The polysialic acid-specific O-acetyltransferase OatC from Neisseria meningitidis serogroup C evolved apart from other bacterial sialate O-acetyltransferases

Neisseria meningitidis serogroup C is a major cause of bacterial meningitis and septicaemia. This human pathogen is protected by a capsule composed of alpha2,9-linked polysialic acid that represents an important virulence factor. In the majority of strains, the capsular polysaccharide is modified by O-acetylation at C-7 or C-8 of the sialic acid residues. The gene encoding the capsule modifying O-acetyltransferase is part of the capsule gene complex and shares no sequence similarities with other proteins. Here, we describe the purification and biochemical characterization of recombinant OatC. The enzyme was found as a homodimer, with the first 34 amino acids forming an efficient oligomerization domain that worked even in a different protein context. Using acetyl-CoA as donor substrate, OatC transferred acetyl groups exclusively onto polysialic acid joined by alpha2,9-linkages and did not act on free or CMP-activated sialic acid. Motif scanning revealed a nucleophile elbow motif (GXS286XGG), which is a hallmark of alpha/beta-hydrolase fold enzymes. In a comprehensive site-directed mutagenesis study, we identified a catalytic triad composed of Ser-286, Asp-376, and His-399. Consistent with a double-displacement mechanism common to alpha/beta-hydrolase fold enzymes, a covalent acetylenzyme intermediate was found. Together with secondary structure prediction highlighting an alpha/beta-hydrolase fold topology, our data provide strong evidence that OatC belongs to the alpha/beta-hydrolase fold family. This clearly distinguishes OatC from all other bacterial sialate O-acetyltransferases known so far because these are members of the hexapeptide repeat family, a class of acyltransferases that adopt a left-handed beta-helix fold and assemble into catalytic trimers.     

Diversity and biocatalytic potential of epoxide hydrolases identified by genome analysis

Epoxide hydrolases play an important role in the biodegradation of organic compounds and are potentially useful in enantioselective biocatalysis. An analysis of various genomic databases revealed that about 20% of sequenced organisms contain one or more putative epoxide hydrolase genes. They were found in all domains of life, and many fungi and actinobacteria contain several putative epoxide hydrolase-encoding genes. Multiple sequence alignments of epoxide hydrolases with other known and putative alpha/beta-hydrolase fold enzymes that possess a nucleophilic aspartate revealed that these enzymes can be classified into eight phylogenetic groups that all contain putative epoxide hydrolases. To determine their catalytic activities, 10 putative bacterial epoxide hydrolase genes and 2 known bacterial epoxide hydrolase genes were cloned and overexpressed in Escherichia coli. The production of active enzyme was strongly improved by fusion to the maltose binding protein (MalE), which prevented inclusion body formation and facilitated protein purification. Eight of the 12 fusion proteins were active toward one or more of the 21 epoxides that were tested, and they converted both terminal and nonterminal epoxides. Four of the new epoxide hydrolases showed an uncommon enantiopreference for meso-epoxides and/or terminal aromatic epoxides, which made them suitable for the production of enantiopure (S,S)-diols and (R)-epoxides. The results show that the expression of epoxide hydrolase genes that are detected by analyses of genomic databases is a useful strategy for obtaining new biocatalysts.     

J1 acylase, a glutaryl-7-aminocephalosporanic acid acylase from Bacillus laterosporus J1, is a member of the alpha/beta-hydrolase fold superfamily

J1 acylase, a glutaryl-7-aminocephalosporanic acid acylase (GCA) isolated from Bacillus laterosporus J1, has been conventionally grouped as the only member of class V GCA, although its amino acid sequence shares less than 10% identity with members of other classes of GCA. Instead, it shows higher sequence similarities with Rhodococcus sp. strain MB1 cocaine esterase (RhCocE) and Acetobacter turbidans alpha-amino acid ester hydrolase (AtAEH), members of the alpha/beta-hydrolase fold superfamily. Homology modeling and secondary structure prediction indicate that the N-terminal region of J1 acylase has an alpha/beta-hydrolase folding pattern. The catalytic triads in RhCocE and AtAEH were identified in J1 acylase as S125, D264 and H309. Mutations to alanine at these positions were found to completely inactivate the enzyme. These results suggest that J1 acylase is a member of the alpha/beta-hydrolase fold superfamily with a serine-histidine-aspartate catalytic triad.     

Identification of the catalytic residues of alpha-amino acid ester hydrolase from Acetobacter turbidans by labeling and site-directed mutagenesis

The alpha-amino acid ester hydrolase from Acetobacter turbidans ATCC 9325 is capable of hydrolyzing and synthesizing the side chain peptide bond in beta-lactam antibiotics. Data base searches revealed that the enzyme contains an active site serine consensus sequence Gly-X-Ser-Tyr-X-Gly that is also found in X-prolyl dipeptidyl aminopeptidase. The serine hydrolase inhibitor p-nitrophenyl-p'-guanidino-benzoate appeared to be an active site titrant and was used to label the alpha-amino acid ester hydrolase. Electrospray mass spectrometry and tandem mass spectrometry analysis of peptides from a CNBr digest of the labeled protein showed that Ser(205), situated in the consensus sequence, becomes covalently modified by reaction with the inhibitor. Extended sequence analysis showed alignment of this Ser(205) with the catalytic nucleophile of some alpha/beta-hydrolase fold enzymes, which posses a catalytic triad composed of a nucleophile, an acid, and a base. Based on the alignments, 10 amino acids were selected for site-directed mutagenesis (Arg(85), Asp(86), Tyr(143), Ser(156), Ser(205), Tyr(206), Asp(338), His(370), Asp(509), and His(610)). Mutation of Ser(205), Asp(338,) or His(370) to an alanine almost fully inactivated the enzyme, whereas mutation of the other residues did not seriously affect the enzyme activity. Circular dichroism measurements showed that the inactivation was not caused by drastic changes in the tertiary structure. Therefore, we conclude that the catalytic domain of the alpha-amino acid ester hydrolase has an alpha/beta-hydrolase fold structure with a catalytic triad of Ser(205), Asp(338), and His(370). This distinguishes the alpha-amino acid ester hydrolase from the Ntn-hydrolase family of beta-lactam antibiotic acylases.     

Structure of an atypical epoxide hydrolase from Mycobacterium tuberculosis gives insights into its function

Epoxide hydrolases are vital to many organisms by virtue of their roles in detoxification, metabolism and processing of signaling molecules. The Mycobacterium tuberculosis genome encodes an unusually large number of epoxide hydrolases, suggesting that they might be of particular importance to these bacteria. We report here the first structure of an epoxide hydrolase from M.tuberculosis, solved to a resolution of 2.5 A using single-wavelength anomalous dispersion (SAD) from a selenomethionine-substituted protein. The enzyme features a deep active-site pocket created by the packing of three helices onto a curved six-stranded beta-sheet. This structure is similar to a previously described limonene-1,2-epoxide hydrolase from Rhodococcus erythropolis and unlike the alpha/beta-hydrolase fold typical of mammalian epoxide hydrolases (EH). A number of changes in the mycobacterial enzyme create a wider and deeper substrate-binding pocket than is found in its Rhodococcus homologue. Interestingly, each structure contains a different type of endogenous ligand of unknown origin bound in its active site. As a consequence of its wider substrate-binding pocket, the mycobacterial EH is capable of hydrolyzing long or bulky lipophilic epoxides such as 10,11-epoxystearic acid and cholesterol 5,6-oxide at appreciable rates, suggesting that similar compound(s) will serve as its physiological substrate(s).     

Crystal structure of yeast YHR049W/FSH1, a member of the serine hydrolase family

Yhr049w/FSH1 was recently identified in a combined computational and experimental proteomics analysis for the detection of active serine hydrolases in yeast. This analysis suggested that FSH1 might be a serine-type hydrolase belonging to the broad functional alphabeta-hydrolase superfamily. In order to get insight into the molecular function of this gene, it was targeted in our yeast structural genomics project. The crystal structure of the protein confirms that it contains a Ser/His/Asp catalytic triad that is part of a minimal alpha/beta-hydrolase fold. The architecture of the putative active site and analogies with other protein structures suggest that FSH1 may be an esterase. This finding was further strengthened by the unexpected presence of a compound covalently bound to the catalytic serine in the active site. Apparently, the enzyme was trapped with a reactive compound during the purification process.     

Molecular basis of formaldehyde detoxification. Characterization of two S-formylglutathione hydrolases from Escherichia coli, FrmB and YeiG

The Escherichia coli genes frmB (yaiM) and yeiG encode two uncharacterized proteins that share 54% sequence identity and contain a serine esterase motif. We demonstrated that purified FrmB and YeiG have high carboxylesterase activity against the model substrates, p-nitrophenyl esters of fatty acids (C2-C6) and alpha-naphthyl acetate. However, both proteins had the highest hydrolytic activity toward S-formylglutathione, an intermediate of the glutathione-dependent pathway of formaldehyde detoxification. With this substrate, both proteins had similar affinity (Km = 0.41-0.43 mM), but FrmB was almost 5 times more active. Alanine replacement mutagenesis of YeiG demonstrated that Ser145, Asp233, and His256 are absolutely required for activity, indicating that these residues represent a serine hydrolase catalytic triad in this protein and in other S-formylglutathione hydrolases. This was confirmed by inspecting the crystal structure of the Saccharomyces cerevisiae S-formylglutathione hydrolase YJG8 (Protein Data Bank code 1pv1), which has 45% sequence identity to YeiG. The structure revealed a canonical alpha/beta-hydrolase fold and a classical serine hydrolase catalytic triad (Ser161, His276, Asp241). In E. coli cells, the expression of frmB was stimulated 45-75 times by the addition of formaldehyde to the growth medium, whereas YeiG was found to be a constitutive enzyme. The simultaneous deletion of both frmB and yeiG genes was required to increase the sensitivity of the growth of E. coli cells to formaldehyde, suggesting that both FrmB and YeiG contribute to the detoxification of formaldehyde. Thus, FrmB and YeiG are S-formylglutathione hydrolases with a Ser-His-Asp catalytic triad involved in the detoxification of formaldehyde in E. coli.     

Biochemical characterization of the Pseudomonas putida 3-hydroxyacyl ACP:CoA transacylase,which diverts intermediates of fatty acid de novo biosynthesis

The 3-hydroxyacyl ACP:CoA transacylase (PhaG) was recently identified in various Pseudomonas species and catalyzes the diversion of ACP thioester intermediates of fatty acid de novo biosynthesis toward the respective CoA thioesters, which serve as precursors for polyester and rhamnolipid biosynthesis. PhaG from Pseudomonas putida was overproduced in Escherichia coli as a C-terminal hexahistidine-tagged (His(6)) fusion protein in high yield. The His(6)-PhaG was purified to homogeneity by refolding of PhaG obtained from inclusion bodies, and a new enzyme assay was established. Kinetic analysis of the 3-hydroxyacyl transfer to ACP, catalyzed by His(6)-PhaG, gave K(0.5) values of 28 microm (ACP) and 65 microm (3-hydroxyacyl-CoA) considering V(max) values of 11.7 milliunits/mg and 12.4 milliunits/mg, respectively. A Hill coefficient of 1.38 (ACP) and 1.32 (3-hydroxyacyl-CoA) indicated a positive substrate cooperativity. Subcellular localization studies showed that PhaG is not attached to polyester granules and resides in the cytosol. Gel filtration chromatography analysis in combination with light scattering analysis indicated substrate-induced dimerization of the transacylase. A threading model of PhaG was developed based on the homology to an epoxide hydrolase (1cqz). In addition, the alignment with the alpha/beta-hydrolase fold region indicated that PhaG belongs to alpha/beta-hydrolase superfamily. Accordingly, CD analysis suggested a secondary structure composition of 29% alpha-helix, 22% beta-sheet, 18% beta-turn, and 31% random coil. Site-specific mutagenesis of seven highly conserved amino acid residues (Asp-60, Ser-102, His-177, Asp-182, His-192, Asp-223, His-251) was used to validate the protein model and to investigate organization of the transacylase active site. Only the D182(A/E) mutation was permissive with about 30% specific activity of the wild type enzyme. Furthermore, this mutation caused a change in substrate specificity, indicating a functional role in substrate binding. The serine-specific agent phenylmethylsulfonyl fluoride (PMSF) or the histidine-specific agent diethylpyrocarbonate (DEPC) caused inhibition of 3-hydroxyacyl transfer to holo-ACP, and the S102(A/T) or H251(A/R) PhaG mutant was incapable of catalyzing 3-hydroxyacyl transfer, suggesting that these residues are part of a catalytic triad.     

Three-dimensional structure of Erwinia chrysanthemi pectin methylesterase reveals a novel esterase active site

Most structures of neutral lipases and esterases have been found to adopt the common alpha/beta hydrolase fold and contain a catalytic Ser-His-Asp triad. Some variation occurs in both the overall protein fold and in the location of the catalytic triad, and in some enzymes the role of the aspartate residue is replaced by a main-chain carbonyl oxygen atom. Here, we report the crystal structure of pectin methylesterase that has neither the common alpha/beta hydrolase fold nor the common catalytic triad. The structure of the Erwinia chrysanthemi enzyme was solved by multiple isomorphous replacement and refined at 2.4 A to a conventional crystallographic R-factor of 17.9 % (R(free) 21.1 %). This is the first structure of a pectin methylesterase and reveals the enzyme to comprise a right-handed parallel beta-helix as seen in the pectinolytic enzymes pectate lyase, pectin lyase, polygalacturonase and rhamnogalacturonase, and unlike the alpha/beta hydrolase fold of rhamnogalacturonan acetylesterase with which it shares esterase activity. Pectin methylesterase has no significant sequence similarity with any protein of known structure. Sequence conservation among the pectin methylesterases has been mapped onto the structure and reveals that the active site comprises two aspartate residues and an arginine residue. These proposed catalytic residues, located on the solvent-accessible surface of the parallel beta-helix and in a cleft formed by external loops, are at a location similar to that of the active site and substrate-binding cleft of pectate lyase. The structure of pectin methylesterase is an example of a new family of esterases.     

Enzymatic activity and protein interactions in alpha/beta hydrolase fold proteins: moonlighting versus promiscuity

Genes coding for members of the alpha/beta hydrolase fold superfamily of proteins are present in all known genomes. Although there is no common and essential function performed by these proteins shared in all living organisms, this fold has been used for a number of diverse functions. The ancestry of both enzymatic and protein-protein interaction capability of this structural scaffold made it an important tinkering tool kit for protein function evolution. Recently, enzymes known since a long time have been found to have a second function in acting promiscuously on alternative substrates, or to be true moonlighting proteins acting also as transporters, receptors, chaperones… The reverse situation has been encountered for adhesion proteins shown to be enzymes. This review, while not exhaustive, surveys some of the best-known examples of multiple functions in alpha/beta hydrolase fold proteins.     

Human microsomal xenobiotic epoxide hydrolase. Complementary DNA sequence, complementary DNA-directed expression in COS-1 cells, and chromosomal localization

A lambda gt11 expression library constructed from human liver mRNA was screened with an antibody against human microsomal xenobiotic epoxide hydrolase. The clone pheh32 contains an insert of 1742 base pairs with an open reading frame coding for a protein of 455 amino acids with a calculated Mr of 52,956. The nucleotide sequence is 77% similar to the previously reported rat xenobiotic epoxide hydrolase cDNA sequence. The deduced amino acid sequence of the human epoxide hydrolase is 80% similar to the previously reported rabbit and 84% similar to the deduced rat protein sequence. The NH2-terminal amino acids deduced from the human xenobiotic epoxide hydrolase cDNA are identical to the published 19 NH2-terminal amino acids of the purified human xenobiotic epoxide hydrolase protein. Northern blot analysis revealed a single mRNA band of 1.8 kilobases. Southern blot analysis indicated that there is only one gene copy/haploid genome. The human xenobiotic epoxide hydrolase gene was assigned to the long arm of human chromosome 1. Several restriction fragment length polymorphisms were observed with the human epoxide hydrolase cDNA. pheh32 was expressed as enzymatically active protein in cultured monkey kidney cells (COS-1).     

Identification of a novel class in the alpha/beta hydrolase fold superfamily: the N-myc differentiation-related proteins

The alpha/beta hydrolases constitute a large protein superfamily that mainly consists of enzymes that catalyze a diverse range of reactions. These proteins exhibit the alpha/beta hydrolase fold, the essential features of which have recently been delineated: the presence of at least five parallel beta-strands, a catalytic triad in a specific order (nucleophile-acid-histidine), and a nucleophilic elbow. Because of the difficulties experimentally in identifying protein structures, we have used a Bayesian computational algorithm (PROBE) to identify the members of this superfamily based on distant sequence relationships. We found that the presence of five sequence motifs, which contain residues important for substrate binding and stabilization of the fold, are required for membership in this superfamily. The superfamily consists of at least 909 members, including the N-myc downstream regulated proteins, which are believed to be involved in cell differentiation. Unlike most of the other superfamily members, the N-myc downstream regulated proteins have never been proposed to possess the alpha/beta hydrolase fold and do not appear to be hydrolases.     

Epoxide hydrolases: their roles and interactions with lipid metabolism

The epoxide hydrolases (EHs) are enzymes present in all living organisms, which transform epoxide containing lipids by the addition of water. In plants and animals, many of these lipid substrates have potent biologically activities, such as host defenses, control of development, regulation of inflammation and blood pressure. Thus the EHs have important and diverse biological roles with profound effects on the physiological state of the host organisms. Currently, seven distinct epoxide hydrolase sub-types are recognized in higher organisms. These include the plant soluble EHs, the mammalian soluble epoxide hydrolase, the hepoxilin hydrolase, leukotriene A4 hydrolase, the microsomal epoxide hydrolase, and the insect juvenile hormone epoxide hydrolase. While our understanding of these enzymes has progressed at different rates, here we discuss the current state of knowledge for each of these enzymes, along with a distillation of our current understanding of their endogenous roles. By reviewing the entire enzyme class together, both commonalities and discrepancies in our understanding are highlighted and important directions for future research pertaining to these enzymes are indicated.     

Structure of Aspergillus niger epoxide hydrolase at 1.8 A resolution: implications for the structure and function of the mammalian microsomal class of epoxide hydrolases

Background: Epoxide hydrolases have important roles in the defense of cells against potentially harmful epoxides. Conversion of epoxides into less toxic and more easily excreted diols is a universally successful strategy. A number of microorganisms employ the same chemistry to process epoxides for use as carbon sources. Results: The X-ray structure of the epoxide hydrolase from Aspergillus niger was determined at 3.5 A resolution using the multiwavelength anomalous dispersion (MAD) method, and then refined at 1.8 A resolution. There is a dimer consisting of two 44 kDa subunits in the asymmetric unit. Each subunit consists of an alpha/beta hydrolase fold, and a primarily helical lid over the active site. The dimer interface includes lid-lid interactions as well as contributions from an N-terminal meander. The active site contains a classical catalytic triad, and two tyrosines and a glutamic acid residue that are likely to assist in catalysis. Conclusions: The Aspergillus enzyme provides the first structure of an epoxide hydrolase with strong relationships to the most important enzyme of human epoxide metabolism, the microsomal epoxide hydrolase. Differences in active-site residues, especially in components that assist in epoxide ring opening and hydrolysis of the enzyme-substrate intermediate, might explain why the fungal enzyme attains the greater speeds necessary for an effective metabolic enzyme. The N-terminal domain that is characteristic of microsomal epoxide hydrolases corresponds to a meander that is critical for dimer formation in the Aspergillus enzyme.     

The alpha/beta hydrolase fold.

We have identified a new protein fold--the alpha/beta hydrolase fold--that is common to several hydrolytic enzymes of widely differing phylogenetic origin and catalytic function. The core of each enzyme is similar: an alpha/beta sheet, not barrel, of eight beta-sheets connected by alpha-helices. These enzymes have diverged from a common ancestor so as to preserve the arrangement of the catalytic residues, not the binding site. They all have a catalytic triad, the elements of which are borne on loops which are the best-conserved structural features in the fold. Only the histidine in the nucleophile-histidine-acid catalytic triad is completely conserved, with the nucleophile and acid loops accommodating more than one type of amino acid. The unique topological and sequence arrangement of the triad residues produces a catalytic triad which is, in a sense, a mirror-image of the serine protease catalytic triad. There are now four groups of enzymes which contain catalytic triads and which are related by convergent evolution towards a stable, useful active site: the eukaryotic serine proteases, the cysteine proteases, subtilisins and the alpha/beta hydrolase fold enzymes.     

The regulation of cytosolic epoxide hydrolase in mice

The concentration of cytosolic epoxide hydrolase in untreated and clofibrate-treated mouse liver extracts was estimated by immunoblotting. Clofibrate treatment of mice was found to increase liver cytosolic epoxide hydrolase concentration by two fold, showing that the increase in cytosolic epoxide hydrolase in mouse liver after clofibrate treatment is primarily due to induction. The induced and uninduced cytosolic epoxide hydrolase, and epoxide hydrolase in the cytosolic and mitochondrial fractions were compared and found to be identical or very similar. Cytosolic epoxide hydrolases in kidney and liver were similar in molecular weight and antigenic properties.     

Isolation and characterization of the epoxide hydrolase-encoding gene from Xanthophyllomyces dendrorhous

The epoxide hydrolase (EH)-encoding gene (EPH1) from the basidiomycetous yeast Xanthophyllomyces dendrorhous was isolated. The genomic sequence has a 1,236-bp open reading frame which is interrupted by eight introns that encode a 411-amino-acid polypeptide with a calculated molecular mass of 46.2 kDa. The amino acid sequence is similar to that of microsomal EH and belongs to the alpha/beta hydrolase fold family. The EPH1 gene was not essential for growth of X. dendrorhous in rich medium under laboratory conditions. The Eph1-encoding cDNA was functionally expressed in Escherichia coli. A sixfold increase in specific activity was observed when we used resting cells rather than X. dendrorhous. The epoxides 1,2-epoxyhexane and 1-methylcyclohexene oxide were substrates for both native and recombinant Eph1. Isolation and characterization of the X. dendrorhous EH-encoding gene are essential steps in developing a yeast EH-based epoxide biotransformation system.     

Characterization of the biotin biosynthesis pathway in Saccharomyces cerevisiae and evidence for a cluster containing BIO5, a novel gene involved in vitamer uptake.

An engineered mutant of Saccharomyces cerevisiae affected in biotin biosynthesis has been isolated. This mutant allowed the characterization of a bio cluster (BIO3-4-5). We demonstrate that BIO3 (YNR058w) and BIO4 (YNR057c) encode, respectively, a 7, 8-diaminopelargonic acid aminotransferase and a dethiobiotin synthase, involved in the biotin biosynthesis pathway. A novel gene, BIO5 (YNR056c), is present immediately downstream from BIO4. This gene encodes Bio5p, a protein with 11 putative transmembrane regions. Uptake experiments performed with labeled 7-keto 8-aminopelargonic acid indicate that Bio5p is responsible for transport into the cell of 7-keto 8-aminopelargonic acid.