Isofunctional Enzymes Research Articles

Improving enzyme functional annotation by integrating in vitro and in silico approaches: The example of histidinol phosphate phosphatases.

Advances in sequencing technologies have led to a rapid growth of public protein sequence databases, whereby the fraction of proteins with experimentally verified function continuously decreases. This problem is currently addressed by automated functional annotations with computational tools, which however lack the accuracy of experimental approaches and are susceptible to error propagation. Here, we present an approach that combines the efficiency of functional annotation by in silico methods with the rigor of enzyme characterization in vitro. First, a thorough experimental analysis of a representative enzyme of a group of homologues is performed which includes a focused alanine scan of the active site to determine a fingerprint of function-determining residues. In a second step, this fingerprint is used in combination with a sequence similarity network to identify putative isofunctional enzymes among the homologues. Using this approach in a proof-of-principle study, homologues of the histidinol phosphate phosphatase (HolPase) from Pseudomonas aeruginosa, many of which were annotated as phosphoserine phosphatases, were predicted to be HolPases. This functional annotation of the homologues was verified by in vitro testing of several representatives and an analysis of the occurrence of annotated HolPases in the corresponding phylogenetic groups. Moreover, the application of the same approach to the homologues of the HolPase from the archaeon Nitrosopumilus maritimus, which is not related to the HolPase from P. aeruginosa and was newly discovered in the course of this work, led to the annotation of the putative HolPase from various archaeal species.

Ecological diversity and associated volatilome of typical mountain Caciotta cheese from Italy

Traditional products are particularly appreciated by consumers and among these products, cheese is a major contributor to the Italian mountainous area economics. In this study, shotgun metagenomics and volatilomics were used to understand the biotic and abiotic factors contributing to mountain Caciotta cheese typicity and diversity. Results showed that the origin of cheese played a significant role; however, curd cooking temperature, pH, salt concentration and water activity also had an impact. Viral communities exhibited higher biodiversity and discriminated cheese origins in terms of production farms. Among the most dominant bacteria, Streptococcus thermophilus showed higher intraspecific diversity and closer relationship to production farm when compared to Lactobacillus delbrueckii. However, despite a few cases in which the starter culture was phylogenetically separated from the most dominant strains sequenced in the cheese, starter cultures and dominant cheese strains clustered together suggesting substantial starter colonization in mountain Caciotta cheese. The Caciotta cheese volatilome contained prominent levels of alcohols and ketones, accompanied by lower proportions of terpenes. Volatile profile not only demonstrated a noticeable association with production farm but also significant differences in the relative abundances of enzymes connected to flavor development. Moreover, correlations of different non-homologous isofunctional enzymes highlighted specific contributions to the typical flavor of mountain Caciotta cheese. Overall, this study provides a deeper understanding of the factors shaping typical mountain Caciotta cheese, and the potential of metagenomics for characterizing and potentially authenticating food products.

The universal Sua5/TsaC family evolved different mechanisms for the synthesis of a key tRNA modification.

TsaC/Sua5 family of enzymes catalyzes the first step in the synthesis of N6-threonyl-carbamoyl adenosine (t6A) one of few truly ubiquitous tRNA modifications important for translation accuracy. TsaC is a single domain protein while Sua5 proteins contains a TsaC-like domain and an additional SUA5 domain of unknown function. The emergence of these two proteins and their respective mechanisms for t6A synthesis remain poorly understood. Here, we performed phylogenetic and comparative sequence and structure analysis of TsaC and Sua5 proteins. We confirm that this family is ubiquitous but the co-occurrence of both variants in the same organism is rare and unstable. We further find that obligate symbionts are the only organisms lacking sua5 or tsaC genes. The data suggest that Sua5 was the ancestral version of the enzyme while TsaC arose via loss of the SUA5 domain that occurred multiple times in course of evolution. Multiple losses of one of the two variants in combination with horizontal gene transfers along a large range of phylogenetic distances explains the present day patchy distribution of Sua5 and TsaC. The loss of the SUA5 domain triggered adaptive mutations affecting the substrate binding in TsaC proteins. Finally, we identified atypical Sua5 proteins in Archaeoglobi archaea that seem to be in the process of losing the SUA5 domain through progressive gene erosion. Together, our study uncovers the evolutionary path for emergence of these homologous isofunctional enzymes and lays the groundwork for future experimental studies on the function of TsaC/Sua5 proteins in maintaining faithful translation.

Substrate Binding in the Allosteric Site Mimics Homotropic Cooperativity in the SIS-fold Glucosamine-6-phosphate Deaminases.

Glucosamine-6-phosphate (GlcN6P) deaminases from E. coli (EcNagBI) and S. denitrificans (SdNagBII) are special examples of what constitute non-homologous isofunctional enzymes due to their convergence, not only in catalysis, but also in cooperativity and allosteric properties. Additionally, we found that the sigmoidal kinetics of SdNagBII cannot be explained by the existing models of homotropic activation. This study describes the regulatory mechanism of SdNagBII using enzyme kinetics, isothermal titration calorimetry (ITC), and X-ray crystallography. ITC experiments revealed two different binding sites with distinctive thermodynamic signatures: a single binding site per monomer for the allosteric activator N-acetylglucosamine 6-phosphate (GlcNAc6P) and two binding sites per monomer for the transition-state analog 2-amino-2-deoxy-D-glucitol 6-phosphate (GlcNol6P). Crystallographic data demonstrated the existence of an unusual allosteric site that can bind both GlcNAc6P and GlcNol6P, implying that the homotropic activation of this enzyme arises from the occupation of the allosteric site by the substrate. In this work we describe the presence of this novel allosteric site in the SIS-fold deaminases, which is responsible for the homotropic and heterotropic activation of SdNagBII by GlcN6P and GlcNAc6P, respectively. This study unveils an original mechanism to generate a high degree of homotropic activation in SdNagBII, mimicking the allosteric and cooperative properties of hexameric EcNagBI but with a reduced number of subunits. This article is protected by copyright. All rights reserved.

Bacterial Dehydrogenases Facilitate Oxidative Inactivation and Bioremediation of Chloramphenicol.

Antimicrobial resistance represents a major threat to human health and knowledge of the underlying mechanisms is therefore vital. Here, we report the discovery and characterization of oxidoreductases that inactivate the broad-spectrum antibiotic chloramphenicol via dual oxidation of the C3-hydroxyl group. Accordingly, chloramphenicol oxidation either depends on standalone glucose-methanol-choline (GMC)-type flavoenzymes, or on additional aldehyde dehydrogenases that boost overall turnover. These enzymes also enable the inactivation of the chloramphenicol analogues thiamphenicol and azidamfenicol, but not of the C3-fluorinated florfenicol. Notably, distinct isofunctional enzymes can be found in Gram-positive (e. g., Streptomyces sp.) and Gram-negative (e. g., Sphingobium sp.) bacteria, which presumably evolved their selectivity for chloramphenicol independently based on phylogenetic analyses. Mechanistic and structural studies provide further insights into the catalytic mechanisms of these biotechnologically interesting enzymes, which, in sum, are both a curse and a blessing by contributing to the spread of antibiotic resistance as well as to the bioremediation of chloramphenicol.

Biologically evolved dual-pathway catalytic pattern indicating an efficient bioremediation strategy for phenol removal

Bioremediation of phenol has been developed for years, however, its actual application is challenged by low efficiency and environmental intolerance. In addition, although multiple phenol degradation pathways have been reported to coexist in bacteria, the potential significance and value have not been clearly revealed. Here, highly efficient phenol catalytic patterns were identified, and multi-pathway catalytic mechanism was investigated. Parageobacillus thermoglucosidasius W-36 employing a dual-pathway pattern exhibited superior degradation performance than P. thermoglucosidasius W-2 that employed a single-pathway pattern. An exogenous copy of degradation pathway in dual-pathway pattern was obtained via horizontal gene transfer. Based on homology modeling and molecular docking, the three-dimensional structures and substrate-binding sites of exogenous key enzymes were distinct from those of isofunctional enzymes encoded by the chromosome. The dual-pathway pattern was expressed in response to substrate levels in a time-dependent manner, indicating that synergistic effect of degradation pathways conferred strain adaptability to high-concentration phenol. Evolutionary analysis showed that the artful single/dual-pathway patterns in this species may be a natural selection phenomenon caused by high-concentration phenol. Collectively, this naturally evolved pattern enlightens the new strategy and feasibility of introducing auxiliary metabolic pathways to improve degradation efficiency of pollutants.

A novel Tetrahymena thermophila sterol C-22 desaturase belongs to the fatty acid hydroxylase/desaturase superfamily

Sterols in eukaryotic cells play important roles in modulating membrane fluidity and in cell signaling and trafficking. During evolution, a combination of gene losses and acquisitions gave rise to an extraordinary diversity of sterols in different organisms. The sterol C-22 desaturase identified in plants and fungi as a cytochrome P-450 monooxygenase evolved from the first eukaryotic cytochrome P450 and was lost in many lineages. Although the ciliate Tetrahymena thermophila desaturates sterols at the C-22 position, no cytochrome P-450 orthologs are present in the genome. Here, we aim to identify the genes responsible for the desaturation as well as their probable origin. We used gene knockout and yeast heterologous expression approaches to identify two putative genes, retrieved from a previous transcriptomic analysis, as sterol C-22 desaturases. Furthermore, we demonstrate using bioinformatics and evolutionary analyses that both genes encode a novel type of sterol C-22 desaturase that belongs to the large fatty acid hydroxylase/desaturase superfamily and the genes originated by genetic duplication prior to functional diversification. These results stress the widespread existence of nonhomologous isofunctional enzymes among different lineages of the tree of life as well as the suitability for the use of T. thermophila as a valuable model to investigate the evolutionary process of large enzyme families.

The roles of diol dehydratase from pdu operon on glycerol catabolism in Klebsiella pneumoniae

The dha operon of Klebsiella pneumoniae is responsible for glycerol catabolism and 1,3-propanediol formation. Subunits of glycerol dehydratase and the large subunit of glycerol dehydratase reactivating factor are encoded by dhaBCE and dhaF, respectively. Proteins of pdu operon form a microcompartment (bacteria organelle) and responsible for 1,2-propanediol catabolism. In this operon, pduCDE and pduG encode subunits of diol dehydratase and its reactivating factor. Diol dehydratase is an isofunctional enzyme of glycerol dehydratase, but its role in glycerol catabolism was not entirely clear. In this study, dhaBCE, pduCDE, dhaF, and pduG in K. pneumoniae were knocked out individually or combinedly. These strains were cultured with glycerol as a substrate, and dehydratase activities in the cytoplasm and microcompartment were detected. Results showed that glycerol dehydratase and diol dehydratase were simultaneously responsible for glycerol catabolism in K. pneumoniae. Besides being packaged in microcompartment, large amounts of diol dehydratase was also presented in the cytoplasm. However, the Pdu microcompartment reduced the accumulation of 3-hydroxypropionaldehyde in the fermentation broth. PduG can cross reactivate glycerol dehydratase instead of DhaF. However, DhaF is not involved in reactivation of diol dehydratase. In conclusion, diol dehydratase and Pdu microcompartment play important roles in glycerol catabolism in K. pneumoniae.

Glycerol as a Substrate and Inactivator of Coenzyme B12 -Dependent Diol Dehydratase.

Diol dehydratase, dependent on coenzyme B12 (B12 -dDDH), displays a peculiar feature of being inactivated by its native substrate glycerol (GOL). Surprisingly, the isofunctional enzyme, B12 -independent glycerol dehydratase (B12 -iGDH), does not undergo suicide inactivation by GOL. Herein we present a series of QM/MM and MD calculations aimed at understanding the mechanisms of substrate-induced suicide inactivation in B12 -dDDH and that of resistance of B12 -iGDH to inactivation. We show that the first step in the enzymatic transformation of GOL, hydrogen abstraction, can occur from both ends of the substrate (either C1 or C3 of GOL). Whereas C1 abstraction in both enzymes leads to product formation, C3 abstraction in B12 -dDDH results in the formation of a low energy radical intermediate, which is effectively trapped within a deep well on the potential energy surface. The long lifetime of this radical intermediate likely enables its side reactions, leading to inactivation. In B12 -iGDH, by comparison, C3 abstraction is an endothermic step; consequently, the resultant radical intermediate is not of low energy, and the reverse process of reforming the reactant is possible.

Structure–activity relationship of ipglycermide binding to phosphoglycerate mutases

Catalysis of human phosphoglycerate mutase is dependent on a 2,3-bisphosphoglycerate cofactor (dPGM), whereas the nonhomologous isozyme in many parasitic species is cofactor independent (iPGM). This mechanistic and phylogenetic diversity offers an opportunity for selective pharmacologic targeting of glycolysis in disease-causing organisms. We previously discovered ipglycermide, a potent inhibitor of iPGM, from a large combinatorial cyclic peptide library. To fully delineate the ipglycermide pharmacophore, herein we construct a detailed structure–activity relationship using 280 substituted ipglycermide analogs. Binding affinities of these analogs to immobilized Caenorhabditis elegans iPGM, measured as fold enrichment relative to the index residue by deep sequencing of an mRNA display library, illuminated the significance of each amino acid to the pharmacophore. Using cocrystal structures and binding kinetics, we show that the high affinity of ipglycermide for iPGM orthologs, from Brugia malayi, Onchocerca volvulus, Dirofilaria immitis, and Escherichia coli, is achieved by a codependence between (1) the off-rate mediated by the macrocycle Cys14 thiolate coordination to an active-site Zn2+ in the iPGM phosphatase domain and (2) shape complementarity surrounding the macrocyclic core at the phosphotransferase–phosphatase domain interface. Our results show that the high-affinity binding of ipglycermide to iPGMs freezes these structurally dynamic enzymes into an inactive, stable complex.

Origin, evolution and functional characterization of the land plant glycoside hydrolase subfamily GH5_11

Colonization of the land by plants was a critical event in the establishment of modern terrestrial ecosystems, and many characteristics of land plants originated during this process, including the emergence of rosette terminal cellulose-synthesizing complexes. Cellulases are non-homologous isofunctional enzymes, encoded by glycosyl hydrolase (GH) gene families. Although the plant GH5_11 gene subfamily is presumed to encode a cell-wall degrading enzyme, its evolutionary and functional characteristics remain unclear. In the present study, we report the evolution of the land plant GH5_11 subfamily, and the functions of its members in terms of cellulase activity, through comprehensive phylogenetic analyses and observation of Arabidopsis mutants. Phylogenetic and sequence similarity analyses reveal that the ancestor of land plants acquired the GH5_11 gene from fungi through a horizontal gene transfer (HGT) event. Subsequently, positive selection with massive gene duplication and loss events contributed to the evolution of this subfamily in land plants. In Arabidopsis and rice, expression of GH5_11 genes are regulated by multiple abiotic stresses, the duplicated genes showing different patterns of expression. The Arabidopsis mutants atgh5_11a and atgh5_11c display low levels of cellulase and endoglucanase activities, with correspondingly high levels of cellulose, implying that the encoded proteins may function as endoglucanases. However, atgh5_11a and atgh5_11c also display an enlarged rosette leaf phenotype, and atgh5_11c is late-flowering under short photoperiods. These observations suggest that plant GH5_11s possess more functions beyond being endonucleases. To summarize, we demonstrate that the ancestor of land plants has acquired GH5_11 gene through HGT, which extends the cellulose degradation complexity. Our investigations illuminate features of part of the molecular framework underlying the origin of land plants and provide a focus on the cellulose degradation pathway.

Mining of potential drug targets through the identification of essential and analogous enzymes in the genomes of pathogens of Glycine max, Zea mays and Solanum lycopersicum.

Pesticides are one of the most widely used pest and disease control measures in plant crops and their indiscriminate use poses a direct risk to the health of populations and environment around the world. As a result, there is a great need for the development of new, less toxic molecules to be employed against plant pathogens. In this work, we employed an in silico approach to study the genes coding for enzymes of the genomes of three commercially important plants, soybean (Glycine max), tomato (Solanum lycopersicum) and corn (Zea mays), as well as 15 plant pathogens (4 bacteria and 11 fungi), focusing on revealing a set of essential and non-homologous isofunctional enzymes (NISEs) that could be prioritized as drug targets. By combining sequence and structural data, we obtained an initial set of 568 cases of analogy, of which 97 were validated and further refined, revealing a subset of 29 essential enzymatic activities with a total of 119 different structural forms, most belonging to central metabolic routes, including the carbohydrate metabolism, the metabolism of amino acids, among others. Further, another subset of 26 enzymatic activities possess a tertiary structure specific for the pathogen, not present in plants, men and Apis mellifera, which may be of importance for the development of specific enzymatic inhibitors against plant diseases that are less harmful to humans and the environment.

Parallel evolution of non-homologous isofunctional enzymes in methionine biosynthesis.

Experimental validation of enzyme function is crucial for genome interpretation, but it remains challenging because it cannot be scaled up to accommodate the constant accumulation of genome sequences. We tackled this issue for the MetA and MetX enzyme families, phylogenetically unrelated families of acyl-L-homoserine transferases involved in L-methionine biosynthesis. Members of these families are prone to incorrect annotation because MetX and MetA enzymes are assumed to always use acetyl-CoA and succinyl-CoA, respectively. We determined the enzymatic activities of 100 enzymes from diverse species, and interpreted the results by structural classification of active sites based on protein structure modeling. We predict that >60% of the 10,000 sequences from these families currently present in databases are incorrectly annotated, and suggest that acetyl-CoA was originally the sole substrate of these isofunctional enzymes, which evolved to use exclusively succinyl-CoA in the most recent bacteria. We also uncovered a divergent subgroup of MetX enzymes in fungi that participate only in L-cysteine biosynthesis as O-succinyl-L-serine transferases.

Functional Analogy in Human Metabolism: Enzymes with Different Biological Roles or Functional Redundancy?

Since enzymes catalyze almost all chemical reactions that occur in living organisms, it is crucial that genes encoding such activities are correctly identified and functionally characterized. Several studies suggest that the fraction of enzymatic activities in which multiple events of independent origin have taken place during evolution is substantial. However, this topic is still poorly explored, and a comprehensive investigation of the occurrence, distribution, and implications of these events has not been done so far. Fundamental questions, such as how analogous enzymes originate, why so many events of independent origin have apparently occurred during evolution, and what are the reasons for the coexistence in the same organism of distinct enzymatic forms catalyzing the same reaction, remain unanswered. Also, several isofunctional enzymes are still not recognized as nonhomologous, even with substantial evidence indicating different evolutionary histories. In this work, we begin to investigate the biological significance of the cooccurrence of nonhomologous isofunctional enzymes in human metabolism, characterizing functional analogous enzymes identified in metabolic pathways annotated in the human genome. Our hypothesis is that the coexistence of multiple enzymatic forms might not be interpreted as functional redundancy. Instead, these enzymatic forms may be implicated in distinct (and probably relevant) biological roles.

Enzyme discovery beyond homology: a unique hydroxynitrile lyase in the Bet v1 superfamily

Homology and similarity based approaches are most widely used for the identification of new enzymes for biocatalysis. However, they are not suitable to find truly novel scaffolds with a desired function and this averts options and diversity. Hydroxynitrile lyases (HNLs) are an example of non-homologous isofunctional enzymes for the synthesis of chiral cyanohydrins. Due to their convergent evolution, finding new representatives is challenging. Here we show the discovery of unique HNL enzymes from the fern Davallia tyermannii by coalescence of transcriptomics, proteomics and enzymatic screening. It is the first protein with a Bet v1-like protein fold exhibiting HNL activity, and has a new catalytic center, as shown by protein crystallography. Biochemical properties of D. tyermannii HNLs open perspectives for the development of a complementary class of biocatalysts for the stereoselective synthesis of cyanohydrins. This work shows that systematic integration of -omics data facilitates discovery of enzymes with unpredictable sequences and helps to extend our knowledge about enzyme diversity.

An assessment of catalytic residue 3D ensembles for the prediction of enzyme function.

BackgroundThe central element of each enzyme is the catalytic site, which commonly catalyzes a single biochemical reaction with high specificity. It was unclear to us how often sites that catalyze the same or highly similar reactions evolved on different, i. e. non-homologous protein folds and how similar their 3D poses are. Both similarities are key criteria for assessing the usability of pose comparison for function prediction.ResultsWe have analyzed the SCOP database on the superfamily level in order to estimate the number of non-homologous enzymes possessing the same function according to their EC number. 89 % of the 873 substrate-specific functions (four digit EC number) assigned to mono-functional, single-domain enzymes were only found in one superfamily. For a reaction-specific grouping (three digit EC number), this value dropped to 35 %, indicating that in approximately 65 % of all enzymes the same function evolved in two or more non-homologous proteins.For these isofunctional enzymes, structural similarity of the catalytic sites may help to predict function, because neither high sequence similarity nor identical folds are required for a comparison. To assess the specificity of catalytic 3D poses, we compiled the redundancy-free set ENZ_SITES, which comprises 695 sites, whose composition and function are well-defined. We compared their poses with the help of the program Superpose3D and determined classification performance. If the sites were from different superfamilies, the number of true and false positive predictions was similarly high, both for a coarse and a detailed grouping of enzyme function. Moreover, classification performance did not improve drastically, if we additionally used homologous sites to predict function.ConclusionsFor a large number of enzymatic functions, dissimilar sites evolved that catalyze the same reaction and it is the individual substrate that determines the arrangement of the catalytic site and its local environment. These substrate-specific requirements turn the comparison of catalytic residues into a weak classifier for the prediction of enzyme function.Electronic supplementary materialThe online version of this article (doi:10.1186/s12859-015-0807-6) contains supplementary material, which is available to authorized users.

Extensive horizontal gene transfer, duplication, and loss of chlorophyll synthesis genes in the algae.

BackgroundTwo non-homologous, isofunctional enzymes catalyze the penultimate step of chlorophyll a synthesis in oxygenic photosynthetic organisms such as cyanobacteria, eukaryotic algae and land plants: the light-independent (LIPOR) and light-dependent (POR) protochlorophyllide oxidoreductases. Whereas the distribution of these enzymes in cyanobacteria and land plants is well understood, the presence, loss, duplication, and replacement of these genes have not been surveyed in the polyphyletic and remarkably diverse eukaryotic algal lineages.ResultsA phylogenetic reconstruction of the history of the POR enzyme (encoded by the por gene in nuclei) in eukaryotic algae reveals replacement and supplementation of ancestral por genes in several taxa with horizontally transferred por genes from other eukaryotic algae. For example, stramenopiles and haptophytes share por gene duplicates of prasinophytic origin, although their plastid ancestry predicts a rhodophytic por signal. Phylogenetically, stramenopile pors appear ancestral to those found in haptophytes, suggesting transfer from stramenopiles to haptophytes by either horizontal or endosymbiotic gene transfer. In dinoflagellates whose plastids have been replaced by those of a haptophyte or diatom, the ancestral por genes seem to have been lost whereas those of the new symbiotic partner are present. Furthermore, many chlorarachniophytes and peridinin-containing dinoflagellates possess por gene duplicates.In contrast to the retention, gain, and frequent duplication of algal por genes, the LIPOR gene complement (chloroplast-encoded chlL, chlN, and chlB genes) is often absent. LIPOR genes have been lost from haptophytes and potentially from the euglenid and chlorarachniophyte lineages. Within the chlorophytes, rhodophytes, cryptophytes, heterokonts, and chromerids, some taxa possess both POR and LIPOR genes while others lack LIPOR. The gradual process of LIPOR gene loss is evidenced in taxa possessing pseudogenes or partial LIPOR gene compliments. No horizontal transfer of LIPOR genes was detected.ConclusionsWe document a pattern of por gene acquisition and expansion as well as loss of LIPOR genes from many algal taxa, paralleling the presence of multiple por genes and lack of LIPOR genes in the angiosperms. These studies present an opportunity to compare the regulation and function of por gene families that have been acquired and expanded in patterns unique to each of various algal taxa.Electronic supplementary materialThe online version of this article (doi:10.1186/s12862-015-0286-4) contains supplementary material, which is available to authorized users.

Isofunctional Enzymes PAD1 and UbiX Catalyze Formation of a Novel Cofactor Required by Ferulic Acid Decarboxylase and 4-Hydroxy-3-polyprenylbenzoic Acid Decarboxylase

The decarboxylation of antimicrobial aromatic acids such as phenylacrylic acid (cinnamic acid) and ferulic acid by yeast requires two enzymes described as phenylacrylic acid decarboxylase (PAD1) and ferulic acid decarboxylase (FDC). These enzymes are of interest for various biotechnological applications, such as the production of chemical feedstocks from lignin under mild conditions. However, the specific role of each protein in catalyzing the decarboxylation reaction remains unknown. To examine this, we have overexpressed and purified both PAD1 and FDC from E. coli. We demonstrate that PAD1 is a flavin mononucleotide (FMN)-containing protein. However, it does not function as a decarboxylase. Rather, PAD1 catalyzes the formation of a novel, diffusible cofactor required by FDC for decarboxylase activity. Coexpression of FDC and PAD1 results in the production of FDC with high levels cofactor bound. Holo-FDC catalyzes the decarboxylation of phenylacrylic acid, coumaric acid and ferulic acid with apparent kcat ranging from 1.4-4.6 s(-1). The UV-visible and mass spectra of the cofactor indicate that it appears to be a novel, modified form of reduced FMN; however, its instability precluded determination of its structure. The E. coli enzymes UbiX and UbiD are related by sequence to PAD1 and FDC respectively and are involved in the decarboxylation of 4-hydroxy-3-octaprenylbenzoic acid, an intermediate in ubiquinone biosynthesis. We found that endogenous UbiX can also activate FDC. This implies that the same cofactor is required for decarboxylation of 4-hydroxy-3-polyprenylbenzoic acid by UbiD and suggests a wider role for this cofactor in metabolism.

Molecular phylogeny and intricate evolutionary history of the three isofunctional enzymes involved in the oxidation of protoporphyrinogen IX.

Tetrapyrroles such as heme and chlorophyll are essential for biological processes, including oxygenation, respiration, and photosynthesis. In the tetrapyrrole biosynthesis pathway, protoporphyrinogen IX oxidase (Protox) catalyzes the formation of protoporphyrin IX, the last common intermediate for the biosynthesis of heme and chlorophyll. Three nonhomologous isofunctional enzymes, HemG, HemJ, and HemY, for Protox have been identified. To reveal the distribution and evolution of the three Protox enzymes, we identified homologs of each along with other heme biosynthetic enzymes by whole-genome clustering across three domains of life. Most organisms possess only one of the three Protox types, with some exceptions. Detailed phylogenetic analysis revealed that HemG is mostly limited to γ-Proteobacteria whereas HemJ may have originated within α-Proteobacteria and transferred to other Proteobacteria and Cyanobacteria. In contrast, HemY is ubiquitous in prokaryotes and is the only Protox in eukaryotes, so this type may be the ancestral Protox. Land plants have a unique HemY homolog that is also shared by Chloroflexus species, in addition to the main HemY homolog originating from Cyanobacteria. Meanwhile, organisms missing any Protox can be classified into two groups; those lacking most heme synthetic genes, which necessarily depend on external heme supply, and those lacking only genes involved in the conversion of uroporphyrinogen III into heme, which would use a precorrin2-dependent alternative pathway. However, hemN encoding coproporphyrinogen IX oxidase was frequently found in organisms lacking Protox enzyme, which suggests a unique role of this gene other than in heme biosynthesis.

The Comparative Genomics and Phylogenomics of Leishmania amazonensis Parasite.

Leishmaniasis is an infectious disease caused by Leishmania species. Leishmania amazonensis is a New World Leishmania species belonging to the Mexicana complex, which is able to cause all types of leishmaniasis infections. The L. amazonensis reference strain MHOM/BR/1973/M2269 was sequenced identifying 8,802 codifying sequences (CDS), most of them of hypothetical function. Comparative analysis using six Leishmania species showed a core set of 7,016 orthologs. L. amazonensis and Leishmania mexicana share the largest number of distinct orthologs, while Leishmania braziliensis presented the largest number of inparalogs. Additionally, phylogenomic analysis confirmed the taxonomic position for L. amazonensis within the “Mexicana complex”, reinforcing understanding of the split of New and Old World Leishmania. Potential non-homologous isofunctional enzymes (NISE) were identified between L. amazonensis and Homo sapiens that could provide new drug targets for development.