The use of an in vitro approach to assess marine invertebrate carboxylesterase responses to chemicals of environmental concern.

Carboxylesterases (CEs) are key enzymes which catalyse the hydrolysis reactions of multiple xenobiotics and endogenous ester moieties. Given their growing interest in the context of marine pollution and biomonitoring, this study focused on the in vitro sensitivity of marine invertebrate CEs to some pesticides, pharmaceuticals, personal care products and plastic additives to assess their potential interaction on this enzymatic system and its suitability as biomarkers. Three bivalves, one gastropod and two crustaceans were used and CEs were quantified following current protocols set for mammalian models. Four substrates were screened for CEs determination and to test their adequacy in the hepatic fraction measures of the selected invertebrates. Two commercial recombinant human isoforms (hCE1 and hCE2) were also included for methodological validation. Among the invertebrates, mussels were revealed as the most sensitive to xenobiotic exposures while gastropods were the least as well as with particular substrate-specific preferences. Among chemicals of environmental concern, the plastic additive tetrabromobisphenol A displayed the highest CE-inhibitory capacity in all species. Since plastic additives easily breakdown from the polymer and may accumulate and metabolise in marine biota, their interaction with the CE key metabolic/detoxification processes may have consequences in invertebrate's physiology, affect bioaccumulation and therefore trophic web transfer and, ultimately, human health as shellfish consumers.

Biochemical evidences for M1-, M17- and M18-like aminopeptidases in marine invertebrates from Cuban coastline.

Metallo-aminopeptidases (mAPs) control many physiological processes. They are classified in different families according to structural similarities. Neutral mAPs catalyze the cleavage of neutral amino acids from the N-terminus of proteins or peptide substrates; they need one or two metallic cofactors in their active site. Information about marine invertebrate's neutral mAPs properties is scarce; available data are mainly derived from genomics and cDNA studies. The goal of this work was to characterize the biochemical properties of the neutral APs activities in eight Cuban marine invertebrate species from the Phyla Mollusca, Porifera, Echinodermata, and Cnidaria. Determination of substrate specificity, optimal pH and effects of inhibitors (1,10-phenanthroline, amastatin, and bestatin) and cobalt on activity led to the identification of distinct neutral AP-like activities, whose biochemical behaviors were similar to those of the M1 and M17 families of mAPs. Additionally, M18-like glutamyl AP activities were detected. Thus, marine invertebrates express biochemical activities likely belonging to various families of metallo-aminopeptidases.

An overview on marine cellulolytic enzymes and their potential applications.

Marine-derived enzymes have recently gained attention particularly for industrial applications. Cellulose-degrading enzymes are among leading biocatalysts with potential utility in biorefineries. This review presents an account of the cellulase production by marine sources from microorganisms including bacteria, yeasts, and molds to marine invertebrates such as protist, rotifer, mollusks, arthropods, and echinoderms. Cellulose-degrading ability of marine invertebrates is attributed to the production of endogenous cellulases and activities by the symbionts. Specialized environments in marine including estuaries and mangroves are rich in lignocellulosic biomass and hence provide a feeding ground for cellulose digesters. Since cellulosic biomass is considered chemical and energy feedstock, therefore, cellulases with the ability to work under extreme environment are much needed to fulfill the demand of modern biotechnological industries. The review also discusses physicochemical parameters of marine-derived cellulases. Key Points: • Cellulolytic ability is widely distributed amongst marine organisms, yet very few have been studied for their biotechnological potential • Cellulase from marine organisms has been demonstrated as a successful agent in degradation of seaweed processing waste to low molecular fragments • Marine derived cellulases can find their application in green processes • Cellulases from marine sources exhibit high specific activity, thermostability, and other important biochemical properties and hence can contend well with the enzymes from terrestrial sources.

Matrix Metalloproteinases in Invertebrates.

Matrix Metalloproteinases (MMPs) belong to a family of metal-dependent endopeptidases which contain a series of conserved pro-peptide domains and catalytic domains. MMPs have been widely found in plants, animals, and microorganisms. MMPs are involved in regulating numerous physiological processes, pathological processes, and immune responses. In addition, MMPs play a key role in disease occurrence, including tumors, cardiovascular diseases, and other diseases. Compared with invertebrate MMPs, vertebrate MMPs have diverse subtypes and complex functions. Therefore, it is difficult to study the function of MMPs in vertebrates. However, it is relatively easy to study invertebrate MMPs because there are fewer subtypes of MMPs in invertebrates. In the present review, the structure and function of MMPs in invertebrates were summarized, which will provide a theoretical basis for investigating the regulatory mechanism of MMPs in invertebrates.

A comprehensive comparison of the metazoan tryptophan degrading enzymes.

Tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO) have an independent origin; however, they have distinctly evolved to catalyze the same reaction. In general, TDO is a single-copy gene in each metazoan species, and TDO enzymes demonstrate similar enzyme activity regardless of their biological origin. In contrast, multiple IDO paralogues are observed in many species, and they display various enzymatic properties. Similar to vertebrate IDO2, invertebrate IDOs generally show low affinity/catalytic efficiency for L-Trp. Meanwhile, two IDO isoforms from scallop (IDO-I and -III) and sponge IDOs show high L-Trp catalytic activity, which is comparable to vertebrate IDO1. Site-directed mutagenesis experiments have revealed that primarily two residues, Tyr located at the 2nd residue on the F-helix (F2nd) and His located at the 9th residue on the G-helix (G9th), are crucial for the high affinity/catalytic efficiency of these 'high performance' invertebrate IDOs. Conversely, those two amino acid substitutions (F2nd/Tyr and G9th/His) resulted in high affinity and catalytic activity in other molluscan 'low performance' IDOs. In human IDO1, G9th is Ser167, whereas the counterpart residue of G9th in human TDO is His76. Previous studies have shown that Ser167 could not be substituted by His because the human IDO1 Ser167His variant showed significantly low catalytic activity. However, this may be specific for human IDO1 because G9th/His was demonstrated to be very effective in increasing the L-Trp affinity even in vertebrate IDOs. Therefore, these findings indicate that the active sites of TDO and IDO are more similar to each other than previously expected.

Review: The structure and function of cellulase (endo-ß-1,4-glucanase) and hemicellulase (ß-1,3-glucanase and endo-ß-1,4-mannase) enzymes in invertebrates that consume materials ranging from microbes, algae to leaf litter.

This review discusses the reaction catalysed, and the structure and function of the cellulase, endo-ß-1,4-glucanase and the hemicellulase enzymes, ß-1,3-glucanase and endo-ß-1,4-mannase that are present in numerous invertebrate groups with a diverse range of feeding specialisations. These range from microbial deposit and filter feeders, micro and macrophagous algal feeders, omnivores to herbivorous leaf litter and wood feeders. Endo-ß-1,4-glucanase from glycosyl hydrolase family 9 (GH9) digests cellulose like ß-1,4-glucans from a range of materials. As it hydrolyses crystalline cellulose very slowly, it is a poor cellulase. Where tested, the enzyme has dual endo-ß-1,4-glucanase and lichenase activity. Its presence does not necessarily indicate the ability of an animal to digest cellulose. It only indicates the ability to digest ß-1,4-glucans and its function, which is discussed in this review, should be considered with reference to the substrates present in the diet. ß-1,3-glucanase (laminarinase) belongs to glycosyl hydrolase family 16 (GH16) and hydrolyses ß-1.3-glucans. These polysaccharides are present in the cell walls of algae, protozoans and yeast, and they also occur as storage polysaccharides within protozoans and algae. Depending on their site of expression, these enzymes may function as a digestive enzyme or may be involved in innate immunity. Enzymes present in the digestive fluids or tissues, would be digestive. Haemolymph GH16 proteins may be involved in innate immunity through the activation of the phenol oxidase system. Insect GH16 proteins expressed within the haemolymph have lost their catalytic residues and function as ß-glucan binding proteins. In contrast, crustacean GH16 proteins expressed within the same tissue, have retained the catalytic residues and thus possibly their ß-1,3-glucanase activity. The potential function of which is discussed. Endo-ß-1,4-mannase from glycosyl hydrolase family 5, subfamily 10 (GH5_10) hydrolyses mannan, glucomannan and galactomannan. These hemicelluloses are present in the cell walls of plants and algae and also function as storage polysaccharides within legume and palm seeds. They are digestive enzymes whose high expression in some species suggests they are a major contributor to hemicellulose digestion. They may also provide the animal with substantial amounts of monosaccharides for energy.

Screening and Immobilization of Interfacial Esterases from Marine Invertebrates as Promising Biocatalyst Derivatives.

Interfacial esterases are useful enzymes in bioconversion and racemic mixture resolution processes. Marine invertebrates are few explored potential sources of these proteins. In this work, aqueous extracts of 41 species of marine invertebrates were screened for esterase, lipase, and phospholipase A activities, being all positive. Five extracts (Stichodactyla helianthus, Condylactis gigantea, Stylocheilus longicauda, Zoanthus pulchellus, and Plexaura homomalla) were selected for their activity values and immobilized on Octyl-Sepharose CL 4B support by interfacial adsorption. The selectivity of this immobilization method for interfacial esterases was evidenced by immobilization percentages ≥ 94% in almost all cases for lipase and phospholipase A activities. Six pharmaceutical-relevant esters (phenylethyl butyrate, ethyl-2-hydroxy-4-phenyl-butanoate, 2-oxyranylmethyl acetate (glycidol acetate), 7-aminocephalosporanic acid, methyl-prostaglandin F2α, and methyl-6-metoxy-α-methyl-2-naphtalen-acetate -naproxen methyl ester-) were bioconverted by at least three of these biocatalysts, with the lowest conversion percentage of 24%. In addition, three biocatalysts were used in the racemic mixture resolution of three previous compounds. The S. helianthus-derived biocatalyst showed the highest enantiomeric ratios for glycidol acetate (2.67, (S)-selective) and naproxen methyl ester (8.32, (R)-selective), and the immobilized extract of S. longicauda was the most resolutive toward the ethyl-2-hydroxy-4-phenyl-butanoate (8.13, (S)-selective). These results indicate the relevance of such marine interfacial esterases as immobilized biocatalysts for the pharmaceutical industry.

AID/APOBEC-like cytidine deaminases are ancient innate immune mediators in invertebrates.

In the course of both innate and adaptive immunity, cytidine deaminases within the activation induced cytidine deaminase (AID)/apolipoprotein B editing complex (APOBEC) family modulate immune responses by mutating specific nucleic acid sequences of hosts and pathogens. The evolutionary emergence of these mediators, however, seems to coincide precisely with the emergence of adaptive immunity in vertebrates. Here, we show a family of genes in species within two divergent invertebrate phyla-the echinoderm Strongylocentrotus purpuratus and the brachiopod Lingula anatina-that encode proteins with similarities in amino acid sequence and enzymatic activities to the vertebrate AID/APOBECs. The expression of these invertebrate factors is enriched in tissues undergoing constant, direct interactions with microbes and can be induced upon pathogen challenge. Our findings suggest that AID/APOBEC proteins, and their function in immunity, emerged far earlier than previously thought. Thus, cytidine deamination is probably an ancient innate immune mechanism that predates the protostome/deuterostome divergence.

Role of the venus kinase receptor in the female reproductive physiology of the desert locust, Schistocerca gregaria.

Venus kinase receptors (VKR) are a subfamily of invertebrate receptor tyrosine kinases, which have only recently been discovered. They contain an intracellular tyrosine kinase domain and an extracellular Venus FlyTrap domain. VKRs have been functionally and pharmacologically characterized in only two invertebrate species, namely the human parasite Schistosoma mansoni and the mosquito Aedes aegypti, where they play a crucial role in oogenesis. Here, we report the characterization of a VKR in the desert locust, Schistocerca gregaria. We performed an in-depth profiling study of the SgVKR transcript levels in different tissues throughout the female adult stage. Using the RNA interference technique, the possible role of SgVKR was investigated. SgVKR knockdown had significant effects on ovarian ecdysteroid levels and on the size of oocytes during the vitellogenic stage. SgVKR is probably involved in the complex cross-talk between several important pathways regulating female reproductive physiology. Contrary to A. aegypti and S. mansoni, we cannot conclude that this receptor is essential for reproduction, since silencing SgVKR did not affect fecundity or fertility. Considering the evolutionary distance between A. aegypti and S. gregaria, as well as the differences in regulation of their female reproductive physiology, this article constitutes a valuable asset in better understanding VKRs.

Phylogenetic analysis of the SINA/SIAH ubiquitin E3 ligase family in Metazoa.

BACKGROUND: The RAS signaling pathway is a pivotal developmental pathway that controls many fundamental biological processes including cell proliferation, differentiation, movement and apoptosis. Drosophila Seven-IN-Absentia (SINA) is a ubiquitin E3 ligase that is the most downstream signaling "gatekeeper" whose biological activity is essential for proper RAS signal transduction. Vertebrate SINA homologs (SIAHs) share a high degree of amino acid identity with that of Drosophila SINA. SINA/SIAH is the most conserved signaling component in the canonical EGFR/RAS/RAF/MAPK signal transduction pathway. RESULTS: Vertebrate SIAH1, 2, and 3 are the three orthologs to invertebrate SINA protein. SINA and SIAH1 orthologs are found in all major taxa of metazoans. These proteins have four conserved functional domains, known as RING (Really Interesting New Gene), SZF (SIAH-type zinc finger), SBS (substrate binding site) and DIMER (Dimerization). In addition to the siah1 gene, most vertebrates encode two additional siah genes (siah2 and siah3) in their genomes. Vertebrate SIAH2 has a highly divergent and extended N-terminal sequence, while its RING, SZF, SBS and DIMER domains maintain high amino acid identity/similarity to that of SIAH1. But unlike vertebrate SIAH1 and SIAH2, SIAH3 lacks a functional RING domain, suggesting that SIAH3 may be an inactive E3 ligase. The SIAH3 subtree exhibits a high degree of amino acid divergence when compared to the SIAH1 and SIAH2 subtrees. We find that SIAH1 and SIAH2 are expressed in all human epithelial cell lines examined thus far, while SIAH3 is only expressed in a limited subset of cancer cell lines. CONCLUSION: Through phylogenetic analyses of metazoan SINA and SIAH E3 ligases, we identified many invariant and divergent amino acid residues, as well as the evolutionarily conserved functional motifs in this medically relevant gene family. Our phylomedicinal study of this unique metazoan SINA/SIAH protein family has provided invaluable evolution-based support towards future effort to design logical, potent, and durable anti-SIAH-based anticancer strategies against oncogenic K-RAS-driven metastatic human cancers. Thus, this method of evolutionary study should be of interest in cancer biology.

Matrix metalloproteinases outside vertebrates.

The matrix metalloproteinase (MMP) family belongs to the metzincin clan of zinc-dependent metallopeptidases. Due to their enormous implications in physiology and disease, MMPs have mainly been studied in vertebrates. They are engaged in extracellular protein processing and degradation, and present extensive paralogy, with 23 forms in humans. One characteristic of MMPs is a ~165-residue catalytic domain (CD), which has been structurally studied for 14 MMPs from human, mouse, rat, pig and the oral-microbiome bacterium Tannerella forsythia. These studies revealed close overall coincidence and characteristic structural features, which distinguish MMPs from other metzincins and give rise to a sequence pattern for their identification. Here, we reviewed the literature available on MMPs outside vertebrates and performed database searches for potential MMP CDs in invertebrates, plants, fungi, viruses, protists, archaea and bacteria. These and previous results revealed that MMPs are widely present in several copies in Eumetazoa and higher plants (Tracheophyta), but have just token presence in eukaryotic algae. A few dozen sequences were found in Ascomycota (within fungi) and in double-stranded DNA viruses infecting invertebrates (within viruses). In contrast, a few hundred sequences were found in archaea and >1000 in bacteria, with several copies for some species. Most of the archaeal and bacterial phyla containing potential MMPs are present in human oral and gut microbiomes. Overall, MMP-like sequences are present across all kingdoms of life, but their asymmetric distribution contradicts the vertical descent model from a eubacterial or archaeal ancestor. This article is part of a Special Issue entitled: Matrix Metalloproteinases edited by Rafael Fridman.

Susceptibility to type 2 diabetes may be modulated by haplotypes in G6PC2, a target of positive selection.

BACKGROUND: The endoplasmic reticulum enzyme glucose-6-phosphatase catalyzes the common terminal reaction in the gluconeogenic/glycogenolytic pathways and plays a central role in glucose homeostasis. In most mammals, different G6PC subunits are encoded by three paralogous genes (G6PC, G6PC2, and G6PC3). Mutations in G6PC and G6PC3 are responsible for human mendelian diseases, whereas variants in G6PC2 are associated with fasting glucose (FG) levels. RESULTS: We analyzed the evolutionary history of G6Pase genes. Results indicated that the three paralogs originated during early vertebrate evolution and that negative selection was the major force shaping diversity at these genes in mammals. Nonetheless, site-wise estimation of evolutionary rates at corresponding sites revealed weak correlations, suggesting that mammalian G6Pases have evolved different structural features over time. We also detected pervasive positive selection at mammalian G6PC2. Most selected residues localize in the C-terminal protein region, where several human variants associated with FG levels also map. This region was re-sequenced in ~560 subjects from Saudi Arabia, 185 of whom suffering from type 2 diabetes (T2D). The frequency of rare missense and nonsense variants was not significantly different in T2D and controls. Association analysis with two common missense variants (V219L and S342C) revealed a weak but significant association for both SNPs when analyses were conditioned on rs560887, previously identified in a GWAS for FG. Two haplotypes were significantly associated with T2D with an opposite effect direction. CONCLUSIONS: We detected pervasive positive selection at mammalian G6PC2 genes and we suggest that distinct haplotypes at the G6PC2 locus modulate susceptibility to T2D.

Proteolysis of HCF-1 by Ser/Thr glycosylation-incompetent O-GlcNAc transferase:UDP-GlcNAc complexes.

In complex with the cosubstrate UDP-N-acetylglucosamine (UDP-GlcNAc),O-linked-GlcNAc transferase (OGT) catalyzes Ser/ThrO-GlcNAcylation of many cellular proteins and proteolysis of the transcriptional coregulator HCF-1. Such a dual glycosyltransferase-protease activity, which occurs in the same active site, is unprecedented and integrates both reversible and irreversible forms of protein post-translational modification within one enzyme. Although occurring within the same active site, we show here that glycosylation and proteolysis occur through separable mechanisms. OGT consists of tetratricopeptide repeat (TPR) and catalytic domains, which, together with UDP-GlcNAc, are required for both glycosylation and proteolysis. Nevertheless, a specific TPR domain contact with the HCF-1 substrate is critical for proteolysis but not Ser/Thr glycosylation. In contrast, key catalytic domain residues and even a UDP-GlcNAc oxygen important for Ser/Thr glycosylation are irrelevant for proteolysis. Thus, from a dual glycosyltransferase-protease, essentially single-activity enzymes can be engineered both in vitro and in vivo. Curiously, whereas OGT-mediated HCF-1 proteolysis is limited to vertebrate species, invertebrate OGTs can cleave human HCF-1. We present a model for the evolution of HCF-1 proteolysis by OGT.

Development of marine biotechnology as a resource for novel proteases and their role in modern biotechnology.

Marine environment consists of the largest sources diversified genetic pool of material with an enormous potential for a wide variety of enzymes including proteases. A protease hydrolyzes the peptide bond and most of proteases possess many industrial applications. Marine proteases differ considerably from those found in internal or external organs of invertebrates and vertebrates. In common with all enzymes, external factors such as temperature, pH and type of media are important for the activity, catalytic efficiency, stability and proper functioning of proteases. In this review valuable characteristics of proteases in marine organisms and their applications are gathered from a wide literature survey. Considering their biochemical significance and their increasing importance in biotechnology, a thorough understanding of marine proteases functioning could be of prime importance.

Structural analysis of the α subunit of Na(+)/K(+) ATPase genes in invertebrates.

The Na(+)/K(+) ATPase is a ubiquitous pump coordinating the transport of Na(+) and K(+) across the membrane of cells and its role is fundamental to cellular functions. It is heteromer in eukaryotes including two or three subunits (α, ß and γ which is specific to the vertebrates). The catalytic functions of the enzyme have been attributed to the α subunit. Several complete α protein sequences are available, but only few gene structures were characterized. We identified the genomic sequences coding the α-subunit of the Na(+)/K(+) ATPase, from the whole-genome shotgun contigs (WGS), NCBI Genomes (chromosome), Genomic Survey Sequences (GSS) and High Throughput Genomic Sequences (HTGS) databases across distinct phyla. One copy of the α subunit gene was found in Annelida, Arthropoda, Cnidaria, Echinodermata, Hemichordata, Mollusca, Placozoa, Porifera, Platyhelminthes, Urochordata, but the nematodes seem to possess 2 to 4 copies. The number of introns varied from 0 (Platyhelminthes) to 26 (Porifera); and their localization and length are also highly variable. Molecular phylogenies (Maximum Likelihood and Maximum Parsimony methods) showed some clusters constituted by (Chordata/(Echinodermata/Hemichordata)) or (Plathelminthes/(Annelida/Mollusca)) and a basal position for Porifera. These structural analyses increase our knowledge about the evolutionary events of the α subunit genes in the invertebrates.

Distribution and evolution of the serine/aspartate racemase family in invertebrates.

Free D-amino acids have been found in various invertebrate phyla, while amino acid racemase genes have been identified in few species. The purpose of this study is to elucidate the distribution, function, and evolution of amino acid racemases in invertebrate animals. We searched the GenBank databases, and found 11 homologous serine racemase genes from eight species in eight different invertebrate phyla. The cloned genes were identified based on their maximum activity as Acropora millepora (Cnidaria) serine racemase (SerR) and aspartate racemase (AspR), Caenorhabditis elegans (Nematoda) SerR, Capitella teleta (Annelida) SerR, Crassostrea gigas (Mollusca) SerR and AspR, Dugesia japonica (Platyhelminthes) SerR, Milnesium tardigradum (Tardigrada) SerR, Penaeus monodon (Arthropoda) SerR and AspR and Strongylocentrotus purpuratus (Echinodermata) AspR. We found that Acropora, Aplysia, Capitella, Crassostrea and Penaeus had two amino acid racemase paralogous genes and these paralogous genes have evolved independently by gene duplication at their recent ancestral species. The transcriptome analyses using available SRA data and enzyme kinetic data suggested that these paralogous genes are expressed in different tissues and have different functions in vivo. Phylogenetic analyses clearly indicated that animal SerR and AspR are not separated by their particular racemase functions and form a serine/aspartate racemase family cluster. Our results revealed that SerR and AspR are more widely distributed among invertebrates than previously known. Moreover, we propose that the triple serine loop motif at amino acid positions 150-152 may be responsible for the large aspartate racemase activity and the AspR evolution from SerR.

Marine invertebrate lipases: Comparative and functional genomic analysis.

Lipases are key enzymes involved in lipid digestion, storage and mobilization of reserves during fasting or heightened metabolic demand. This is a highly conserved process, essential for survival. The genomes of five marine invertebrate species with distinctive digestive system were screened for the six major lipase families. The two most common families in marine invertebrates, the neutral an acid lipases, are also the main families in mammals and insects. The number of lipases varies two-fold across analyzed genomes. A high degree of orthology with mammalian lipases was observed. Interestingly, 19% of the marine invertebrate lipases have lost motifs required for catalysis. Analysis of the lid and loop regions of the neutral lipases suggests that many marine invertebrates have a functional triacylglycerol hydrolytic activity as well as some acid lipases. A revision of the expression profiles and functional activity on sequences in databases and scientific literature provided information regarding the function of these families of enzymes in marine invertebrates.

First comparative insight into the architecture of COI mitochondrial minicircle molecules of dicyemids reveals marked inter-species variation.

Dicyemids, poorly known parasites of benthic cephalopods, are one of the few phyla in which mitochondrial (mt) genome architecture departs from the typical ~16 kb circular metazoan genome. In addition to a putative circular genome, a series of mt minicircles that each comprises the mt encoded units (I-III) of the cytochrome c oxidase complex have been reported. Whether the structure of the mt minicircles is a consistent feature among dicyemid species is unknown. Here we analyse the complete cytochrome c oxidase subunit I (COI) minicircle molecule, containing the COI gene and an associated non-coding region (NCR), for ten dicyemid species, allowing for first time comparisons between species of minicircle architecture, NCR function and inferences of minicircle replication. Divergence in COI nucleotide sequences between dicyemid species was high (average net divergence = 31.6%) while within species diversity was lower (average net divergence = 0.2%). The NCR and putative 5' section of the COI gene were highly divergent between dicyemid species (average net nucleotide divergence of putative 5' COI section = 61.1%). No tRNA genes were found in the NCR, although palindrome sequences with the potential to form stem-loop structures were identified in some species, which may play a role in transcription or other biological processes.

Aldehyde dehydrogenase homologous folate enzymes: Evolutionary switch between cytoplasmic and mitochondrial localization.

Cytosolic and mitochondrial 10-formyltetrahydrofolate dehydrogenases are products of separate genes in vertebrates but only one such gene is present in invertebrates. There is a significant degree of sequence similarity between the two enzymes due to an apparent origin of the gene for the mitochondrial enzyme (ALDH1L2) from the duplication of the gene for the cytosolic enzyme (ALDH1L1). The primordial ALDH1L gene originated from a natural fusion of three unrelated genes, one of which was an aldehyde dehydrogenase. Such structural organization defined the catalytic mechanism of these enzymes, which is similar to that of aldehyde dehydrogenases. Here we report the analysis of ALDH1L1 and ALDH1L2 genes from different species and their phylogeny and evolution. We also performed sequence and structure comparison of ALDH1L enzymes possessing aldehyde dehydrogenase catalysis to those lacking this feature in an attempt to explain mechanistic differences between cytoplasmic ALDH1L1 and mitochondrial ALDH1L2 enzymes and to better understand their functional roles.

Substrate-dependent evolution of cytochrome P450: rapid turnover of the detoxification-type and conservation of the biosynthesis-type.

Members of the cytochrome P450 family are important metabolic enzymes that are present in all metazoans. Genes encoding cytochrome P450s form a multi-gene family, and the number of genes varies widely among species. The enzymes are classified as either biosynthesis- or detoxification-type, depending on their substrates, but their origin and evolution have not been fully understood. In order to elucidate the birth and death process of cytochrome P450 genes, we performed a phylogenetic analysis of 710 sequences from 14 vertebrate genomes and 543 sequences from 6 invertebrate genomes. Our results showed that vertebrate detoxification-type genes have independently emerged three times from biosynthesis-type genes and that invertebrate detoxification-type genes differ from vertebrates in their origins. Biosynthetic-type genes exhibit more conserved evolutionary processes than do detoxification-type genes, with regard to the rate of gene duplication, pseudogenization, and amino acid substitution. The differences in the evolutionary mode between biosynthesis- and detoxification-type genes may reflect differences in their respective substrates. The phylogenetic tree also revealed 11 clans comprising an upper category to families in the cytochrome P450 nomenclature. Here, we report novel clan-specific amino acids that may be used for the qualitative definition of clans.