The evolutionary history of the first three enzymes in pyrimidine biosynthesis

Some metabolic pathways are nearly ubiquitous among organisms: the genes encoding the enzymes for such pathways must therefore be ancient and essential. De novo pyrimidine biosynthesis is an example of one such metabolic pathway. In animals a single protein called CAD

1 Abbreviations: CAD, trifunctional protein catalyzing the first three steps of de novo pyrimidine biosynthesis in higher eukaryotes; CPS, carbamyl phosphate synthetase domain; CPSase, carbamyl phosphate synthetase activity; ATC, aspartate transcarbamylase domain; ATCase, aspartate transcarbamylase activity; DHO, dihydroorotase domain; DHOase, dihydroorotase activity; GLN, glutaminase subdomain or subunit of carbamyl phosphate synthetase, GL Nase, glutaminase activity; SYN, synthetase subdomain or subunit of carbamyl phosphate synthetase; SYNase, synthetase activity.

carries the first three steps of this pathway. The same three enzymes in prokaryotes are associated with separate proteins. The CAD gene appears to have evolved through a process of gene duplication and DNA rearrangement, leading to an in-frame gene fusion encoding a chimeric protein. A driving force for the creation of eukaryotic genes encoding multienzymatic proteins such as CAD may be the advantage of coordinate expression of enzymes catalyzing steps in a biosynthetic pathway. The analogous structure in bacteria is the operon. Differences in the translational mechanisms of eukaryotes and prokaryotes may have dictated the different strategies used by organisms to evolve coordinately regulated genes.     

Evolutionary relationships of the carbamoylphosphate synthetase genes

Carbamoylphosphate is a common intermediate in the metabolic pathways leading to the biosynthesis of arginine and pyrimidines. The amino acid sequences of all available proteins that catalyze the formation of carbamoylphosphate were retrieved from Genbank and aligned to estimate their mutual phylogenetic relations. In gram-negative bacteria carbamoylphosphate is synthesized by a two-subunit enzyme with glutamiriase and carbamoylphosphate synthetase (CPS) activity, respectively. In gram-positive bacteria and lower eukaryotes this two-subunit CPS has become dedicated to arginine biosynthesis, while in higher eukaryotes the two subunits fused and subsequently lost the glutaminase activity. The CPS dedicated to pyrimidine synthesis is part of a multifunctional enzyme (CPS II), encoding in addition dihydroorotase and aspartate transcarbamoylase. Evidence is presented to strengthen the hypothesis that the two kinas subdomains of all CPS isozymes arose from a duplication of an ancestral gene in the progenote. A further duplication of the entire CPS gene occurred after the divergence of the plants and before the divergence of the fungi from the eukaryotec root, generating the two isoenzymes involved in either the synthesis of arginine or that of pyrimidines. The mutation rate was found to be five- to tenfold higher after the duplication than before, probably reflecting optimization of the enzymes for their newly acquired specialized function. We hypothesize that this duplication arose from a need for metabolic channeling for pyrimidine biosynthesis as it was accompanied by the tagging of the CPS gene with the genes for dihydroorotase and aspartate transcarbamoylase, and as the duplication occurred independently also in gram-positive bacteria. Analysis of the exon-intron organization of the two kinase subdomains in CPS I and II suggests that ancient exons may have comprised approx. 19 amino acids, in accordance with the prediction of the intron-early theory. Correspondence to: M.J.B. van den Hoff     

Identification of New Members of the GS ADP-Forming Family from the De Novo Purine Biosynthesis Pathway

Most living organisms can synthesize isosinate from 5-phosphoribosyl 1-pyrophosphate in the de novo purine biosynthesis pathway, which is basically composed of 10 reaction steps. Phosphoribosylglycinamide synthetase (GARS) catalyzes the second step of the pathway. We found that the enzyme shows weak, but significant, sequence similarity to phosphoribosylglycinamide formyltransferase 2 (GART2) and the ATPase domain of phosphoribosylaminoimidazole carboxylase (AIRCA), which catalyze the third and sixth steps of the pathway, respectively. In addition, the three enzymes were similar in amino acid sequence to biotin carboxylase (BC) and carbamoylphosphate synthetase (CPS), which are the members of the GS ADP-forming family. This family has been identified through a tertiary structure comparison and includes glutathione synthetase, d-alanine:d-alanine ligase, BC, succinyl-CoA synthetase β-chain, and phosphoribosylaminoimidazole-succinocarboxamide synthase. Molecular phylogenetic analysis based on a multiple alignment of GARS, GART2, AIRCA, BC, and CPS suggests that GART2 is more closely related to AIRCA than to GARS among the three enzymes from the pathway, though the three enzymes are relatively close to each other within the GS ADP-forming family. Moreover, the analysis showed that archaeal GARS had diverged before the speciation between bacteria and eucarya. Received: 3 June 1998 / Accepted: 8 September 1998     

Global Patterns of Protein Domain Gain and Loss in Superkingdoms

Domains are modules within proteins that can fold and function independently and are evolutionarily conserved. Here we compared the usage and distribution of protein domain families in the free-living proteomes of Archaea, Bacteria and Eukarya and reconstructed species phylogenies while tracing the history of domain emergence and loss in proteomes. We show that both gains and losses of domains occurred frequently during proteome evolution. The rate of domain discovery increased approximately linearly in evolutionary time. Remarkably, gains generally outnumbered losses and the gain-to-loss ratios were much higher in akaryotes compared to eukaryotes. Functional annotations of domain families revealed that both Archaea and Bacteria gained and lost metabolic capabilities during the course of evolution while Eukarya acquired a number of diverse molecular functions including those involved in extracellular processes, immunological mechanisms, and cell regulation. Results also highlighted significant contemporary sharing of informational enzymes between Archaea and Eukarya and metabolic enzymes between Bacteria and Eukarya. Finally, the analysis provided useful insights into the evolution of species. The archaeal superkingdom appeared first in evolution by gradual loss of ancestral domains, bacterial lineages were the first to gain superkingdom-specific domains, and eukaryotes (likely) originated when an expanding proto-eukaryotic stem lineage gained organelles through endosymbiosis of already diversified bacterial lineages. The evolutionary dynamics of domain families in proteomes and the increasing number of domain gains is predicted to redefine the persistence strategies of organisms in superkingdoms, influence the make up of molecular functions, and enhance organismal complexity by the generation of new domain architectures. This dynamics highlights ongoing secondary evolutionary adaptations in akaryotic microbes, especially Archaea.     

Evolution of cytochrome oxidase, an enzyme older than atmospheric oxygen.

Cytochrome oxidase is a key enzyme in aerobic metabolism. All the recorded eubacterial (domain Bacteria) and archaebacterial (Archaea) sequences of subunits 1 and 2 of this protein complex have been used for a comprehensive evolutionary analysis. The phylogenetic trees reveal several processes of gene duplication. Some of these are ancient, having occurred in the common ancestor of Bacteria and Archaea, whereas others have occurred in specific lines of Bacteria. We show that eubacterial quinol oxidase was derived from cytochrome c oxidase in Gram-positive bacteria and that archaebacterial quinol oxidase has an independent origin. A considerable amount of evidence suggests that Proteobacteria (Purple bacteria) acquired quinol oxidase through a lateral gene transfer from Gram-positive bacteria. The prevalent hypothesis that aerobic metabolism arose several times in evolution after oxygenic photosynthesis, is not sustained by two aspects of the molecular data. First, cytochrome oxidase was present in the common ancestor of Archaea and Bacteria whereas oxygenic photosynthesis appeared in Bacteria. Second, an extant cytochrome oxidase in nitrogen-fixing bacteria shows that aerobic metabolism is possible in an environment with a very low level of oxygen, such as the root nodules of leguminous plants. Therefore, we propose that aerobic metabolism in organisms with cytochrome oxidase has a monophyletic and ancient origin, prior to the appearance of eubacterial oxygenic photosynthetic organisms.     

Evolutionary implications of the mosaic pyrimidine-biosynthetic pathway in eukaryotes

The de-novo pyrimidine biosynthetic pathway involves six enzymes, in order from the first to the sixth step, carbamoyl-phosphate synthetase II (CPS II) comprising glutamine amidotransferase (GAT) and carbamoyl-phosphate synthetase (CPS) domains or subunits, aspartate carbamoyltransferase (ACT), dihydroorotase (DHO), dihydroorotate dehydrogenase (DHOD), orotate phosphoribosyltransferase (OPRT), and orotidine-5'-monophosphate decarboxylase (OMPDC). In contrast with reports on molecular evolution of the individual enzymes, we attempted to draw an evolutionary picture of the whole pathway using the protein phylogeny. We demonstrate highly mosaic organizations of the pyrimidine biosynthetic pathway in eukaryotes. During evolution of the eukaryotic pathway, plants and fungi (or their ancestors) in particular may have secondarily acquired the characteristic enzymes. This is consistent with the fact that the organization of plant enzymes is highly chimeric: (1) two subunits of CPS II, GAT and CPS, cluster with a clade including cyanobacteria and red algal chloroplasts, (2) ACT not with a cyanobacterium, Synechocystis spp., irrespective of its putative signal sequence targeting into chloroplasts, and (3) DHO with a clade of proteobacteria. In fungi, DHO and OPRT cluster respectively with the corresponding proteobacterial counterparts. The phylogenetic analyses of DHOD and OMPDC also support the implications of the mosaic pyrimidine biosynthetic pathway in eukaryotes. The potential importance of the horizontal gene transfer(s) and endosymbiosis in establishing the mosaic pathway is discussed.     

Phylogeny Based on Whole Genome as inferred from Complete Information Set Analysis

Previous molecular phylogeny algorithms mainly rely onmulti-sequence alignments of cautiously selected characteristic sequences,thus not directly appropriate for whole genome phylogeny where eventssuch as rearrangements make full-length alignments impossible. Weintroduce here the concept of Complete Information Set (CIS) and itsmeasurement implementation as evolution distance without reference tosizes. As method proof-test, the 16s rRNA sequences of 22 completelysequenced Bacteria and Archaea species are used to reconstruct aphylogenetic tree, which is generally consistent with the commonlyaccepted one. Based on whole genome, our further efforts yield a highlyrobust whole genome phylogenetic tree, supporting separate monophyleticcluster of species with similar phenotype as well as the early evolution ofthermophilic Bacteria and late diverging of Eukarya. The purpose of thiswork is not to contradict or confirm previous phylogeny standards butrather to bring a brand-new algorithm and tool to the phylogeny researchcommunity. The software to estimate the sequence distance and materialsused in this study are available upon request to corresponding author.     

The Evolutionary History of the Structure of 5S Ribosomal RNA

5S rRNA is the smallest nucleic acid component of the large ribosomal subunit, contributing to ribosomal assembly, stability, and function. Despite being a model for the study of RNA structure and RNA–protein interactions, the evolution of this universally conserved molecule remains unclear. Here, we explore the history of the three-domain structure of 5S rRNA using phylogenetic trees that are reconstructed directly from molecular structure. A total of 46 structural characters describing the geometry of 666 5S rRNAs were used to derive intrinsically rooted trees of molecules and molecular substructures. Trees of molecules revealed the tripartite nature of life. In these trees, superkingdom Archaea formed a paraphyletic basal group, while Bacteria and Eukarya were monophyletic and derived. Trees of molecular substructures supported an origin of the molecule in a segment that is homologous to helix I (α domain), its initial enhancement with helix III (β domain), and the early formation of the three-domain structure typical of modern 5S rRNA in Archaea. The delayed formation of the branched structure in Bacteria and Eukarya lends further support to the archaeal rooting of the tree of life. Remarkably, the evolution of molecular interactions between 5S rRNA and associated ribosomal proteins inferred from a census of domain structure in hundreds of genomes established a tight relationship between the age of 5S rRNA helices and the age of ribosomal proteins. Results suggest 5S rRNA originated relatively quickly but quite late in evolution, at a time when primordial metabolic enzymes and translation machinery were already in place. The molecule therefore represents a late evolutionary addition to the ribosomal ensemble that occurred prior to the early diversification of Archaea.     

Determining the Relative Rates of Change for Prokaryotic and Eukaryotic Proteins with Anciently Duplicated Paralogs

The relative rates of change for eight sets of ubiquitous proteins were determined by a test in which anciently duplicated paralogs are used to root the universal tree and distances are calculated between each taxonomic group and the last common ancestor. The sets included ATPase subunits, elongation factors, signal recognition particle and its receptor, three sets of tRNA synthetases, transcarbamoylases, and an internal duplication in carbamoyl phosphate synthase. In each case phylogenetic trees were constructed and the distances determined for all pairs. Taken over the period of time since their last common ancestor, average evolutionary rates are remarkably similar for Bacteria and Eukarya, but Archaea exhibit a significantly slower average rate. Received: 30 December 1999 / Accepted: 5 April 2000     

Genomics and early cellular evolution. The origin of the DNA world.

The sequencing of several genomes from each of the three domains of life (Archaea, Bacteria and Eukarya) has provided a huge amount of data that can be used to gain insight about early cellular evolution. Some features of the universal tree of life based on rRNA polygenies have been confirmed, such as the division of the cellular living world into three domains. The monophyly of each domain is supported by comparative genomics. However, the hyperthermophilic nature of the 'last universal common ancestor' (LUCA) is not confirmed. Comparative genomics has revealed that gene transfers have been (and still are) very frequent in genome evolution. Nevertheless, a core of informational genes appears more resistant to transfer, testifying for a close relationship between archaeal and eukaryal informational processes. This observation can be explained either by a common unique history between Archaea and Eukarya or by an atypical evolution of these systems in Bacteria. At the moment, comparative genomics still does not allow to choose between a simple LUCA, possibly with an RNA genome, or a complex LUCA, with a DNA genome and informational mechanisms similar to those of Archaea and Eukarya. Further comparative studies on informational mechanisms in the three domains should help to resolve this critical question. The role of viruses in the origin and evolution of DNA genomes also appears an area worth of active investigations. I suggest here that DNA and DNA replication mechanisms appeared first in the virus world before being transferred into cellular organisms.     

Phylogenetic and phyletic studies of informational genes in genomes highlight existence of a 4 domain of life including giant viruses

The discovery of Mimivirus, with its very large genome content, made it possible to identify genes common to the three domains of life (Eukarya, Bacteria and Archaea) and to generate controversial phylogenomic trees congruent with that of ribosomal genes, branching Mimivirus at its root. Here we used sequences from metagenomic databases, Marseillevirus and three new viruses extending the Mimiviridae family to generate the phylogenetic trees of eight proteins involved in different steps of DNA processing. Compared to the three ribosomal defined domains, we report a single common origin for Nucleocytoplasmic Large DNA Viruses (NCLDV), DNA processing genes rooted between Archaea and Eukarya, with a topology congruent with that of the ribosomal tree. As for translation, we found in our new viruses, together with Mimivirus, five proteins rooted deeply in the eukaryotic clade. In addition, comparison of informational genes repertoire based on phyletic pattern analysis supports existence of a clade containing NCLDVs clearly distinct from that of Eukarya, Bacteria and Archaea. We hypothesize that the core genome of NCLDV is as ancient as the three currently accepted domains of life.     

The differentially conserved residues of carbamoyl-phosphate synthetase

Carbamoyl-phosphate synthetase (CPS) from Escherichia coli is a heterodimeric protein. The larger of the two subunits (M(r) approximately 118,000) contains a pair of homologous domains of approximately 400 residues each that are approximately 40% identical in amino acid sequence. The carboxy phosphate (residues 1-400) and carbamoyl phosphate domains (residues 553-933) also contain approximately 79 differentially conserved residues. These are residues that are conserved throughout the bacterial evolution of CPS in one of these homologous domains but not the other. The role of these differentially conserved residues in the structural and catalytic properties of CPS was addressed by swapping segments of these residues from one domain to the other. Nine of these chimeric mutant enzymes were constructed, expressed, purified, and characterized. A majority of the mutants were unable to synthesize any carbamoyl phosphate and the rest were severely crippled. True tandem repeat chimeric proteins were constructed by the complete substitution of one homologous domain sequence for the other. Neither of the two possible chimeric proteins was structurally stable. These results have been interpreted to demonstrate that the two homologous domains in the large subunit of CPS are functionally and structurally nonequivalent. This nonequivalence is a direct result of the specific functions each of these domains must perform during the overall synthesis of carbamoyl phosphate in the wild type enzyme and the specific structural alterations imposed by the differentially conserved residues.     

Coenzyme A biosynthesis: reconstruction of the pathway in archaea and an evolutionary scenario based on comparative genomics

Coenzyme A (CoA) holds a central position in cellular metabolism and therefore can be assumed to be an ancient molecule. Starting from the known E. coli and human enzymes required for the biosynthesis of CoA, phylogenetic profiles and chromosomal proximity methods enabled an almost complete reconstruction of archaeal CoA biosynthesis. This includes the identification of strong candidates for archaeal pantothenate synthetase and pantothenate kinase, which are unrelated to the corresponding bacterial or eukaryotic enzymes. According to this reconstruction, the topology of CoA synthesis from common precursors is essentially conserved across the three domains of life. The CoA pathway is conserved to varying degrees in eukaryotic pathogens like Giardia lamblia or Plasmodium falciparum, indicating that these pathogens have individual uptake-mechanisms for different CoA precursors. Phylogenetic analysis and phyletic distribution of the CoA biosynthetic enzymes suggest that the enzymes required for the synthesis of phosphopantothenate were recruited independently in the bacterial and archaeal lineages by convergent evolution, and that eukaryotes inherited the genes for the synthesis of pantothenate (vitamin B5) from bacteria. Homologues to bacterial enzymes involved in pantothenate biosynthesis are present in a subset of archaeal genomes. The phylogenies of these enzymes indicate that they were acquired from bacterial thermophiles through horizontal gene transfer. Monophyly can be inferred for each of the enzymes catalyzing the four ultimate steps of CoA synthesis, the conversion of phosphopantothenate into CoA. The results support the notion that CoA was initially synthesized from a prebiotic precursor, most likely pantothenate or a related compound.     

Phylogenetic Analyses Reveal Ancient Duplication of Estrogen Receptor Isoforms

To determine the origin and evolutionary significance of a recently discovered isoform of the estrogen receptor (ERβ), we examined the phylogenetic relationship of ERβ to the well-known α isoform (ERα) and other steroid receptors. Our phylogenetic analyses traced the origin of ERβ to a single duplication event at least 450 million years ago. Since this duplication, the evolution of both ER isoforms has apparently been constrained such that 80% of the amino acid positions in the DNA binding domain (DBD) and 53% of the ligand binding domain (LBD) have remained unchanged. Using the phylogenetic tree, we determined the amount of evolutionary change that had occurred in two ER isoforms. The DBD and the LBD had lower rates of evolutionary change compared to the NH₂ terminal domain. However, even with strong selective constraints on the DBD and LBD, our phylogenetic analyses demonstrate two clearly separate phylogenetic histories for ERα and ERβ dating back several hundred million years. The ancient duplication of ER and the parallel evolution of the two ER isoforms suggest that, although ERα and ERβ share a substantial degree of sequence identity, they play unique roles in vertebrate physiology and reproduction. Received: 19 January 1999 / Accepted: 26 May 1999     

Origins and evolution of isoprenoid lipid biosynthesis in archaea

A characteristic feature of the domain archaea are the lipids forming the hydrophobic core of their cell membrane. These unique lipids are composed of isoprenoid side-chains stereospecifically ether linked to sn-glycerol-1-phosphate. Recently, considerable progress has been made in characterizing the enzymes responsible for the synthesis of archaeal lipids. However, little is known about their evolution. To better understand how this unique biosynthetic apparatus came to be, large-scale database surveys and phylogenetic analyses were performed. All characterized enzymes involved in the biosynthesis of isoprenoid side-chains and the glycerol phosphate backbone along with their assembly in ether lipids were included in these analyses. The sequence data available in public databases was complemented by an in-depth sampling of isoprenoid lipid biosynthesis genes from multiple genera of the archaeal order Halobacteriales, allowing us to look at the evolution of these enzymes on a smaller phylogenetic scale. This investigation of the isoprenoid biosynthesis apparatus of archaea on small and large phylogenetic scales reveals that it evolved through a combination of evolutionary processes, including the co-option of ancestral enzymes, modification of enzymatic specificity, orthologous and non-orthologous gene displacement, integration of components from eukaryotes and bacteria and lateral gene transfer within and between archaeal orders.     

The Evolution of Histidine Biosynthesis in Archaea: Insights into the <Emphasis Type="Italic">his</Emphasis> Genes Structure and Organization in LUCA

The available sequences of genes encoding the enzymes associated with histidine biosynthesis suggest that this is an ancient metabolic pathway that was assembled prior to the diversification of Bacteria, Archaea, and Eucarya. Paralogous duplication, gene elongation, and fusion events of several different his genes have played a major role in shaping this biosynthetic route. We have analyzed the structure and organization of histidine biosynthetic genes from 55 complete archaeal genomes and combined it with phylogenetic inference in order to investigate the mechanisms responsible for the assembly of the his pathway and the origin of his operons. We show that a wide variety of different organizations of his genes exists in Archaea and that some his genes or entire his (sub-)operons have been likely transferred horizontally between Archaea and Bacteria. However, we show that, in most Archaea, his genes are monofunctional (except for hisD) and scattered throughout the genome, suggesting that his operons might have been assembled multiple times during evolution and that in some cases they are the result of recent evolutionary events. An evolutionary model for the structure and organization of his genes in LUCA is proposed.     

A study of archaeal enzymes involved in polar lipid synthesis linking amino acid sequence information, genomic contexts and lipid composition

Cellular membrane lipids, of which phospholipids are the major constituents, form one of the characteristic features that distinguish Archaea from other organisms. In this study, we focused on the steps in archaeal phospholipid synthetic pathways that generate polar lipids such as archaetidylserine, archaetidylglycerol, and archaetidylinositol. Only archaetidylserine synthase (ASS), from Methanothermobacter thermautotrophicus, has been experimentally identified. Other enzymes have not been fully examined. Through database searching, we detected many archaeal hypothetical proteins that show sequence similarity to members of the CDP alcohol phosphatidyltransferase family, such as phosphatidylserine synthase (PSS), phosphatidylglycerol synthase (PGS) and phosphatidylinositol synthase (PIS) derived from Bacteria and Eukarya. The archaeal hypothetical proteins were classified into two groups, based on the sequence similarity. Members of the first group, including ASS from M. thermautotrophicus, were closely related to PSS. The rough agreement between PSS homologue distribution within Archaea and the experimentally identified distribution of archaetidylserine suggested that the hypothetical proteins are ASSs. We found that an open reading frame (ORF) tends to be adjacent to that of ASS in the genome, and that the order of the two ORFs is conserved. The sequence similarity of phosphatidylserine decarboxylase to the product of the ORF next to the ASS gene, together with the genomic context conservation, suggests that the ORF encodes archaetidylserine decarboxylase, which may transform archaetidylserine to archaetidylethanolamine. The second group of archaeal hypothetical proteins was related to PGS and PIS. The members of this group were subjected to molecular phylogenetic analysis, together with PGSs and PISs and it was found that they formed two distinct clusters in the molecular phylogenetic tree. The distribution of members of each cluster within Archaea roughly corresponded to the experimentally identified distribution of archaetidylglycerol or archaetidylinositol. The molecular phylogenetic tree patterns and the correspondence to the membrane compositions suggest that the two clusters in this group correspond to archaetidylglycerol synthases and archaetidylinositol synthases. No archaeal hypothetical protein with sequence similarity to known phosphatidylcholine synthases was detected in this study.