Fungal catalases: Function, phylogenetic origin and structure

Most fungi have several monofunctional heme-catalases. Filamentous ascomycetes (Pezizomycotina) have two types of large-size subunit catalases (L1 and L2). L2-type are usually induced by different stressors and are extracellular enzymes; those from the L1-type are not inducible and accumulate in asexual spores. L2 catalases are important for growth and the start of cell differentiation, while L1 are required for spore germination. In addition, pezizomycetes have one to four small-size subunit catalases. Yeasts (Saccharomycotina) do not have large-subunit catalases and generally have one peroxisomal and one cytosolic small-subunit catalase. Small-subunit catalases are inhibited by substrate while large-subunit catalases are activated by H(2)O(2). Some small-subunit catalases bind NADPH preventing inhibition by substrate. We present a phylogenetic analysis revealing one or two events of horizontal gene transfers from Actinobacteria to a fungal ancestor before fungal diversification, as the origin of large-size subunit catalases. Other possible horizontal transfers of small- and large-subunit catalases genes were detected and one from bacteria to the fungus Malassezia globosa was analyzed in detail. All L2-type catalases analyzed presented a secretion signal peptide. Mucorales preserved only L2-type catalases, with one containing a secretion signal if two or more are present. Basidiomycetes have only L1-type catalases, all lacking signal peptide. Fungal small-size catalases are related to animal catalases and probably evolved from a common ancestor. However, there are several groups of small-size catalases. In particular, a conserved group of fungal sequences resemble plant catalases, whose phylogenetic origin was traced to a group of bacteria. This group probably has the heme orientation of plant catalases and could in principle bind NADPH. From almost a hundred small-subunit catalases only one fourth has a peroxisomal localization signal and in fact many fungi lack a peroxisomal catalase. Catalases have a deep buried active site and H(2)O(2) has to go through a long passage to reach it. In all known structures of catalases, the major channel has common features, particularly in the straight and narrow final section that is positioned perpendicular to the heme. Besides, other conserved channels are present in catalases whose function remains to be elucidated. One of these channels intercommunicates the major channels from the two R-related subunits. In three of the four known large-subunits catalase structures, the heme b is partially transformed into heme d. In Neurospora crassa, this occurs in vivo and is related to oxidative stress conditions in which singlet oxygen is produced. A pure source of singlet oxygen oxidizes catalases purified from different sources and singlet oxygen quenchers prevent oxidation. A second modification is observed in N. crassa catalase-1, in which the tyrosine that forms the fifth coordination bound to the heme iron makes a covalent bond with a vicinal cysteine, similarly to the tyrosine-histidine bonding found in Escherichia coli hydroperoxidase II. Molecular dynamics has been used to determine how H(2)O(2) reaches the enzyme active site and how products exit the protein. We found that the bottleneck of the major channel seems to disappear in water and is wide open in the presence of substrate. Amino acid residues exhibiting an increased residence time for H(2)O(2) are abundant at the protein surface and at the entrances to the major channel. The net effect of this is an increased H(2)O(2)/H(2)O ratio in the major channel. Once in the final section of this channel, H(2)O(2) is retained and tends to occupy specific sites while water molecules have a higher turnover rate and occupy different sites. Despite the intense study of catalases our knowledge of this enzyme is still limited and in need of new studies and different approaches.     

Unique presence of a manganese catalase in a hyperthermophilic archaeon,Pyrobaculum calidifontis VA1

We had previously isolated a facultatively anaerobic hyperthermophilic archaeon, Pyrobaculum calidifontis strain VA1. Here, we found that strain VA1, when grown under aerobic conditions, harbors high catalase activity. The catalase was purified 91-fold from crude extracts and displayed a specific activity of 23,500 U/mg at 70 degrees C. The enzyme exhibited a K(m) value of 170 mM toward H(2)O(2) and a k(cat) value of 2.9 x 10(4) s(-1).subunit(-1) at 25 degrees C. Gel filtration chromatography indicated that the enzyme was a homotetramer with a subunit molecular mass of 33,450 Da. The purified catalase did not display the Soret band, which is an absorption band particular to heme enzymes. In contrast to typical heme catalases, the catalase was not strongly inhibited by sodium azide. Furthermore, with plasma emission spectroscopy, we found that the catalase did not contain iron but instead contained manganese. Our biochemical results indicated that the purified catalase was not a heme catalase but a manganese (nonheme) catalase, the first example in archaea. Intracellular catalase activity decreased when cells were grown anaerobically, while under aerobic conditions, an increase in activity was observed with the removal of thiosulfate from the medium, or addition of manganese. Based on the N-terminal amino acid sequence of the purified protein, we cloned and sequenced the catalase gene (kat(Pc)). The deduced amino acid sequence showed similarity with that of the manganese catalase from a thermophilic bacterium, Thermus sp. YS 8-13. Interestingly, in the complete archaeal genome sequences, no open reading frame has been assigned as a manganese catalase gene. Moreover, a homology search with the sequence of kat(Pc) revealed that no orthologue genes were present on the archaeal genomes, including those from the "aerobic" (hyper)thermophilic archaea Aeropyrum pernix, Sulfolobus solfataricus, and Sulfolobus tokodaii. Therefore, Kat(Pc) can be considered a rare example of a manganese catalase from archaea.     

Comparative interactomics analysis of protein family interaction networks using PSIMAP (protein structural interactome map)

MOTIVATION: Many genomes have been completely sequenced. However, detecting and analyzing their protein-protein interactions by experimental methods such as co-immunoprecipitation, tandem affinity purification and Y2H is not as fast as genome sequencing. Therefore, a computational prediction method based on the known protein structural interactions will be useful to analyze large-scale protein-protein interaction rules within and among complete genomes. RESULTS: We confirmed that all the predicted protein family interactomes (the full set of protein family interactions within a proteome) of 146 species are scale-free networks, and they share a small core network comprising 36 protein families related to indispensable cellular functions. We found two fundamental differences among prokaryotic and eukaryotic interactomes: (1) eukarya had significantly more hub families than archaea and bacteria and (2) certain special hub families determined the topology of the eukaryotic interactomes. Our comparative analysis suggests that a very small number of expansive protein families led to the evolution of interactomes and seemed to have played a key role in species diversification. SUPPLEMENTARY INFORMATION: http://interactomics.org.     

Evolution of ATP synthase subunit c and cytochrome c gene families in selected Metazoan classes

To investigate the integrated evolution of mitochondrial and nuclear genomes in the eukaryotic cell, we have focused our attention on OXPHOS (oxidative phosphorylation) gene families which encode proteins involved in the main mitochondrial function. The present study reports the phylogenetic analysis of two OXPHOS gene families: ATP synthase subunit c (or lipid binding protein, LBP) and Cytochrome c (Cytc). Both gene families possess a higher expansion trend than the typically low duplication rate of OXPHOS genes in Metazoa, but follow a completely different evolutionary history, especially in mammals. LBP is represented by three well conserved isoforms in all mammals (P1, P2, P3): only P3 possesses a clearly conserved isoform in all Vertebrates, P1 and P2 were already present before the bird-mammal divergence and there are preliminary evidence from the in silico analysis that P1, the most evolutionary divergent isoform, is poorly expressed and not regulated by NRF1. In contrast, Cytc family presents at least two duplicated genes in all the analysed Vertebrates, is subject to a high expansion trend, especially of processed pseudogenes in mammals, and some events of gain and loss of function can be supposed.     

Identification of two highly divergent catalase genes in the fungal tomato pathogen, Cladosporium fulvum.

Catalases of pathogenic micro-organisms have attracted attention as potential virulence factors. Homology-based screens were performed to identify catalase genes in the fungal tomato pathogen Cladosporium fulvum. Two highly divergent genes, Cat1 and Cat2, were isolated and characterized. Cat1 codes for a putative 566-amino-acid catalase subunit and belongs to the gene family that also encodes the mainly peroxisome-localized catalases of animal and yeast species. Cat2 codes for a putative catalase subunit of 745 amino acids and belongs to a different gene family coding for the large-subunit catalases similar to ones found in bacteria and filamentous fungi. Neither catalase had an obvious secretory signal sequence. A search for an extracellular catalase was unproductive. The Cat1 and Cat2 genes showed differential expression, with the Cat1 mRNA preferentially accumulating in spores and the Cat2 mRNA preferentially accumulating in response to external H(2)O(2). With Cat2-deleted strains, activity of the Cat2 gene product (CAT2) was identified among four proteins with catalase activity separated on non-denaturing gels. The CAT2 activity represented a minor fraction of the catalase activity in spores and H(2)O(2)-stressed mycelium, and no phenotype was observed for Cat2-deleted strains, which showed a normal response to H(2)O(2) treatment. These results indicate the existence of a complex catalase system in C. fulvum, with regard to both the structure and regulation of the genes involved. In addition, efficient C. fulvum gene-replacement technology has been established.     

The RND permease superfamily: an ancient, ubiquitous and diverse family that includes human disease and development proteins

A previous report identified and classified a small family of gram-negative bacterial drug and heavy metal efflux permeases, now commonly referred to as the RND family (TC no. 2.6). We here show that this family is actually a ubiquitous superfamily with representation in all major kingdoms. We report phylogenetic analyses that define seven families within the RND superfamily as follows: (1) the heavy metal efflux (HME) family (gram negative bacteria), (2) the hydrophobe/amphiphile efflux-1 (HAE1) family (gram negative bacteria), (3) the nodulation factor exporter (NFE) family (gram negative bacteria), (4) the SecDF protein-secretion accessory protein (SecDF) family (gram negative and gram positive bacteria as well as archaea), (5) the hydrophobe/amphiphile efflux-2 (HAE2) family (gram positive bacteria), (6) the eukaryotic sterol homeostasis (ESH) family, and (7) the hydrophobe/amphiphile efflux-3 (HAE3) family (archaea and spirochetes). Functionally uncharacterized proteins were identified that are members of the RND superfamily but fall outside of these seven families. Some of the eukaryotic homologues function as enzymes and receptors instead of (or in addition to) transporters. The sizes and topological patterns exhibited by members of all seven families are shown to be strikingly similar, and statistical analyses establish common descent. Multiple alignments of proteins within each family allow derivation of family-specific signature sequences. Structural, functional, mechanistic and evolutionary implication of the reported results are discussed.     

Patterns and processes in the evolution of the eukaryotic endomembrane system

Abstract

The eukaryotic endomembrane system (ES) is served by hundreds of dedicated proteins. Experimental characterization of the ES-associated molecular machinery in several model eukaryotes complemented by a recent progress in phylogenomics and comparative genomics have revealed a conserved complex core of the machinery that appears to have been established before the last eukaryotic common ancestor (LECA). At the same time, modern eukaryotes exhibit a huge variation in the ES resulting from a multitude of evolutionary processes operating along the ever-branching paths from the LECA to its descendants. The most important source of evolutionary novelty in the ES functioning has undoubtedly been gene duplication followed by divergence of the gene copies, responsible not only for the pre-LECA establishment of many multi-paralog families of proteins in the very core of the ES-associated machinery, but also for post-LECA lineage-specific elaborations via family expansions and the origin of novel components. Extreme sequence divergence has obscured actual homologous relationships between potentially many components of the machinery, even between orthologous proteins, as illustrated by the yeast Vps51 subunit of the vesicle tethering complex GARP hypothesized here to be a highly modified ortholog of a conserved eukaryotic family typified by the zebrafish Fat-free (Ffr) protein. A dynamic evolution of many ES-associated proteins, especially those centred around RAB and ARF GTPases, seems to take place at the level of their domain architectures. Finally, reductive evolution and recurrent gene loss are emerging as pervasive factors shaping the ES in all phylogenetic lineages.     

Patterns and processes in the evolution of the eukaryotic endomembrane system

The eukaryotic endomembrane system (ES) is served by hundreds of dedicated proteins. Experimental characterization of the ES-associated molecular machinery in several model eukaryotes complemented by a recent progress in phylogenomics and comparative genomics have revealed a conserved complex core of the machinery that appears to have been established before the last eukaryotic common ancestor (LECA). At the same time, modern eukaryotes exhibit a huge variation in the ES resulting from a multitude of evolutionary processes operating along the ever-branching paths from the LECA to its descendants. The most important source of evolutionary novelty in the ES functioning has undoubtedly been gene duplication followed by divergence of the gene copies, responsible not only for the pre-LECA establishment of many multi-paralog families of proteins in the very core of the ES-associated machinery, but also for post-LECA lineage-specific elaborations via family expansions and the origin of novel components. Extreme sequence divergence has obscured actual homologous relationships between potentially many components of the machinery, even between orthologous proteins, as illustrated by the yeast Vps51 subunit of the vesicle tethering complex GARP hypothesized here to be a highly modified ortholog of a conserved eukaryotic family typified by the zebrafish Fat-free (Ffr) protein. A dynamic evolution of many ES-associated proteins, especially those centred around RAB and ARF GTPases, seems to take place at the level of their domain architectures. Finally, reductive evolution and recurrent gene loss are emerging as pervasive factors shaping the ES in all phylogenetic lineages.     

Genomic characterization of the human heterotrimeric G protein alpha, beta, and gamma subunit genes.

Heterotrimeric guanine nucleotide binding proteins (G proteins) transduce extracellular signals received by transmembrane receptors to effector proteins. Each subunit of the G protein complex is encoded by a member of one of three corresponding gene families. Currently, 16 different members of the alpha subunit family, 5 different members of the beta subunit family, and 11 different members of the gamma subunit family have been described in mammals. Here we have identified and characterized Bacterial Artificial Chromosomes (BACs) containing the human homologs of each of the alpha, beta, and gamma subunit genes as well as a G alpha11 pseudogene and a previously undiscovered G gamma5-like gene. The gene structure and chromosome location of each gene was determined, as were the orientations of paired genes. These results provide greater insight into the evolution and functional diversity of the mammalian G protein subunit genes.     

Slow Co-Evolution of the MAGO and Y14 Protein Families Is Required for the Maintenance of Their Obligate Heterodimerization Mode

The exon junction complex (EJC) plays important roles in RNA metabolisms and the development of eukaryotic organisms. MAGO (short form of MAGO NASHI) and Y14 (also Tsunagi or RBM8) are the EJC core components. Their biological roles have been well investigated in various species, but the evolutionary patterns of the two gene families and their protein-protein interactions are poorly known. Genome-wide survey suggested that the MAGO and Y14 two gene families originated in eukaryotic organisms with the maintenance of a low copy. We found that the two protein families evolved slowly; however, the MAGO family under stringent purifying selection evolved more slowly than the Y14 family that was under relative relaxed purifying selection. MAGO and Y14 were obliged to form heterodimer in a eukaryotic organism, and this obligate mode was plesiomorphic. Lack of binding of MAGO to Y14 as functional barrier was observed only among distantly species, suggesting that a slow co-evolution of the two protein families. Inter-protein co-evolutionary signal was further quantified in analyses of the Tol-MirroTree and co-evolution analysis using protein sequences. About 20% of the 41 significantly correlated mutation groups (involving 97 residues) predicted between the two families was clade-specific. Moreover, around half of the predicted co-evolved groups and nearly all clade-specific residues fell into the minimal interaction domains of the two protein families. The mutagenesis effects of the clade-specific residues strengthened that the co-evolution is required for obligate MAGO-Y14 heterodimerization mode. In turn, the obliged heterodimerization in an organism serves as a strong functional constraint for the co-evolution of the MAGO and Y14 families. Such a co-evolution allows maintaining the interaction between the proteins through large evolutionary time scales. Our work shed a light on functional evolution of the EJC genes in eukaryotes, and facilitates to understand the co-evolutionary processes among protein families.     

Purification and physical-chemical characterization of the three hydroperoxidases from the symbiotic bacterium Sinorhizobium meliloti

Three genes encoding heme hydroperoxidases (katA, katB, and katC) have been identified in the soil bacterium Sinorhizobium meliloti. The recombinant proteins were overexpressed in Escherichia coli and purified in order to achieve a spectral and kinetic characterization. The three proteins contain heme b with high-spin Fe(III). KatB is an acidic bifunctional homodimeric catalase-peroxidase exhibiting both catalase (k(cat) = 2400 s(-1)) and peroxidase activity and having a high affinity for hydrogen peroxide (apparent K(M) = 1.6 mM). KatA and KatC are acidic monofunctional homotetrameric catalases. Although different in size (KatA is a small subunit catalase while KatC is a large subunit catalase) both enzymes exhibit the same heme type and a similar affinity for H(2)O(2) (apparent K(M) values of 160 and 150 mM). However, the turnover rate of KatA (k(cat) = 279000 s(-1)) exceeds that of KatC (k(cat) = 3100 s(-1)) significantly. The kinetic parameters are in good agreement with the physiological role of these heme proteins. KatB is the housekeeping hydroperoxidase exhibiting the highest affinity for hydrogen peroxide, while KatA has the lowest H(2)O(2) affinity but the highest k(cat)/K(M) value (1.75 x 10(6) M(-1) s(-1)), in agreement with the hydrogen peroxide inducibility of the encoding gene. Moreover, the lower catalytic efficiency of KatC (2.1 x 10(4) M(-1) s(-1)) appears to be enough for growing in the stationary phase and/or under heat or salt stress (conditions that are known to favor katC expression).     

Lipopolysaccharide phosphorylating enzymes encoded in the genomes of Gram-negative bacteria are related to the eukaryotic protein kinases

By means of profile-matching procedures, conservation of functionally important residues, and fold-recognition techniques, we show that two distinct families of lipopolysaccharide kinases encoded in the genomes of Gram-negative bacteria are related to each other and to two distinct classes of proteins, namely eukaryotic protein kinases and right open reading frame (RIO1). Members of one of the lipopolysaccharide kinase families are identified only in pathogenic bacteria. Phosphorylation by these enzymes is relevant in the construction of outer membrane, immune response, and pathogenic virulence. The class of proteins called RIO1, also related to eukaryotic protein kinases and previously known to occur only in archaea and eukaryotes, are now identified in eubacteria as well. It has been suggested here that RIO1 proteins are intermediately related to lipopolysaccharide kinases and eukaryotic protein kinases implying an evolutionary relationship between the three classes of proteins.     

Protein length in eukaryotic and prokaryotic proteomes

We analyzed length differences of eukaryotic, bacterial and archaeal proteins in relation to function, conservation and environmental factors. Comparing Eukaryotes and Prokaryotes, we found that the greater length of eukaryotic proteins is pervasive over all functional categories and involves the vast majority of protein families. The magnitude of these differences suggests that the evolution of eukaryotic proteins was influenced by processes of fusion of single-function proteins into extended multi-functional and multi-domain proteins. Comparing Bacteria and Archaea, we determined that the small but significant length difference observed between their proteins results from a combination of three factors: (i) bacterial proteomes include a greater proportion than archaeal proteomes of longer proteins involved in metabolism or cellular processes, (ii) within most functional classes, protein families unique to Bacteria are generally longer than protein families unique to Archaea and (iii) within the same protein family, homologs from Bacteria tend to be longer than the corresponding homologs from Archaea. These differences are interpreted with respect to evolutionary trends and prevailing environmental conditions within the two prokaryotic groups.     

Ancestral paralogs and pseudoparalogs and their role in the emergence of the eukaryotic cell

Gene duplication is a crucial mechanism of evolutionary innovation. A substantial fraction of eukaryotic genomes consists of paralogous gene families. We assess the extent of ancestral paralogy, which dates back to the last common ancestor of all eukaryotes, and examine the origins of the ancestral paralogs and their potential roles in the emergence of the eukaryotic cell complexity. A parsimonious reconstruction of ancestral gene repertoires shows that 4137 orthologous gene sets in the last eukaryotic common ancestor (LECA) map back to 2150 orthologous sets in the hypothetical first eukaryotic common ancestor (FECA) [paralogy quotient (PQ) of 1.92]. Analogous reconstructions show significantly lower levels of paralogy in prokaryotes, 1.19 for archaea and 1.25 for bacteria. The only functional class of eukaryotic proteins with a significant excess of paralogous clusters over the mean includes molecular chaperones and proteins with related functions. Almost all genes in this category underwent multiple duplications during early eukaryotic evolution. In structural terms, the most prominent sets of paralogs are superstructure-forming proteins with repetitive domains, such as WD-40 and TPR. In addition to the true ancestral paralogs which evolved via duplication at the onset of eukaryotic evolution, numerous pseudoparalogs were detected, i.e. homologous genes that apparently were acquired by early eukaryotes via different routes, including horizontal gene transfer (HGT) from diverse bacteria. The results of this study demonstrate a major increase in the level of gene paralogy as a hallmark of the early evolution of eukaryotes.     

Origin and evolution of eukaryotic chaperonins: phylogenetic evidence for ancient duplications in CCT genes

Chaperonins are oligomeric protein-folding complexes which are divided into two distantly related structural classes. Group I chaperonins (called GroEL/cpn60/hsp60) are found in bacteria and eukaryotic organelles, while group II chaperonins are present in archaea and the cytoplasm of eukaryotes (called CCT/TriC). While archaea possess one to three chaperonin subunit-encoding genes, eight distinct CCT gene families (paralogs) have been characterized in eukaryotes. We are interested in determining when during eukaryotic evolution the multiple gene duplications producing the CCT subunits occurred. We describe the sequence and phylogenetic analysis of five CCT genes from TRICHOMONAS: vaginalis and seven from GIARDIA: lamblia, representatives of amitochondriate protist lineages thought to have diverged early from other eukaryotes. Our data show that the gene duplications producing the eight CCT paralogs took place prior to the organismal divergence of TRICHOMONAS: and GIARDIA: from other eukaryotes. Thus, these divergent protists likely possess completely hetero-oligomeric CCT complexes like those in yeast and mammalian cells. No close phylogenetic relationship between the archaeal chaperonins and specific CCT subunits was observed, suggesting that none of the CCT gene duplications predate the divergence of archaea and eukaryotes. The duplications producing the CCTdelta and CCTepsilon subunits, as well as CCTalpha, CCTbeta, and CCTeta, are the most recent in the CCT gene family. Our analyses show significant differences in the rates of evolution of archaeal chaperonins compared with the eukaryotic CCTs, as well as among the different CCT subunits themselves. We discuss these results in light of current views on the origin, evolution, and function of CCT complexes.     

The general protein secretory pathway: phylogenetic analyses leading to evolutionary conclusions

We have identified all homologues in the current databases of the ubiquitous protein constituents of the general secretory (Sec) pathway. These prokaryotic/eukaryotic proteins include (1) SecY/Sec61alpha, (2) SecE/Sec61gamma, (3) SecG/Sec61beta, (4) Ffh/SRP54 and (5) FtsY/SRP receptor subunit-alpha. Phylogenetic and sequence analyses lead to major conclusions concerning (1) the ubiquity of these proteins in living organisms, (2) the topological uniformity of some but not other Sec constituents, (3) the orthologous nature of almost all of them, (4) a total lack of paralogues in almost all organisms for which complete genome sequences are available, (5) the occurrence of two or even three paralogues in a few bacteria, plants, and yeast, depending on the Sec constituent, and (6) a tremendous degree of sequence divergence in bacteria compared with that in archaea or eukaryotes. The phylogenetic analyses lead to the conclusion that with a few possible exceptions, the five families of Sec constituents analyzed generally underwent sequence divergence in parallel but at different characteristic rates. The results provide evolutionary insights as well as guides for future functional studies. Because every organism with a fully sequenced genome exhibits at least one orthologue of each of these Sec proteins, we conclude that all living organisms have relied on the Sec system as their primary protein secretory/membrane insertion system. Because most prokaryotes and many eukaryotes encode within their genomes only one of each constituent, we also conclude that strong evolutionary pressure has minimized gene duplication events leading to the establishment of Sec paralogues. Finally, the sequence diversity of bacterial proteins as compared with their archaeal and eukaryotic counterparts is in agreement with the suggestion that bacteria were the evolutionary predecessors of archaea and eukaryotes.     

An evolutionary analysis of the helix-hairpin-helix superfamily of DNA repair glycosylases

The helix-hairpin-helix (HhH) superfamily of base excision repair DNA glycosylases is composed of multiple phylogenetically diverse enzymes that are capable of excising varying spectra of oxidatively and methyl-damaged bases. Although these DNA repair glycosylases have been widely studied through genetic, biochemical, and biophysical approaches, the evolutionary relationships of different HhH homologs and the extent to which they are conserved across phylogeny remain enigmatic. We provide an evolutionary framework for this pervasive and versatile superfamily of DNA glycosylases. Six HhH gene families (named AlkA: alkyladenine glycosylase; MpgII: N-methylpurine glycosylase II; MutY/Mig: A/G-specific adenine glycosylase/mismatch glycosylase; Nth: endonuclease III; OggI: 8-oxoguanine glycosylase I; and OggII: 8-oxoguanine glycosylase II) are identified through phylogenetic analysis of 234 homologs found in 94 genomes (16 archaea, 64 bacteria, and 14 eukaryotes). The number of homologs in each gene family varies from 117 in the Nth family (nearly every genome surveyed harbors at least one Nth homolog) to only five in the divergent OggII family (all from archaeal genomes). Sequences from all three domains of life are included in four of the six gene families, suggesting that the HhH superfamily diversified very early in evolution. The phylogeny provides evidence for multiple lineage-specific gene duplication events, most of which involve eukaryotic homologs in the Nth and AlkA gene families. We observe extensive variation in the number of HhH superfamily glycosylase genes present in different genomes, possibly reflecting major differences among species in the mechanisms and pathways by which damaged bases are repaired and/or disparities in the basic rates and spectra of mutation experienced by different genomes.     

Testing the neutral fixation of hetero-oligomerism in the archaeal chaperonin CCT

The evolutionary transition from homo-oligomerism to hetero-oligomerism in multimeric proteins and its contribution to function innovation and organism complexity remain to be investigated. Here, we undertake the challenge of contributing to this theoretical ground by investigating the hetero-oligomerism in the molecular chaperonin cytosolic chaperonin containing tailless complex polypeptide 1 (CCT) from archaea. CCT is amenable to this study because, in contrast to eukaryotic CCTs where sub-functionalization after gene duplication has been taken to completion, archaeal CCTs present no evidence for subunit functional specialization. Our analyses yield additional information to previous reports on archaeal CCT paralogy by identifying new duplication events. Analyses of selective constraints show that amino acid sites from 1 subunit have fixed slightly deleterious mutations at inter-subunit interfaces after gene duplication. These mutations have been followed by compensatory mutations in nearby regions of the same subunit and in the interface contact regions of its paralogous subunit. The strong selective constraints in these regions after speciation support the evolutionary entrapment of CCTs as hetero-oligomers. In addition, our results unveil different evolutionary dynamics depending on the degree of CCT hetero-oligomerism. Archaeal CCT protein complexes comprising 3 distinct classes of subunits present 2 evolutionary processes. First, slightly deleterious and compensatory mutations were fixed neutrally at inter-subunit regions. Second, sub-functionalization may have occurred at substrate-binding and adenosine triphosphate-binding regions after the 2nd gene duplication event took place. CCTs with 2 distinct types of subunits did not present evidence of sub-functionalization. Our results provide the 1st in silico evidence for the neutral fixation of hetero-oligomerism in archaeal CCTs and provide information on the evolution of hetero-oligomerism toward sub-functionalization in archaeal CCTs.     

Phylogenetic relationships in class I of the superfamily of bacterial, fungal, and plant peroxidases.

Molecular phylogeny among catalase-peroxidases, cytochrome c peroxidases, and ascorbate peroxidases was analysed. Sixty representative sequences covering all known subgroups of class I of the superfamily of bacterial, fungal, and plant heme peroxidases were selected. Each sequence analysed contained the typical peroxidase motifs evolved to bind effectively the prosthetic heme group, enabling peroxidatic activity. The N-terminal and C-terminal domains of catalase-peroxidases matching the ancestral tandem gene duplication event were treated separately in the phylogenetic analysis to reveal their specific evolutionary history. The inferred unrooted phylogenetic tree obtained by three different methods revealed the existence of four clearly separated clades (C-terminal and N-terminal domains of catalase-peroxidases, ascorbate peroxidases, and cytochrome c peroxidases) which were segregated early in the evolution of this superfamily. From the results, it is obvious that the duplication event in the gene for catalase-peroxidase occurred in the later phase of evolution, in which the individual specificities of the peroxidase families distinguished were already formed. Evidence is presented that class I of the heme peroxidase superfamily is spread among prokaryotes and eukaryotes, obeying the birth-and-death process of multigene family evolution.     

Solution structure of Archaeglobus fulgidis peptidyl-tRNA hydrolase (Pth2) provides evidence for an extensive conserved family of Pth2 enzymes in archea, bacteria, and eukaryotes

The solution structure of protein AF2095 from the thermophilic archaea Archaeglobus fulgidis, a 123-residue (13.6-kDa) protein, has been determined by NMR methods. The structure of AF2095 is comprised of four alpha-helices and a mixed beta-sheet consisting of four parallel and anti-parallel beta-strands, where the alpha-helices sandwich the beta-sheet. Sequence and structural comparison of AF2095 with proteins from Homo sapiens, Methanocaldococcus jannaschii, and Sulfolobus solfataricus reveals that AF2095 is a peptidyl-tRNA hydrolase (Pth2). This structural comparison also identifies putative catalytic residues and a tRNA interaction region for AF2095. The structure of AF2095 is also similar to the structure of protein TA0108 from archaea Thermoplasma acidophilum, which is deposited in the Protein Data Bank but not functionally annotated. The NMR structure of AF2095 has been further leveraged to obtain good-quality structural models for 55 other proteins. Although earlier studies have proposed that the Pth2 protein family is restricted to archeal and eukaryotic organisms, the similarity of the AF2095 structure to human Pth2, the conservation of key active-site residues, and the good quality of the resulting homology models demonstrate a large family of homologous Pth2 proteins that are conserved in eukaryotic, archaeal, and bacterial organisms, providing novel insights in the evolution of the Pth and Pth2 enzyme families.