Planetary change and biochemical adaptation: molecular evolution of corrinoid and heme biosyntheses

Abstract

This review examines the evidence suggesting that the anaerobic biosynthesis of cobalamin (vitamin B12) evolved during early stages of cell evolution and was quickly recruited in the pathway leading to deoxyribonucleotides, the building blocks of DNA genomes. Biochemical evolution preceding the synthesis of the heme group and related molecules is discussed within the framework of geological evolution in which the appearance and accumulation of an oxygen-rich atmosphere stands as one of the major events in the evolution of the planet and the biosphere.

Keywords:

• Adenosylcobalamin
• Porphyrin biosynthesis
• Ribonucleotide reduction
• Globins
• Heme group

Introduction

Deeply disturbed by the sight of blood, in 1827 the young Charles Darwin left the prestigious Edinburgh medical school and moved to Cambridge, before devoting himself in full to the study of natural history and biological evolution. He would have never guessed that less than 50 years after the publication of the Origin of Species, the distinguished physician George H. F. Nuttall would realize the significance of blood as a phylogenetic marker using immunological methods. With admirable intuition, Nuttall wrote in his 1904 Blood Immunity and Blood Relationship that ‘…a common has persisted in the bloods of certain groups of animals throughout the ages which have elapsed during their evolution from a common ancestor, and this in spite of differences of food and habits of life’, adding that ‘the persistence of the chemical blood-relationship between the various groups of animals serves to carry us back into geological times, and I believe we have but begun the work along these lines, and that it will lead to valuable results in the study of various problems of evolution’.1

As shown by the detailed reconstructions of the evolutionary history of hemoglobin genes and homologous sequences found in distantly related eukaryotic and prokaryotic species,2,3 Nuttall’s insight has been fulfilled beyond any expectations. Molecular phylogenies demonstrate that the evolution of globin-like hemeproteins goes back well before the origin of animal blood, and can now be traced back to more than 2×10⁹ years ago.2^–4 As suggested by Moens et al.,3 all globins may have evolved from an ancestral 17 kDa redox protein already endowed with the eight helices typical of the globin-fold. What is rarely realized is that the heme prosthetic group, which plays a key role in the binding and/or transport of diatomic oxygen, is in fact much older than globins themselves. The biosynthetic precursor of the Fe-bearing heme group is uroporphyrinogen III (UroIII), which is also an intermediate in the biosynthesis of other porphyrins, including siroheme, Mg chlorophylls, Ni-F430 and cobalt cobalamin or coenzyme B12, a cofactor in the synthesis of DNA components.5 As summarized here, comparative biochemistry and molecular phyogenies indicate that one of the first outcomes of the biosynthesis of corrinoids, which stops just short of porphyrin formation, was the reduction of ribose to deoxyribose, i.e. it is a pathway almost as old as DNA cellular genomes that probably appeared 3·8 to 4×10⁹ years ago.

Coenzymes as Molecular Fossils of the RNA World

The independent discovery of ribozymes by Sidney Altman and Thomas Cech gave considerable credibility to the idea that primordial organisms were based on RNA as both the genetic material and as catalyst, a possibility first suggested in the late 1960s by Carl Woese, Leslie Orgel and Francis Crick.6 The surprising ability of RNA molecules to catalyze an increasingly large number of chemical reactions has lent strong support to the likelihood of the so-called RNA world, and greatly simplifies the understanding of the origin of protein biosynthesis and of the genetic code. Although how the RNA world itself originated is an open question, the experimental evidence demonstrating that ribozymes can mediate amino acid activation, aminoacyl-RNA synthesis, peptide-bond formation, and RNA-based coding, suggests ribosome-mediated protein synthesis first evolved in an RNA world.7

Coenzymes play a pivotal role in extant biosynthetic routes, and it is widely accepted that ribonucleotide-like coenzymes and other cofactors are the oldest metabolic fossils. The ribonucleotide moieties of many coenzymes may reflect recruitment processes that diversified the catalytic abilities of ribozymes, providing new venues for an increasingly complex RNA world metabolism, i.e. they are holdovers of a primitive RNA-based metabolic apparatus.8,9 This possibility is strongly supported by the easiness of prebiotic synthesis of several cofactor components, including adenine, ribose, and nicotinamide derivatives,10 as well as by the in vitro selection of ribozymes capable of synthesizing coenzymes and to use them to perform chemical catalysis of protein enzymes.11

The presence of an adenosyl moiety in ATP, FADH, NAD(P)H, coenzyme A, S-adenosylmethionine and adenosylcobalamine, i.e. vitamin B12 coenzyme, links these cofactors with RNA biochemistry.12 As argued forcefully by Eschenmoser,13 simple corrinoids may have formed non-enzymatically, and the energetically favorable reactions that underlie the in vitro self-assembly potential of porphyrins and corrins may explain its ubiquity as a cofactor.

However, coenzymes are not all equally ancient. It is likely that the highly reactive adenosyl radical produced by the single-electron reduction of S-adenosylmethionine preceded the evolutionary emergence of the more complex heme-related porphyrin adenosylcobalamine or coenzyme B12,14 which may have originally had fermentative functions.5 The corrinoid moiety of coenzyme B₁₂ is formed from uroporphyrinogen III (UroIII) via a series of complex enzyme-mediated chemical changes. Few additional enzymatic steps from it produce chlorophyll, the heme group and siroheme, suggesting the possibility that anoxygenic and oxygen-producing photosynthesis, aerobic respiration, methyl transfer, and transport of electrons to NO₂ and SO₃, are linked with an ancient metabolic pathway that evolved under the anoxic conditions of the primitive Earth soon after the appearance of life.

DNA Genomes as Latecomers in Molecular Evolution

Since all extant cells are endowed with DNA genomes, the most parsimonious conclusion is that this genetic polymer was already present in the last common ancestor (LCA) or cenancestor that existed prior to the divergence of the three primary domains, i.e. the Bacteria, Archaea, and Eucarya. A significant number of the highly conserved genes common to all three lineages are sequences involved in the synthesis, degradation and binding of RNA, including transcription and translation.15 Although estimates of the gene complement of the cenancestor include sequences that may have originated in different epochs, the extraordinary conservation of this core of RNA-related sequences supports the hypothesis that the LCA was an evolutionary outcome of the so-called RNA/protein world.

The sequence similarities shared by many ancient large proteins found in all three domains suggest that considerable fidelity existed in the operative genetic system of the LCA. Despite claims to the contrary,16 such fidelity is unlikely to be found in RNA-based genetic systems,17,18 which do not replicate using the multiunit cellular DNA-dependent RNA polymerases, but are based on RNA replicases lacking editing mechanisms. It is thus likely that double-stranded DNA genomes had become firmly established prior to the divergence of the three primary domains.

The molecular takeover that led to DNA genomes was the outcome of the metabolic evolution of involving: (i) the reductive elimination of the 2′-hydroxyl group in ribonucleotides; (ii) the replacement of uracil by thymine; and (iii) the selection of editing mechanisms. The presence of uracil in DNA and ribothymidine in tRNA, are well documented.19 However, all known cellular and viral genomes have either ribo- or deoxyribonucleotides in the same chain, but not a mixed composition. In other words, the key event in the evolutionary transition to DNA genomes was not the development of thymine anabolism, but of deoxyribose biosynthesis. In sharp contrast with other energetically favorable biochemical reactions (such as phosphodiester backbone hydrolysis or the transfer of amino groups), the direct removal of the oxygen from the 2′-C ribonucleotide pentose ring to form the corresponding deoxy-equivalents is a thermodynamically much less-favored reaction, suggesting that biological ribonucleotide reduction evolved only once.

The three known classes of ribonucleotide reductases (RNRs) that can mediate the reduction of ribonucleotides do so by generating the substrate 3′-radical species required for the removal of the 2′-OH group.20,21 Each class of RNRs requires different cofactors: the strictly aerobic class I RNR uses a diferric iron center, while cobalamin and S-adenosylmethionine (SAM) are used by RNR II and III, respectively. Sequence analysis and biochemical characterization have confirmed their ultimate monophyletic origin.20,22 The hypothesis that RNRs diverged from an ancestral SAM-dependent class III-like enzyme20 is supported in part by the non-enzymatic synthesis of the cofactor components22 and the wide distribution of SAM-synthetizing enzymatic machinery, as well as by reports of SAM riboswitches that appear to be molecular holdovers from the RNA World.23 In other words, several key steps of porphyrin biosynthesis appeared soon after the emergence of SAM-dependent RNRs, but prior to the divergence of the three classes of RNRs.

Concluding Remarks

The evolving chemistry of the planet due to the cyanobacterial production of oxygen had extraordinary consequences on the evolution of the biosphere and the Earth itself. The accumulation of significant amounts of free oxygen in the Proterozoic atmosphere and its hazardous effects on microbial evolution became a powerful selection pressure that led first to the appearance of manifold polyphyletic protection and repair mechanisms. It is possible that a number of ancestral lineages became extinct, while other anoxic prokaryotes were forced either to adapt to aerobic conditions or to become restricted to anaerobic environments, leading to a spatial redistribution of microbial ecosystems. However, the inventory of microbial mechanisms that offset oxygen poisoning is an extraordinary demonstration of manifold intracellular molecular processes to detect, detoxify and, in many cases, exploit the properties of oxygen. This is exemplified, for instance, in the diversification of the 2-oxoglutarate-Fe (II) oxygenase superfamily, that led to the evolution of the AlkB protein involved in the repair of DNA nucleophilic centers susceptible to alkylating agents.24

Biochemical evolution preceding the synthesis of the heme group and related molecules can thus be understood against an axis representing geological time in which the appearance and accumulation of an oxygen-rich atmosphere stands as one of the major events in the evolution of the planet and the biosphere. It is remarkable how several defense mechanisms against the highly deleterious effects of free oxygen led to more efficient energy producing mechanisms like aerobic respiration, as well as to the replacement of anaerobic processes by more effective O₂-dependent pathways. For instance, the appearance of free oxygen not only led to the two different radical generating mechanisms found in class I and class II RNRs,25 but also had a major impact in the biosynthesis of coenzyme B12.5 Although the older anaerobic pathway has been retained in many prokaryotes, its comparison with the aerobic route indicates that the appearance of the oxygen-dependent CobG protein led to the irreversible and simpler aerobic cobalamin biosynthesis.

The synthesis of UroIII and the subsequent production of chlorophyll and the heme group also led to novel mechanisms of O₂-detection and detoxification and, eventually, in its transport and utilization in more efficient ways of energy production. Because of their ability to form metal complexes, the heme prosthetic groups are particularly important in biological systems, as shown by their ample distribution as coenzymes of catalases, peroxidases, cytochromes, myoglobins, and hemoglobins. These examples not only demonstrate that biological traits that arose by natural selection did not necessarily evolve for their present purpose, but also that in the course of evolution, that which does not kill us makes us, at least in some cases, stronger.

The author is indebted to Ricardo Hernández-Morales and Mario Rivas for help with the manuscript.