corresponding splicing reaction. If these unstructured spacer RNAs were preferentially utilized as “guiding rails” by the rudimentary protoribosomes (Sect. 12.12), they could have adopted the role of mRNAs in the emerging world of coded protein synthesis. In turn, as coded proteins took over from uncoded peptides and ribozymes, the role of mighty-intron RNAs subsided and was superseded by exon-encoded proteins. Here, for a coherent view of this section, I take the early prevalence of introns for granted and consider introns-late alternatives later on (Sects. 12.15 and 12.16). After the discovery of split genes in eukaryotes, the intriguing possibility of exon shuffling led to seminal hypotheses of gene proliferation at various evolutionary stages (Gilbert et al. 1997; Patthy 1999; Roy and Gilbert 2006). This notion posits that introns may preferentially reside between functionally consolidated protein domains, so that occasional recombination events between introns in different genes would connect the flanking exons – as well as their encoded protein domains – in novel combinations. Alas, the restricted range of spliceosomal introns to eukaryotes has prevented many scholars from accepting a general relevance of such proposals, but a reappraisal of fundamental traits, as to their primitive or derived condition (Sects. 12.15 and 12.16), should add significance to exon shuffling at the RNA world stage already. As for eukaryotes in general, and metazoa in particular, there is ample evidence that exon shuffling has indeed occurred, providing for networks of physical protein–protein interactions on a prolific scale (Cancherini et al. 2010). If similar mechanisms already prevailed at the RNA world stage (Lehman 2003), the transition from uncoded peptides and ribozymes to RNAencoded proteins should have been greatly facilitated. Inasmuch as intron splicing is a kind of genetic recombination at the RNA level, which can also act in trans between different molecules (Kooter et al. 1984; G€unzl 2010), the partaking ribozymes have inevitably had a significant impact on RNA diversification whenever they first appeared on the evolutionary scene. While the advanced mode of exon shuffling in metazoa concerns the generation of modular multidomain proteins (Patthy 1999), primordial protein evolution by similar mechanisms would have involved considerably smaller entities, encoding ~20 amino acid residues per shuffling event or even less (Dorit et al. 1990). In fact, there is evidence that introns were generally more abundant in the past (Roy 2006; Cs€ur€os et al. 2008). Also, where rates of intron loss and gain could be compared in representative data sets, loss rates exceeded gain >20 fold (Roy and Gilbert 2005). Intron loss, in turn, has led to exon fusion, yielding longer contiguous coding regions.

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It was argued above that the first interactive peptide motifs congregated from stochastic amino acid oligomers, physically stabilizing complementary fit among a-helix and/or b-hairpin segments (Sect. 12.10). Subsequently, the formation of similar uncoded peptides was facilitated by oligoribonucleotides (Sect. 12.11), to be followed by sequence-encoded protein synthesis (Sect. 12.12). All this complexity had to emerge from random-hit kinetics and preferential retention of physically coherent semi-stable complexes. To start with, all sequences had similar likelihood of forming, but all products were not equally fit for being retained. With increasing length of functionally relevant sequences, some memory of what had worked in the past became mandatory before longer functional sequences could evolve. Thus, the first mini-exons became the memory of interactively “useful” peptide motifs. Such “useful” segments – among many other possible sequences – point out local or global optima in a virtually limitless sequence space. The most powerful mode of exploring the vast range of sequence space for local optima at increasing the chain length of product is to use functionally established small modular units and recombine them in various combinations (Lehman and Unrau 2005; Lehman 2008). The shuffling of mini-exons, which originally occurred as a byproduct from the processing and maturation of intronic ribozymes, is an expedient mechanism for such primordial recombination in a genetic sense. Moreover, the gathering of functionally related genes on contiguous entities is itself a self-supportive evolutionary strategy. While it is reasonable to assume that primordial genes emerged as a growing community of short linear molecules, collecting an entire cell’s essential genes on a single chromosome could not have happened all at once. As ribosomes are central to all life as we know it and consist of numerous physically interacting components, it is not surprising that the clustering of genes for rRNAs and/or ribosomal proteins has very ancient roots, preceding the split into bacterial and archaeal lineages (Siefert et al. 1997; Dandekar et al. 1998). Next in line are genes required for particular metabolic pathways, where overall function depends on many components being present in close proximity. Collecting interactive genes in so-called operons can have led to the assembly of plasmid-like entities (Glansdorff 1999; Fondi et al. 2009; Emiliani et al. 2010). While early RNA genes presumably started out as linear molecules, it is less obvious whether emerging chromosomal concatenates of several genes necessarily remained linear as well. Circular RNA genomes are certainly rare, but not impossible. Among animal viruses, only hepatitis D virus has circular RNA; several other examples are known in plants. The combination of rolling-circle replication and hammerhead-mediated self-cleavage of monomeric subunits in certain plant viruses (Song et al. 1999) may point at ancient connections to an RNA world, but so does the maintenance of eukaryotic chromosome ends by RNA-carrying telomerase as well (Blackburn 2005; Blackburn and Collins 2010). Due to the inherent instability of RNA, together with the constraints of the limiting Eigen threshold on the size of contiguous genomes as a function of error rate (Eigen 1993; Poole et al. 1998, 1999), large chromosomes – capable of carrying the entire genome of a quasi-autonomous cell – could not realistically arise before the advent of more stable DNA as genetic material. Modern prokaryotic cells – of

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archaea and bacteria alike – carry essentially all their genes on a single circular chromosome, but this is not the only possible evolutionarily stable strategy. In plants, animals, and other eukaryotic cells, nuclear genomes are actually distributed over several linear chromosomes – as opposed to the circular relics from bacterial endosymbionts in organelles. Possibilities for bypassing the constraints of low replicative Eigen thresholds are based on genetic redundancy (Reanney 1986), collecting many small entities in the same compartment and/or allowing recombination between larger molecules.

12.15

Organizing Protocells

Individuality emerging

How a primordial molecular ecosystem of protometabolic interactions began to partition into protocellular subsystems and how these subsystems organized themselves and functioned with many individual genes to start with is still anybody’s guess. Scientific approaches to rationalize this guesswork are deeply divided on a primal controversy in origin-of-life research (Pereto´ 2005), as to when cell-like entities first emerged on the trajectory from geochemical circumstances to biochemical conditions. Did cells appear quite early on and were they simple? or, rather, Did cells appear late and were they complex to begin with? While prevailing views take for granted that early protocells were simple (bacteria-like, or simpler yet), this intuitive axiomatic assertion lacks factual support and may be fundamentally flawed. Putting a few simple genes inside a simple vesicle, so as to watch or contemplate what happens next, is rather naive, indicating some lack of appreciation of how self-organizing stochastic processes arise and interact. Also, the tacit assumption that primordial protocells resembled a bacterial model in any way severely underestimates the evolutionary efforts that must have preceded the shaping of the bacteria-type model into a viable and evolutionarily stable strategy. To put it bluntly, modern bacteria can afford to appear tiny and simple in maintaining highly specific and efficient enzymes for all essential functions and carrying most of their essential genes exactly once on a single circular molecule of double-stranded DNA, which is accurately doubled up and segregated to self-similar daughter cells at each division. Yet, none of these prerequisites can have prevailed in primordial protocells already. The mutual entanglement of self-complication and self-simplification, which characterizes Darwinian evolution (Conrad 1990), likely goes back to precellular origins. All current life is organized around long and complex sequences of interactive chain-like macromolecules: proteins and nucleic acids. While present sequences represent highly selected configurations from a combinatorial sequence space of vast numbers and horizons, their evolutionary history must ultimately connect back to stochastic beginnings (Eigen 1971). Presumably, therefore, the precellular molecular ecosystem accumulated large numbers of quasi-stochastic

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sequences, still relatively short, from which certain functionally interactive combinations (“hypercycles”) were progressively amplified and, in turn, diversified at longer chain length. To satisfy various conditions for adaptive walks through sequence space, the emerging system had many redundant components with multiple weak interactions (Conrad 1990). As adaptive evolution gradually reduced the number of low-specificity components and, in turn, increased the length of sequences, expressing higher specificity and/or activity of system-supportive reactions, complementary tendencies for simplification and complication went hand in hand. The primordial redundancy of components stabilized both the entire system and quasi-autonomous subsystems, against potentially destructive mutational, metabolic, and environmental perturbations. Among all error-minimizing mechanisms in general, genetic redundancy is the most widespread prerequisite (Reanney 1986), allowing proofreading and recombination. At the precellular stage, when the first generations of “meaningful” proteins and their RNA genes were singled out among more or less random sequences of comparable length, the stochastic problems were enormous. As redundancy is required for many components at various levels, a simple genes-inside-vesicles model faces depletion by stochastic losses at each division and would hardly be viable early on. Instead, before long multi-genic plasmids and/or chromosomes had been established, together with effective proof reading, damage repair systems and segregation mechanisms, extensive masses of proto-cytoplasmic hydrogels had to remain connected in a state of confluence, spanning volumes much larger than presently seen in bacteria. Presumably, therefore, proto-cytoplasm and vesicular membranes coevolved for a long period, irrespective whether most of the membranes occurred inside or outside the proto-cytoplasm (Griffiths 2007). To confine the space available for the formation and premordial evolution of proto-cytoplasmic aggregates, I herein favor the pore space between mineral grains in temporally flooded sedimentary layers, and considerations discussed in various chapters of this book are certainly compatible with this inclination. Connected spaces surrounded by mineral precipitates at deep-sea hydrothermal vents have likewise been suggested (Martin and Russell 2007; Lane et al. 2010), but such abyssal settings lack the synthetic potential of sunlight and wet/drying cycles in surface-exposed environments. If indeed the precellular proto-cytoplasmic conglomerates exceeded bacterial dimensions substantially a priori, it should be most reasonable to assume that the internal organization of these colloid masses by fibrous cytoskeleton components, internal membrane complexes, and interactive motor proteins had high evolutionary priority early on (Griffiths 2007) – much higher than commonly appreciated. External wall-like boundaries or regular cell division mechanisms would only come in later, after genetic identities had been established more firmly. Membrane-bending protein complexes, in particular, are crucial in many ways for complex eukaryotic cell organization (Ungewickell and Hinrichsen 2007; Dawson et al. 2009), but also are increasingly recognized in prokaryotic cells. Hence, the ability to form membrane-bounded organelles may have existed before the

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divergence of eukaryotes from prokaryotes (Shively 2006; Murat et al. 2010; Sect. 12.17). An intrinsic principle of internal self-organization in mixed populations of proteins and nucleic acids is observed as visco-elastic phase separation, both for bacterial nucleoids (Woldringh and Nanninga 2006) and in eukaryotic nuclei (Iborra 2007; Rippe 2007). In fact, whenever the nuclear envelope dissolves in uttinger et al. 2009), this principle “open mitosis” of higher plants and animals22 (G€ is responsible in keeping the numerous chromosomes together, before the envelope reassembles from the endoplasmatic reticulum (ER) after nuclear division (Anderson and Hetzer 2008). In general, two principal models are under discussion to rationalize the emergence of eukaryotic nuclei – from karyogenic or endokaryotic origins (Lake and Rivera 1994). Together with the afore-mentioned principle of protein-assisted membrane bending at the characteristic nuclear pore complexes (Antonin and Mattaj 2005), the Karyogenic Hypothesis, resembling the regularly recurring regeneration of the nuclear envelope, appears to represent a more facile model to rationalize the primordial emergence of genome-enclosing nuclear envelopes (de Roos 2006), than the currently prevailing Endokaryotic Hypothesis, viewing nuclei as tentative relics from endosymbiotic origins (Martin 2005). While driving forces for retaining nuclear membranes are unclear if the posited prokaryotic donor already had a unified circular genome (and endosymbiotic events among prokaryotes are rare in the first place), primordial karyogenesis from endomembranes would represent a rational means of collecting highly fragmented genomes as heritable and selectable units in a common proto-cytoplasm. To conclude this section, I am led by the notion that precellular systems were highly organized internally, before they could ever become miniaturized as genuine cells in the modern sense. In this scenario, precellular life is more concerned with sessile growth and spatial organization, than with periodic division at the earliest possible time. Such priorities are more related to the molecular ecology of biofilms (Wolfaardt et al. 1994), than to free-living, suspended individuals and populations. Clearly, the earliest viable full-fledged cell had to come from somewhere – from a differently organized system that could not qualify as a genuine cell itself. On the other hand, this not-yet-a-cell system had to be viable on its own beforehand, which is the crux of every evolutionary intermediate – or else, not being viable itself would terminally disconnect the chain of continuity. How could a viable organismal system, at the height of a tentative RNA world scenario, conceivably be organized at all? To my mind, at least, the sessile origins of prebiotic interactions would most naturally lead to an amoeboid/plasmodial life style initially, where flat masses of proto-cytoplasm are appressed to mineral surfaces. Freely suspended cells, however, would only be generated much later.

22

This differs from "closed mitosis" in fungi and many protists, where the nuclear envelope remains intact throughout the entire cell division cycle.

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Pertinent to the problem of living in an RNA world, a partial model has been described for the genome organization of a conceptual breakthrough ribo-organism (Riborgis eigensis), at the stage when genetically encoded protein translation evolved (Jeffares et al. 1998; Poole et al. 1998), although spatial concerns had no part in these considerations. This model assumes fragmented genomes of many linear chromosomes, present in multiple copies each. As judged by RNA viruses, even contemporary RNA genomes are rather short (~10 kb) and highly mutable (Belshaw et al. 2008), which cannot provide for a contiguous RNA genome sufficient for an autonomously functioning organism. High gene copy number per primitive cell are not as such prohibitive for evolution to proceed (Koch 1984), but to maintain so many genes in a common entity, the average volume should be considerably larger than in contemporary prokaryotic cells. Moreover, as reliable segregation mechanisms to cope with highly fragmented genomes were still lacking, regular division cycles had probably not yet developed either. In dedication to Otto Kandler’s pre-cell theory (Kandler 1994a; Sect. 12.16), a spatially extended model is sketched out here, denoted as Riboplasmodium kandleri.23 To briefly motivate the advantage of its plasmodial characteristics, the flatness of amoeboid lumps of proto-cytoplasm combines large overall volume with a small diffusional distance in at least one of three dimensions.24 If an amoeboid cell mass keeps growing in the two dimensions of the substrate, but does not regularly split up, its general organization is described as plasmodial, syncytial or coenocytic, referring to high genome copy numbers in a common cytoplasm.25 Gene copy multiplicity is generally referred to as polyploidy, although strictly speaking, this term implies the existence of multiple sets of integral genomes, which were not yet established at the precellular stage considered here. To indicate the stochastic nature of gene or chromosome distributions at that primordial stage, heteroploidy has been suggested instead (Dougherty 1955). Internally, the many gene copies would likely assemble in lumps by visco-elastic phase separation. With time, functionally related genes would be clustered on longer molecules first, so that ribosomes, for example, could be assembled in multiple proto-nucleoli. To organize the large volume of such plasmodia efficiently, the differentiation of various

23

The nominal patronage for either model appeals to more than symbolic affiliations. While Eigen’s thresholds against inevitable error catastrophy set limits to the length of integral genomes, the complex pre-cells of Kandler’s model bypass the Eigen limits with highly fragmented and multiply redundant genomes, so as to allow sampling variation of overlapping subsets in different descendant lineages. 24 A small diffusional distance facilitates exchange of reactants in metabolic processes. In comparison, bacteria are small in three dimensions, and inbetween, hyphal mycelia are small in two of three dimensions. 25 This notion is modelled after the plasmodial slime mold Physarum polycephalum, where reticulate streams of multi-nucleate cytoplasm can spread on a surface in various directions, pinching off occasionally, but also fusing again, where two approaching streams happen to meet.

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internal membrane systems would likely have a high evolutionary incentive (Griffiths 2007). Not inconceavably, therefore, enclosing of proto-nucleoli and other gene-rich clusters by curved internal membrane envelopes could have formed multiple proto-nuclei in a common cytoplasm, well before the emergence of regular cell division cycles. Moreover, linear minichromosomes and circular plasmids can have coexisted in these proto-syncytial masses for a considerable time. To distinguish the primordially redundant states from true plasmodia, syncytia or coenocytes, which all are genuinely eukaryotic, proto-coenocytic, and protocoenocytes, is suggested to replace the pre-cells (sensu Kandler) term. Due to the initially quasi-stochastic distributions of genes and chromosomes, the posited proto-nuclei would not in general contain balanced genome sets in the beginning. With increasingly accurate segregation mechanisms evolving, however, nuclear division cycles could graduately favor and stabilize the maintenance of balanced genomes. Only thereafter could the coupling of cell division to the nuclear cycle ensure the regular generation of uninuclear cells with balanced genomes as well. One way of driving miniaturization from complex plasmodial layers would be pinching off of cyst- or spore-like propagules at certain intervals, so as to facilitate the spreading to potential distant habitats or survival during adverse periods in a dormant state. Frequent repetition of budding processes without falling into dormancy could subsequently evolve into a stabilized mini-cycle of cell division in suspension. Inasmuch as encapsulated dormant cysts and spores are often selected for resistance against desiccation and/or heat exposure, such stages could have served as preadaptations in the colonization of hydrothermal environments, which the original proto-organisms not necessarily were able to tolerate. This view is in line with the fact that complex eukaryotic cells have never adapted to hyperthermophilic growth conditions, whereas both archaea and bacteria have been subject to thermoreductive selection early on (Forterre 1995). Moreover, the sessile precellular systems assumed here and unicellular microorganisms in suspension would be subject to fundamentally different regimens of population dynamics, commonly referred to as K- and r-selection,26 respectively (MacArthur and Wilson 1967; Pianka 1970).

12.16

Common Rooting of the Tree of Life

Relating a simple trefoil to a complex source

Intriguingly, the central conjecture of this section – that proto-cellular evolution depended on collective sharing in a large communal ecosystem – has gained vital support from a rather unexpected source. As corroborated by computer simulations, the observed universality and exquisite optimization of the genetic code (Sect.

26

K refers to carrying capacity and r to the maximal intrinsic rate of natural increase. Accordingly, K-selection favors limited growth, maintenance and replacement, while r-selection drives unlimited multiplication and dispersal, which occasionally is truncated by widespread collapse.

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12.12) are best rationalized as resulting from communal innovation-sharing on the broadest basis (Vetsigian et al. 2006). The general notion of a common origin of living creatures on Earth goes back to Charles Darwin’s Origin of Species (Darwin 1859; Haeckel 1866),27 since every tree-like evolutionary branching pattern should have a root. The modern revival of universal-tree phylogeny, however, dates back to systematic sequence comparisons of ribosomal RNA (Woese and Fox 1977; Woese 1998; Pace 2006). This led to the recognition of a primal dichotomy, in that simple prokaryotic cells, in fact, are deeply divided into two subtrees – the superkingdoms or phylodomains of Bacteria and Archaea – with Eucarya (the eukaryotes) being more closely associated with archaea than with bacteria. The same – universal – phylogenetic Tree of Life is recognizable for a relatively narrow core set of protein-coding genes, most of which have functionally coevolved with protein translation (Harris et al. 2003; Puigbo` et al. 2009; Goldman et al. 2010). The nature of the association of archaea with eukaryotes, however, has not yet been resolved (Cox et al. 2008; Gribaldo et al. 2010), and secondary contributions of endosymbiotic, bacteria-derived organelles compound the issue.28 At any rate, the deeper roots of archaeal phylogeny are intimately intertwined with eukaryotic origins (Fournier et al. 2011). Ancient records of bacterial phylogeny, too, are shrouded with ambiguity by evolutionarily frequent lateral gene transfer (Dagan and Martin 2006; Bapteste and Boucher 2008; Puigbo` et al. 2010). Some 3.2-billion-year-old globular microfossils (50–300 mm) are surprisingly big (Javaux et al. 2010). They either resembled eukaryotes already, or prokaryotes of that era were substantially larger than modern bacteria. About the same time as inferred from large-scale comparison of gene families, a surge of genetic innovation – Archaean Expansion – was accompanied by, first, a formidable burst of new gene families, as closely followed by a similar counter-spike of gene loss events in specializing lineages (David and Alm 2010). This major expansion presumbly coincided with the basal radiation of bacterial lineages. As for complexity in modern prokaryotic cells, quite elaborate internal membrane systems are actually occurring in a corner of the bacterial world (Fuerst 2005; Forterre and Gribaldo 2010). Phylogenetically, the bacteria in question belong to the planctobacterial superphylum, comprising Planctomycetes, Verrucomicrobia, Chlamydiae Lentispherae, and Poribacteria. When enveloping membranes around certain bacterial nucleoids were first discovered (Fuerst and Webb 1991), this was merely considered as a curiosity. Yet, comparative evidence is mounting that similarities to eukaryotic characteristics are deeply engrained in protein structure and function, showing fold-level resemblance to vesicle coat protein complexes

27

Charles Darwin himself was very cautious, at least in public, not to speculate about the ultimate beginning(s) of life on Earth. Not so reluctant was Ernst Haeckel, a fervent promoter of Darwinian principles, deliberately combining the major evolutionary branches in a single tree (Haeckel 1866; Wikipedia 2011). 28 Mitochondria of essentially all eukaryotes (for respiratory electron transfer chain and oxidative phosphorylation) relate to a-proteobacteria (Clements et al. 2009), while plastids of green plants (for photosynthesis) relate to cyanobacteria (Gross et al. 2008).

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(Santarella-Mellwig et al. 2010), as well as functional equivalence to receptormediated endocytosis (Lonhienne et al. 2010). Incongruent trajectories of different genes can have more than a single explanation. While lateral (horizontal) gene transfer between divergent lineages is by far the most widely assumed mechanism, massive lineage-specific losses and selective retention of certain paralogs – originating from redundant gene families present in common ancestors – can likewise obscure the underlying ‘true’ phylogeny (Glansdorff 2000). By that token, modern birds are genomically streamlined relative to mammals and other vertebrates (Hughes and Friedman 2008). Among eukaryotic microbial lineages, too, genomic streamlining can occur to a great extent, as documented for microsporidia and budding yeast. In fact, microsporidia were once considered “extremely ancient eukaryotes” (Vossbrinck et al. 1987), but later recognized as being related to zygomycete fungi instead (Hirt et al. 1999; Keeling 2003). In budding yeast, genomic streamlining has resulted in intron loss at an enormous scale (Bon et al. 2003; Garfinkel 2005; Dujon 2010), as compared to most of its fungal relatives. This streamlining is a convergent trait in yeast and microbial evolution in general (Lynch 2006). As argued above (Sect. 12.15), highly redundant, fragmented genomes – encoding many families of low-specificity components, and changing rapidly, due to high error rates – were abundant in the universal-ancestor lineage. If massive loss of ancestral protein-coding genes can be tolerated (or evolutionarily promoted) at the advanced level separating birds from other higher vertebrates (Hughes and Friedman 2008), yet higher loss rates have certainly accompanied the genomic streamlining to be expected when error rates were drastically reduced with the breakthrough of full-fledged DNA-based organisms. The degree of streamlining, however, was not necessarily the same in all the lineages descending from a genomically redundant universal ancestor. Just as the genomically streamlined birds did not eradicate the less streamlined (more redundant) mammals, the highly streamlined prokaryotic cells of a modern kind need not have outcompeted all other descendants from a more sluggishly evolving stem lineage ( or stemline) – especially if life styles were radically different. As for the still prevailing mantra that prokaryotic cells exclusively comprised Earth’s biosphere for ~50% or more of Life’s existence, alternative views are rare but not unheard of. Founding advocates of a complex primordial stemline – leading to eukaryotes more directly – are Darryl Reanney, Carl Woese, Ford Doolittle, and Otto Kandler, accompanied by David Penny, Patrick Forterre, Nicolas Glansdorff and other colleagues. In addition to these overt supporters, Eugene Koonin has provided some complementary considerations (see below). The various arguments are framed as follows: • Theoretical implications from a primordial surplus of genetic redundancy at all levels, together with RNA splicing as a primordial mechanism of functional reconstitution from accumulating errors (Reanney 1974); • “Urkaryotes”, as represented by their (eukaryotic) 18 S rRNA – the engulfing host organisms of endosymbiotic proto-mitochondria (Woese and Fox 1977);

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• Very early divergence into three lineages – the “nuclear-cytoplasmic lineage” (~urkaryotes) retaining split genes as a primordial trait (Doolittle 1978, 1980); • Quasi-random sampling in the three superkingdoms, drawn from a pool of traits in a promiscuous and multiphenotypical population of precellular proto-organisms, or “pre-cells” (Kandler 1994a, b, c; Moreira and Lo´pez-Garcı´a 2007); • Continuity of function in complex RNA-based traits from a tentative RNP world to eukaryotes (Jeffares et al. 1998; Poole et al. 1998, 1999; Penny and Poole 1999; Kurland et al. 2006; Poole and Penny 2007b; Collins and Penny 2005, 2009; Collins et al. 2009; Penny et al. 2009; Poole 2010); • Unrecognized paralogies from primordial redundancy, reshuffling, and recombination, rather than pervasive lateral gene transfer at later stages (Glansdorff 2000; Glansdorff et al. 2008, 2009a,b); • Thermoreduction hypothesis of prokaryotic evolution (Forterre 1995; Gribaldo and Forterre 2005; Gribaldo et al. 2010); • Cellular escape, including giant pox-type viruses, from a Primordial Virus World Scenario, originally embedded in interconnected networks of inorganic compartments29 (Koonin 2009). Seen from the vantage point of a coherent precellular molecular ecosystem, a direct path to eukaryotic cell organization poses no mystifying conundrum at any step. Conversely, quite serious objections can be raised on various issues about a tentative prokaryote-to-eukaryote transition at any later evolutionary stage (Poole et al. 2003; Kurland et al. 2006; Poole 2006; de Nooijer et al. 2009; Poole and Neumann 2011). Advocates of primacy-of-prokaryotes models, however, tend to neglect or underestimate the pitfalls and inconsistencies of their assumptions (Lake and Rivera 1994; Martin 2005; Embley and Martin 2006; Martin and Koonin 2006), and molecular phylogenomics has hitherto failed to discriminate between the opposing views (Koonin 2010). In fact, the recently proposed Cellular Escape Model from a Primordial Virus World Scenario (Fig. 3 of Koonin 2009) has many ingredients in parallel with the views promoted here, except for its inherent assumption that the “advancedmembrane protocells” in interconnected “networks of inorganic compartments” at the base of the model did not leave any other direct descendants than the prokaryotic “escaping cells” of archaea and bacteria. This inadvertant neglect, however, should be up for revision, considering the validity of host continuity as a guiding principle. Giant viruses (Mimi- and pox-type), in particular, have much in common with incipient cellular escape (Claverie et al. 2006; Raoult and Forterre 2008). These viral giants, however, rely on yet larger eukaryotic cells as hosts, including the free-living amoeba Acanthamoeba polyphaga. Conceptually connecting Mimiviridae to primordial origins, therefore, presupposes a correspondingly primordial proto-eukaryotic host lineage as well. It is beyond the scope of this chapter

29

to be equated with porespace in sedimentary layers, as promoted in the current paper.

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to fully discern the range of problems arising from any prokaryotes-first scenario, but I should like to stress two major points, introns and engulfment. There is no doubt that nuclear spliceosomal introns and organellar group II self-splicing introns are interrelated (Cech 1986; Copertino and Hallick 1993), yet their origin is uncertain (Bonen and Vogel 2001). It is merely hypothetical as yet, that the multipartite spliceosomal machinery might have originated from compact self-splicing elements (Cech 1986) and the acquisition of bacterial protomitochondrial endosymbionts in an archaeal host might have triggered a chain reaction to that effect (Martin and Koonin 2006). More likely, the self-splicing transposable elements themselves are derived from preexisting multipartite components. Not only is the massive intron expansion in the chimeric fusant clone as such unlikely (Poole 2006), but the correlated assumptions of inventing both spliceosomes and nuclear envelopes – respectively to limitate intron expansion and to separate splicing from translation (Martin and Koonin 2006) – remain mechanistically a “Deus ex Machina” illusion. It is one thing to presume a precellular genetic melting pot from which the first genomic associations can have coalesced (Woese 1998), which requires a quite large promiscuous population or molecular ecosystem to be robustly self-supportive; but starting a secondary genomic meltdown from a single cell (the first host cell of the intron-bearing endosymbiont) appears acutely self-destructive. Moreover, curing the intrusive threat by ad hoc invention of spliceosomes as the largest ever RNP complexes in an established proteinsynthesizing organism is not the most straightforward evolutionary possibility. Rather, the composite spliceosomes bear all the hallmarks of relating to the same type of precellular (proto-coenocytic) genetic melting pot that also gave rise to ribosomes and other RNP machines (Penny et al. 2009). A particular trait of eukaryotic cells is no longer contested by any party – that mitochondria are of bacterial descent. In fact, modern eukaryotes have attained other endosymbionts repeatedly in various lineages, as commonly mediated by engulfment – phagocytosis without digestion. Why shouldn’t the ancient host to receive the mitochondrial ancestor have done the same? While smooth extrapolations into the past are perfectly admissible as a scientific tool for model building, discontinuous ad hoc assumptions are more questionable, especially when they have little mechanistic precedence or justification. By this token, assuming by default that a protoeukaryote host engulfed the mitochondrial ancestor is less presumptious than invoking more imaginary mechanisms (Poole and Penny 2007a). Phagocytosis is indeed a very ancient trait of eukaryote in general (Cavalier-Smith 2002; Hartman and Fedorov 2002). Also, the recognition of endocytosis-like protein uptake in planctobacteria (Lonhienne et al. 2010) is readily compatible with the notion that such endomembrane-facilitated uptake precedes the primal dichotomy of the phylogenetic Tree of Life. Moreover, extended evolutionary periods without any feeding predators are quite unlikely (de Nooijer et al. 2009). What mechanism may have led to the primal dichotomy in the universal Tree of Life, separating bacteria from the archaea–eukaryotic lineage? The most effective and arguably the oldest barrier against convective gene flows is physical or geographic separation (Darwin 1859), which should be considered first to this effect

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(Kandler 1994a). In fact, if this deepest split developed already at the stage of sessile precellular systems, effective distances of physical separation need not have been very large. Conceivably, bacterial lineages may have developed from the bulk of the precellular ecosystem, remaining under the influence of sunlight exposure, whereas a subsystem was translocated to more secluded places (to slightly deeper sediments or deeper waters). From there, the archaea may have diverted in two waves; the extremophilic Euryarchaeota diverged to habitats where protoeukaryotes could not follow, whereas the Crenarchaeota (eocytes) and eukaryotes developed from the remaining lineage (Lake et al. 1984; Cox et al. 2008). The mere existence of eukaryotic introns, as well as their enormous variability, is one of the most vexing aspects of molecular evolution. The dynamical records of intron loss and gain are manifold and heterogeneous (Jeffares et al. 2006), so that no single theory can explain all the different trends. All this diversification notwithstanding, there seems to exist a minor subset of conserved intron sites, the origin of which precedes the common eukaryotic ancestor (Rogozin et al. 2003; de Roos 2005, 2007), and perhaps the last universal ancestor all the same. Most lineages have indeed experienced more intron losses than gains (Roy and Gilbert 2005; Roy 2006) – Why are any introns retained at all? Some introns, at least, cannot be deleted without loss of essential functions (Parenteau et al. 2008). Since eukaryotic snoRNAs, involved in essential rRNA modification, are mostly derived from intron-encoded transcripts (Collins and Penny 2009; Hoeppner et al. 2009), such introns are essential for survival. Many other introns encode micro-RNAs of less specific function. Perhaps belonging to this class, a functional role of conspicuously retained mini-introns in the reduced genomes of algal endosymbiont “nucleoids” (Slamovits and Keeling 2009) has not yet been ascertained. Certain archaeal guide RNAs, too, are intron-encoded, albeit by tRNA introns in this case (Clouet d’Orval et al. 2001; Tang et al. 2002). This points at a direct link to a pre-coding RNA world, which at least archaea and eukaryotes have retained. In contrast, bacteria modify their stable RNAs by pure protein enzymes exclusively, although structural homology of such bacterial enzymes with their guide-RNA-dependent archaeal/eukaryotic counterparts is still significant. As implied by the continuity theorem of RNA functionality (Jeffares et al. 1998; Penny et al. 2009), the loss of guide RNA dependence in bacteria is more likely than gaining such functions at a protein-dominated advanced stage in the archaeal/eukaryotic lineage. In other words, bacteria could afford to lose their introns after their protein enzymes had become sophisticated enough to function without a need for guiding RNAs, while archaea have gathered most of their remaining guide RNA on intron-independent transcripts. To summarize the overall framework of the universal phylogenetic tree, I assume organizational continuity in a complex stemline, from which less complex lineages have sprouted off repeatedly. To distinguish between the decisive intervals along its evolutionary course, I propose to define “urkaryotic” for the earliest common stem before bacterial divergence, “archkaryotic” for the archaea–eukaryotic stem lineage, and “protokaryotic” for eukaryotic ancestors before the acquisition or mitochondrial endosymbionts. Irrespective of the polyphyletic origins of archaea and

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bacteria, the common description as prokaryotes still serves an important purpose as an organizational discriminator, similar to the utility of the yeast term within the fungal kingdom. The multiple appearance of a particular life style by convergent adaptation to more general selective constraints is commonplace elsewhere in evolutionary biology. Thus, it is not presumptious to suppose that unicellular microorganisms can have emerged from sessile precellular systems more than once. Such multiple cellular escape events from geographically separated protocells might not only have given rise to bacterial and archaeal lineages independently, but also to different archaeal branches from bifurcation in the tentative archkaryotic lineage, as well as to different branches of the highly diversified bacterial radiation, such as gram-positive vs. gram-negative bacteria, from now extinct urkaryotic remnants. As seen from this perspective, the planctobacterial superphylum has retained the highest degrees of intracellular compartmentalization from the complex proto-coenocytic stemline and, therefore, may represent the latest round of cellular escape on the bacterial frontier of the primal dichotomy.

12.17

Concluding Remarks

Making ends meet

The essence of Life is not only about organizing cells and organisms, but arguably even more about entire ecosystems. Just as hardly any cell exists completely on its own in any environment, no individual gene can survive without interactions with other genes in any protoplasm. Accordingly, selective trends act at various levels to bundle and reinforce self-supportive interactions – in turn accumulating organic molecules on mineral-surrounded pore space surfaces, bundling genes on common chains, and associating chromosomal chains in sub-cellular compartments, before cell-like entities can successfully escape with a considerable chance of propagative stability and survival. All the while, the overarching ecosystems become more complex, and individual cells, too, can eventually be integrated into endosymbiotically merged hypercells or multicellular organisms. Presumably, the long transition from geochemical origins to organismal life has passed through several stages (Fig. 12.2): • Carboxylic acids, aldol phosphates, amino acids, heterocyclics, and other organic compounds accumulated in the multi-connected pore space of mineralcatalytic, and probably photon-activated, geochemical reactors. • Self-stabilizing proto-metabolic networks coalesced as a mineral-cofactor world scenario. • Peptide-like amino acid polymers added catalytic potential to water–hydrophobic interfaces, at proto-membranes of hydrogels in a peptide-cofactor world scenario. • From a dual role of ribose phosphates, acting both in amino acid activation and in ribonucleotide polymerization, ribozymes and RNPd complexes took over in a cofactor-assisted RNPd world scenario.

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Fig. 12.2 Overview of transitional stages from primordial beginnings toward modern life

• By speeding up the generation of stochastic peptides, as followed by the adoption of sequence-specifying coding rules, the ever more sophisticated protoribosomes ushered in the currently prevailing regimen of RNA-encoded protein synthesis. • The initially self-sufficient RNP world regimen was subsequently backed up by genomic DNA for higher genetic stability – the modern RNA- and proteinassisted DNA world. • Presumably up to the RNP world level, a pervasive communal precellular system organized itself as a primarily photoautotrophic molecular ecosystem, mostly subject to K-selection. • With the generation of autonomously viable and propagative cell-like systems (cellular escape), free-living populations of r-selected organisms could enrich the evolutionary scene, which quickly differentiated into multiple ecological niches, comprising both autotrophic producers and heterotrophic recyclers of various kinds.

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As for the sluggish, sessile, and communal precellular systems of the LUCAS era, we have no phylogenetic indication that any complex (non-bacterial) descendents survived on the bacterial side of the primal dichotomy. Only the planctobacterial superphylum may come closest to such an evolutionary relic. On the archaeal side, however, complex remnants were not necessarily wiped out altogether by the newly appearing r-selected prokaryotic cells. Instead, a mutual adjustment process let other precellular remnants specialize in recycling of particulate organic matter, including the engulfment of free-living cells. Eventually, some complex communal remnants organized themselves as more slowly evolving proto-eukaryotic macro-cells. The prokaryotic micro-cells, in turn, became smaller in size but more prolific and ubiquitous by sheer numbers. Mainly relying on phagocytosis of prokaryotic cells for a living, the complex macro-cells had no need to miniaturize. Instead, they could retain and perfect much higher degrees of cytoskeletal infrastructure and compartmentalization at subcellular levels. At least once in such engulfing cells, bacterial cells were retained as pepetuating proto-mitochondrial endosymbionts. These compound cells gave rise to all the eukaryotes of the modern era.

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Index

A Abiotic, 143 Abiotic organic chemistry, 38 Achiral, 64 Activate, 133 Adenosine, 58, 61, 67, 73 Adenosine triphosphate (ATP), 58, 60–64, 68, 70, 78, 131, 254 ADP-ribose, 188, 201 AFM. See Atomic force microscopy (AFM) Alabandite, 6 Alkylation of nucleobases, 12 Alu elements, 262 Amino acids, 39, 111–114, 116–122 Amyloid, 161–162 Anaplerotic, 91 Anatase/rutile, 6 Anthropic, 227, 229, 231, 237, 240, 260, 275 Anthropic principle, 227, 230 Anticodon, 268, 270, 271, 273 Antisense RNA, 232–234 Aquifex aeolicus, 234 Arabidopsis, 234 Archaea, 234, 240, 247, 249, 250, 253, 255 Archkaryotic, 341 Argonaut, 233, 234, 238, 239, 241 Arsenate, 77 Ascobolus immersus, 236 a-sheet, 161 Atmospheric pressure, 5 Atomic force microscopy (AFM), 170, 178, 179 ATP. See Adenosine triphosphate (ATP) ATPases, 9, 16, 17, 21, 23 ATP synthases, 16, 23

Autocatalysis, 88, 95 feedback, 315 networks, 291, 312, 316 Autotrophic, 94

B Bayes’ theorem, 260 Beadle-Tatum, 268 Beilstein, 229 Beilstein/Gmelin database, 230 Belozersky, A.N., 11 Biofilms, 334 Bottom-up approach, 230 Breakthrough ribo-organism, 335 Brenner, S., 272 Broad-band semiconductors, 6

C Caenorhabditis, 234 Calvin cycle, 313 Carbonaceous chondrites, 38, 39 Carbon fixation, 96, 101, 102 Catalysis, 93, 98, 133 substrate-assisted, 320 Cell membrane, 16, 23 Cellular escape model, 339, 342 Channel, 163–164 Chaperones, 211 Chemical evolution, 110, 111, 113, 114, 119–122 Chemo, 133 Chlamydomonas, 249 Chloroplasts, 16

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361

362 Chondrites, 75 Circularly polarized light, 41 Clay, 19, 93 colloidal, 307 minerals, 113, 116, 121, 122 Coenzyme A (CoA), 254 Coenzymes, 186–187, 194–198, 200, 203 ribonucleotide-like, 319 world, 195 Coevolution, 98, 202, 203, 293, 294, 309, 311, 326 Compartmentalization, 16, 18, 19 Conformation, 135 Coribozymes, 187 Crick, F.H., 11, 14, 228, 232, 261, 268, 270, 272, 275 Cu(II), 114–116, 118, 119 Cyanate, 68, 71 Cyanohydrin synthesis, 45 2’,3’ Cyclophosphate, 258 Cyclotrimetaphosphate (cTMP), 67, 68, 75

D Darwin, C., 11, 229, 230, 266, 274 Darwinian, 86, 87 Darwinian selection, 18 Definition of life, 228–229 Dehydration reaction, 66 Delbr€uck, M., 263 Dendrigraft a-amino acids, 146 Dendritic, 140 Deoxyriboaldolase reaction, reverse, 216–218 Deoxyribonucleotides, 210, 211, 216–218 salvage, 216 Dicer, 234, 235, 239, 240 Dissipative structures, 299 DNA, 211–213, 216–219 Driving force, 149 Drosha, 235, 240 Dynamic, 130

E E. coli, 261, 265 Eigen, M., 11, 13, 262, 264–268, 270, 272, 274 Eigen thresholds, 332 Enantio, 133 Enantiomeric enrichments, 40, 41 Endokaryotic hypothesis, 334 Energizing, 129 Energy, 4–8, 10, 12–16, 19, 20, 23, 24 Enzymatic luciferin/luciferase system, 145

Index Enzymes, 90, 92, 93, 101 Eocytes, 341 Epigenetic inheritance, 236 Epimerization, 134 Error threshold, 266, 267 Eukaryota, 240, 247 Evolution, 86, 87, 89, 93–95, 101 Evolutionary transition, 210, 211, 219 Exapted, 144 Exon shuffling, 330, 331 Extraterrestrial organic material, 38

F Faint young Sun puzzle, 257 FeS, 8, 18, 22 clusters, 310 FeS2, 8 Feynman, R., 257 Fire, A., 234 Flavin adenine dinucleotide (FAD), 254 Fluorescence resonance energy transfer (FRET), 246, 274 Foldable, 135 Formamide, 9, 11, 67, 73 Fossils, 96 Fractal objects, 301 FRET. See Fluorescence resonance energy transfer (FRET) Fumarole, 68

G Gell-Mann, M., 229 Gene ago1, 234 Gene conversion, 236, 249 Genetic code, 322, 324 Genetic takeover, 215, 295 Gene transfer lateral or horizontal, 338 Genomic streamlining, 338 Geochemical reactor, 303 Geothermal pool, 10 Geothermal waters, 10, 19 Gilbert, W., 11, 228, 232 Glyceric acid, 41, 45, 46, 48, 49, 51, 52 Glycerol, 41, 46–49 Glycolaldehyde, 41, 47 Glycolic acid, 45 Glycolonitrile, 45 Gmelin, 229 Golden spike, 228, 229, 231, 247, 275 Gould, S.J., 247

Index Guide RNA, 327, 341 Gypsy, 242

H Habitability zone, 296 Hazen, 229 HCHO, 40, 43–45, 47–51 HCN, 40, 43–45, 47, 48, 50 Helix, 178, 179 Heterochromatin protein 1 (HP1), 236 Heteroploidy, 335 Hexamethylene tetramine (HMT), 47 Histone methylation, 236 Histone methyltransferases (HMTs), 236 HIV, 239, 241, 242 H3K9, 236 HMT. See Hexamethylene tetramine (HMT); Histone methyltransferases (HMTs) Homochirality, 40, 133 Honeycomb micro compartment, 18, 20, 21 H2S, 7, 9, 11, 22 Hydantoin, 141 Hydrolysis, 62 Hydrothermal, 5, 7–11, 18 Hydrothermal vents, 7, 9, 18, 93, 94, 97, 159 Hydroxy acids, 39 Hydroxyl radicals, 18 Hypercycles, 270, 272, 274, 333 Hypophosphite, 73–76

I I-CreI homing mega-endonuclease, 249 Inert, 131 Information, 12, 14, 294 Infrabiological system, 219 Initial Darwinian ancestor (IDA), 228, 260, 261, 267 Integrase, 239–241, 252 Introns, 326–330, 340, 341 group I, 249 group II, 250 mobile, 249 Introns-first theory, 295, 330 Iron-sulphur centers, 160 Irradiation, 6, 13 Irreducible complexity trap, 247, 260 Irving-Williams series, 317

J J€ackle, H., 232

363 K Karyogenic hypothesis, 334 Klug, A., 272 Kornberg, A., 275

L Lactic acid, 46 Landauer principle, 14 Lariat-RNA, 251 Last Universal Common Ancestor (LUCA), 7, 16, 21, 23, 86, 295 Last Universal Common Ancestor State (LUCAS), 228, 229, 241, 247, 250, 296, 308 Life NASA definition of, 291 Lightning, 75 Light quantum, 5 LINEs, 237, 238, 241, 251, 253, 262 Linne’s revenge, 246–249 Lipid membranes, 17–19, 22, 24 Lipids, 87, 97, 101 Lipids late hypothesis, 17 Long terminal repeat (LTR), 238, 241–245, 251, 253 LUCA. See Last Universal Common Ancestor LUCAS. See Last Universal Common Ancestor State (LUCAS)

M Maillard reaction, 47 Mello, C., 234 Membrane channels, 60 Membranes, 162, 301, 302, 308–310, 333, 334, 337 Mer, 135 Meselson, M., 231 Metabolism, 85, 87, 88, 90–97, 99–102 Metalliferous fluids, 8, 19 Meteorite, 74, 75 Micropia, 233, 234, 236, 239, 242, 243, 249, 273 Mighty introns model, 330 Miller paradox, 47 Miller, S., 227, 228 Miller, S.L., 38 Miller-Urey, 229 Minerals catalytic, 309 compartments, 21

364 Mitochondria, 16 MnS, 6–8, 10 Molecular immune system, 233, 236 Monophyletic groups, 246 Murchison meteorite, 38, 39, 41, 43, 45, 46, 51 Mutation, 93

N NAD, 186–188, 190, 192–201, 203, 204 NADH. See Nicotinamide adenine dinucleotide NADP, 188, 190 Nash-equilibrium, 269 Nests, 157–160, 164 Neurospora crassa, 236 Niches, 163 Nicotinamide adenine dinucleotide (NADH), 91–92 Nicotinate phosphoribosyltransferase, 190, 192 NMN adenylyltransferase, 190, 195 Nonenzymatic, 86, 93, 94 Non-ribosomal peptide synthesis (NRPS), 213–216, 219 Nucleic acids, 211, 213, 215, 216, 218 modified, 218 Nucleosides, 58, 66 Nucleotides, 58, 78

O Oligomer, 13 Orgel, L.E., 6, 11, 15, 228, 232, 272 Origin of life, 38, 39, 47, 51, 85–87, 90, 92–94, 96, 101, 102

P PAHs, 132 Pairing annealing, 14 Parity violation, 119, 120 Penelope, 249 Peptide-Cofactor world, 197, 200 Peptides, 111–116, 118–122, 176–179, 211–216, 219 lipopeptide, 214 oligopeptide, 213, 215 uncoded, 308, 318–321, 326, 328, 330, 331 world, 215 Peptide world, 111, 120, 122, 197, 200, 204 Peptidyl transferase, 210

Index Peptidyl transferase center (PTC), 320–322, 325 pH, 132 Phase transition, 266, 267 Phosphate bonds organic, 305 Phosphates, 58–62, 64, 67–71, 73–76, 78 Phosphite, 10, 13, 73–76, 78 Phospho-aldol, 76 Phospholipids, 66, 77 Phosphonates, 59, 74 Phosphor, 6, 9, 11 Phosphorescence, 7 Phosphorus, 58, 62, 63, 70–73 high-energy compounds, 318 Phosphorylation, 58, 61, 62, 66, 69, 72, 73, 75–78 Photo-activation ZnS-mediated, 303 Photocatalysis, 98, 100, 101 Photocatalysts, 7, 19 Photochemical reaction centers, 5, 23 Photo-dissociation, 12, 14, 15 Photostability, 12–14, 19 Photostable, 13–15, 19 Photosynthesis, 85, 86, 101 Phylogenetic systematics, 230, 246 Phylogenetic tree, 246, 248, 274 Phylogenomic analysis, 16, 17 Pieczenik, 272 Planctobacterial superphylum, 337, 342 Plasmid, 331, 333, 335 Plate tectonics, 300 P-loop, 159 Polyanionic, 142 Polycationic, 142 Polyhydroxy acids, 46 Polyols, 41, 42, 46, 51 Polyphosphates, 60, 61, 63, 69, 70, 72, 161, 165 Polyploidy, 335 Popper, 227, 232, 246, 254, 256, 275 Pore space, 303, 333 Prebiotic, 89–91, 93–96, 98, 101, 102 Precells, 86 Pre-cell theory, 335 Precipitates mineral, 8, 15, 18–21, 23 Primal dichotomy, 337, 340, 342 Primary pump, 317 Primordial atmosphere, 5, 6, 8 Primordial Earth, 7, 10, 12, 22 Primordial ocean, 110, 113, 114

Index Primordial pizza, 302 Primordial RNA-like polymers, 13, 14 Primordial soup, 302 Primordial stemline complex, 338, 341 Proofreading, 333 Protein synthesis gene-encoded, 293, 294, 328, 330 Protocell, 87, 101 Proto-coenocytic, 336, 340, 342 Proto-enzymes, 187, 197, 201 Protokaryotic, 341 Protometabolism prebiotic, 313 Protoribosomes, 203, 231, 255–257, 259, 260, 263, 265, 266, 268–270, 272, 274, 320–322, 325, 330 Provirus, 232, 242, 245 PTC. See Peptidyl transferase center (PTC) Pyridoxal–5’-phosphate, 198 Pyrite (FeS2), 258 Pyrophosphate, 60, 160 Pyruvate, 62, 76, 91, 94, 98, 99

Q Qb minivariant, 262, 265 Qb replicase, 261, 264 Quantum yield, 6 Quasispecies, 260 Quinolinate phosphoribosyltransferase, 190, 199

R Racemic, 132 RdRP, 234 Recombination, 333 Redfield ratio, 59 Redox reactions, 8–10, 22, 23 Reducing CO2, 5 Reducing potential, 5, 8 Reductionist, 133 Redundancy, 329, 332, 333 genetic, 333 Replicating entities, 18–21 Replicating systems, 11 Replication, 87, 93 Replication/repair/recombination (RRR) factories, 231 Replicators, 18, 19, 24 Retroids, 241, 242, 247–249, 251

365 Retrotransposons, 232, 233, 236, 238, 241, 242, 244, 247, 249, 251, 253, 273 Reverse transcriptase, 232, 233, 241, 250 Reverse transcription, 242–246 Ribonucleases, 239–241 Ribonucleoprotein (RNP), 211 Ribonucleotides, 143, 210–212 ribonucleotide reductase, 210, 212, 216, 218 ribonucleotide reduction, 211, 212, 216–218 Ribosomes, 15, 210, 211, 213, 230, 238–240, 254–256, 270, 272, 274 Riboswitches, 11 Ribozymes, 194, 195, 200, 202, 203, 210, 211, 215, 216, 319, 320, 324, 326, 329, 330 intronic, 330, 331 RNA, 209–213, 215, 216, 218, 219 replicase, 210, 211 RNA world, 209–211, 213, 215, 219 RNA-first world, 90–92, 101 RNAi. See RNA interference (RNAi) RNA-induced silencing complex (RISC), 234, 235, 238–242, 245, 251, 269 RNA interference (RNAi), 233, 234, 236–239, 241, 245, 251 RNaseH, 239–241, 244, 245, 247, 250, 253 RNase III, 234, 235, 240 RNA world, 11, 13, 58, 78, 110, 111, 122, 156, 187, 188, 194, 195, 197, 200, 201, 203, 232, 254 RNA world scenario, 294, 319, 324, 326, 334, 335 RNY, genetic code, 269 rRNA, 241, 249, 255, 274 RRR-factory, 231 rTCA cycle, 91, 92, 94–98, 100, 312, 313, 316 RT/RNaseH, 241, 242, 245, 251, 269, 273

S Salt-induced peptide formation (SIPF) reaction, 109, 113, 114, 122, 316 Schreibersite, 74, 75 SDSA. See Synthesis dependent strand annealing (SDSA) Self-organization, 292, 293, 299, 301–303, 314, 334 Self-replication, 13, 19 Semiconductors, 95, 99, 100 Sequence-homology search engine, 234 Serpentinization, 5, 6, 9, 10 SHREC, 236

366 Silencing-effector complex, 234, 236 SINEs, 262 SIPF. See Salt-induced peptide formation Sodium chloride, 113, 114, 120 Solar radiation, 6 Sphalerite, 6, 257, 260 Spiegelman Monster, 261, 262, 264 Spiegelman, S., 261, 262, 264, 270 Spliceosomes, 327, 328, 340 Splicing, 121, 122 16S rRNA, 229, 274 23S rRNA, 255 28S rRNA, 255 Stabilizes, 137 Stahl, F.W., 231 Stem group, LUCAS as, 247, 253 Stem lineage, 338, 341 Stereoselectivity, 120 Strecker synthesis, 40 Strong stop DNA, 245 Struvite, 58 Sugar acids, 41, 47, 50 Sugar-phosphate units, 13 Sunlight, 96, 98, 102 Surface metabolism, 295, 302, 310 Synthesis dependent strand annealing (SDSA), 237, 245, 246, 249–256, 258, 268, 269, 272, 274, 275

T Tamme, D., 230 TCA cycle, reverse. See rTCA cycle Tetrahymena thermophila, 236 Theory of life, 227, 228 Thermoreduction hypothesis, 339 Thermoreductive selection, 336 Thio-amino acids, 142 Thioester world, 317 Thymidylate synthase, 212 Thymine, 211

Index Top-down approach, 228, 230, 247, 293 Transacting homology signal, 234 Transferred, 130 Transition metals, 304 Transmembrane potentials, 169, 177 Tree of life, 296, 337, 340 Trimetaphosphate, 69, 75

U Ulysses, 249 Uracil, 211, 212 uracil-DNA, 212 Urey, H.C., 38 Urkaryotes, 338, 341 UV, 6, 12–14, 16, 19, 21, 24 dissociation, 13 protection, 13, 259 stability, 14

V Vesicles, 88, 93, 101 Vicinal OH groups, 318, 319 Virus like particles (VLPs), 242, 244–245 Volcanoes, 73

W W€achersh€auser, G., 258 Watson-Crick, 231, 252 Weissmann, C., 264 Woese, C.R., 11, 18, 228, 232, 268 Wolframite, 6

Z Zinc sulfide, 6–8, 10, 13, 15, 18–21, 23, 24, 75–76, 97–99 ZnS. See Zinc sulfide Zn world hypothesis, 257

Origins of Life: The Primal Self-Organization