Cleavage Specificity of Enterococcus faecalis EnpA (EF1473), a Peptidoglycan Endopeptidase Related to the LytM/Lysostaphin Family of Metallopeptidases

Enterococcus faecalis EnpA (EF1473) is a 1721-residue predicted protein encoded by prophage 03 that displays similarity to the staphylolytic glycyl-glycyl endopeptidases lysostaphin and LytM. We purified a catalytically active fragment of the protein, EnpA_C, comprising residues 1374-1505 and showed that the recombinant polypeptide efficiently cleaved cross-linked muropeptides generated by muramidases, but was poorly active in intact sacculi. Analysis of the products of digestion of purified dimers by mass spectrometry indicated that EnpA_C cleaves the d-Ala-l-Ala bond formed by the d,d-transpeptidase activity of penicillin-binding proteins in the last cross-linking step of peptidoglycan synthesis. Synthetic d was identified as the minimum substrate of EnpA_C indicating that interaction of the enzyme with the donor peptide stem of cross-linked dimers is sufficient for its activity. Peptidoglycan was purified from various bacterial species and digested with mutanolysin and EnpA_C to assess enzyme specificity. EnpA_C did not cleave direct cross-links, but tolerated extensive variation in cross-bridges with respect to both their length (one to five residues) and their amino acid sequence. Recognition of the donor stem of cross-linked dimers could account for the substrate specificity of EnpA_C, which is significantly broader in comparison to endopeptidases belonging to the lysostaphin family.     

Erwinia chrysanthemi iron metabolism: the unexpected implication of the inner membrane platform within the type II secretion system

The type II secretion (T2S) system is an essential device for Erwinia chrysanthemi virulence. Previously, we reported the key role of the OutF protein in forming, along with OutELM, an inner membrane platform in the Out T2S system. Here, we report that OutF copurified with five proteins identified by matrix-assisted laser desorption ionization-time of flight analysis as AcsD, TogA, SecA, Tsp, and DegP. The AcsD protein was known to be involved in the biosynthesis of achromobactin, which is a siderophore important for E. chrysanthemi virulence. The yeast two-hybrid system allowed us to gain further evidence for the OutF-AcsD interaction. Moreover, we showed that lack of OutF produced a pleiotropic phenotype: (i) altered production of the two siderophores of E. chrysanthemi, achromobactin and chrysobactin; (ii) hypersensitivity to streptonigrin, an iron-activated antibiotic; (iii) increased sensitivity to oxidative stress; and (iv) absence of the FbpA-like iron-binding protein in the periplasmic fraction. Interestingly, outE and outL mutants also exhibited similar phenotypes, but, outD and outJ mutants did not. Moreover, using the yeast two-hybrid system, several interactions were shown to occur between components of the T2S system inner membrane platform (OutEFL) and proteins involved in achromobactin production (AcsABCDE). The OutL-AcsD interaction was also demonstrated by Ni(2+) affinity chromatography. These results fully confirm our previous view that the T2S machinery is made up of three discrete blocks. The OutEFLM-forming platform is proposed to be instrumental in two different processes essential for virulence, protein secretion and iron homeostasis.     

Evidence for a novel phosphopantetheinyl transferase domain in the polyketide synthase for enediyne biosynthesis

The polyketide synthase associated with the biosynthesis of enediyne-containing calicheamicin contains a putative phosphopantetheinyl transferase (PPTase) domain. By cloning and expressing the C-terminal region of the polyketide synthase and in vitro phosphopantetheinylation assay, we found that the PPTase domain exhibits preferred substrate specificity towards acyl and peptidyl carrier proteins in fatty acid and non-ribosomal peptide synthesis over its cognate partner. We also found evidence suggesting that the PPTase domain adopts a pseudo-trimeric structure, distinct from the pseudo-dimeric structure of type II PPTases. The results revealed a novel type of PPTase with unique structure and substrate specificity, and suggested that the polyketide synthase probably acquired the PPTase domain from a primary metabolic pathway in evolution.     

Pyruvate Formate-Lyase Interacts Directly with the Formate Channel FocA to Regulate Formate Translocation

The FNT (formate-nitrite transporters) form a superfamily of pentameric membrane channels that translocate monovalent anions across biological membranes. FocA (formate channel A) translocates formate bidirectionally but the mechanism underlying how translocation of formate is controlled and what governs substrate specificity remains unclear. Here we demonstrate that the normally soluble dimeric enzyme pyruvate formate-lyase (PflB), which is responsible for intracellular formate generation in enterobacteria and other microbes, interacts specifically with FocA. Association of PflB with the cytoplasmic membrane was shown to be FocA dependent and purified, Strep-tagged FocA specifically retrieved PflB from Escherichia coli crude extracts. Using a bacterial two-hybrid system, it could be shown that the N-terminus of FocA and the central domain of PflB were involved in the interaction. This finding was confirmed by chemical cross-linking experiments. Using constraints imposed by the amino acid residues identified in the cross-linking study, we provide for the first time a model for the FocA–PflB complex. The model suggests that the N-terminus of FocA is important for interaction with PflB. An in vivo assay developed to monitor changes in formate levels in the cytoplasm revealed the importance of the interaction with PflB for optimal translocation of formate by FocA. This system represents a paradigm for the control of activity of FNT channel proteins.     

Molecular characterization of staphyloferrin B biosynthesis in Staphylococcus aureus

Siderophores are iron-scavenging molecules produced by many microbes. In general, they are synthesized using either non-ribosomal peptide synthetase (NRPS) or NRPS-independent siderophore (NIS) pathways. Staphylococcus aureus produces siderophores, of which the structures of staphyloferrin A and staphyloferrin B are known. Recently, the NIS biosynthetic pathway for staphyloferrin A was characterized. Here we show that, in S. aureus , the previously identified sbn ( s iderophore b iosy n thesis) locus encodes enzymes required for the synthesis of staphyloferrin B, an α-hydroxycarboxylate siderophore comprised of l -2,3-diaminopropionic acid, citric acid, 1,2-diaminoethane and α-ketoglutaric acid. Staphyloferrin B NIS biosynthesis was recapitulated in vitro , using purified recombinant Sbn enzymes and the component substrates. In vitro synthesized staphyloferrin B readily promoted the growth of iron-starved S. aureus , via the ABC transporter SirABC. The SbnCEF synthetases and a decarboxylase, SbnH, were necessary and sufficient to produce staphyloferrin B in reactions containing component substrates l -2,3-diaminopropionic acid, citric acid and α-ketoglutaric acid. Since 1,2-diaminoethane was not required, this component of the siderophore arises from the SbnH-dependent decarboxylation of a 2,3-diaminoproprionic acid-containing intermediate. Liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) analyses of a series of enzyme reactions identified mass ions corresponding to biosynthetic intermediates, allowing for the first proposed biosynthetic pathway for staphyloferrin B.     

Phosphinothricin-tripeptide biosynthesis: An original version of bacterial secondary metabolism?

Streptomyces viridochromogenes Tü494 produces the herbicide phosphinothricyl-alanyl-alanine (phosphinothricin-tripeptide = PTT; bialaphos). Its bioactive moiety phosphinothricin competitively inhibits bacterial and plant glutamine synthetases. The biosynthesis of PTT includes the synthesis of the unusual amino acid N-acetyl-demethyl-phosphinothricin and a three step condensation via non-ribosomal peptide synthetases. Two characteristics within the PTT biosynthesis make it suitable to study the evolution of secondary metabolism biosynthesis. First, PTT biosynthesis represents the only known system where all peptide synthetase modules are located on separate proteins. This ‘single enzyme system’ might be an archetype of the multimodular and multienzymatic non-ribosomal peptide synthetases in evolutionary terms. The second interesting feature of PTT biosynthesis is the pathway-specific aconitase Pmi that is involved in the supply of N-acetyl-demethyl-phosphinothricin. Pmi is highly similar to the tricarboxylic acid aconitase AcnA. They share 64% identity at the DNA level and both belong to the Iron-Regulatory-Protein/AcnA family. Despite their high sequence similarity, AcnA and Pmi catalyze different reactions and are not able to substitute for each other. Thus, the enzyme pair AcnA/Pmi presents an example of the evolution of a secondary metabolite-specific enzyme from a primary metabolism enzyme.     

黄独微型块茎低温离体保存的GC/MS代谢组学分析

GC/MS检测方法采用初步探明黄独低温离体保存微型块茎的差异代谢物。与黄独微型块茎25℃离体保存相比较,黄独微型块茎4℃离体保存的差异性代谢物有丙氨酸（Alanine）、儿茶素（Catechin）、N,N-双（2-羟乙基）甲胺（N,N-Di-（2-Hydroxyethyl）-methanamine）、水杨酸（Salicylic acid）、柠檬酸（Citric acid）和山梨糖（Sorbose）等。在黄独微型块茎4℃离体保存中,丙氨酸（Alanine）参与氰基氨基酸代谢;儿茶素（Catechin）参与次生代谢产物生物合成、黄酮类化合物的生物合成和苯丙素的生物合成;水杨酸（Salicylic acid）参与多环芳烃降解、微生物在不同环境中的代谢、植物激素信号转导、次生代谢产物生物合成、二恶英降解、苯丙氨酸代谢、芳烃降解、植物激素生物合成、铁载体组非核糖体肽合成和苯丙素的生物合成等。柠檬酸（Citric acid）参与来自鸟氨酸、赖氨酸和烟酸的生物碱生物合成、组氨酸和嘌呤的生物碱生物合成、微生物在不同环境中的代谢、植物次生代谢产物的生物合成、2-氧代羧酸代谢、萜类和类固醇的生物合成、原核生物固碳途径、次生代谢产物生物合成、来自莽草酸途径的生物碱生物合成、来自萜类化合物和聚酮的生物碱生物合成、柠檬酸循环（TCA循环）、植物激素生物合成、乙醛酸和二羧酸代谢、双组分系统、苯丙素的生物合成以及来自鸟氨酸,赖氨酸和烟酸的生物碱生物合成等。黄独低温离体保存微型块茎差异代谢物的初步发现为进一步了解其低温离体保存的分子机制奠定了基础,也为低温离体保存黄独微型块茎的破除休眠以及其后续萌发提供了理论依据。     

Characterisation of Desulfovibrio vulgaris haem b synthase,a radical SAM family member

An alternative route for haem b biosynthesis is operative in sulfate-reducing bacteria of the Desulfovibrio genus and in methanogenic Archaea. This pathway diverges from the canonical one at the level of uroporphyrinogen III and progresses via a distinct branch, where sirohaem acts as an intermediate precursor being converted into haem b by a set of novel enzymes, named the alternative haem biosynthetic proteins (Ahb). In this work, we report the biochemical characterisation of the Desulfovibrio vulgaris AhbD enzyme that catalyses the last step of the pathway. Mass spectrometry analysis showed that AhbD promotes the cleavage of S-adenosylmethionine (SAM) and converts iron-coproporphyrin III via two oxidative decarboxylations to yield haem b, methionine and the 5′-deoxyadenosyl radical. Electron paramagnetic resonance spectroscopy studies demonstrated that AhbD contains two [4Fe–4S]^2 +/1 + centres and that binding of the substrates S-adenosylmethionine and iron-coproporphyrin III induces conformational modifications in both centres. Amino acid sequence comparisons indicated that D. vulgaris AhbD belongs to the radical SAM protein superfamily, with a GGE-like motif and two cysteine-rich sequences typical for ligation of SAM molecules and iron-sulfur clusters, respectively. A structural model of D. vulgaris AhbD with putative binding pockets for the iron-sulfur centres and the substrates SAM and iron-coproporphyrin III is discussed.     

A Novel Mechanism for Azoreduction

Azoreductases are important due to their ability to activate anti-inflammatory azo pro-drugs and to detoxify azo dyes. Three genes encoding azoreductases have been identified in Pseudomonas aeruginosa. We describe here a comparison of the three enzymes. The pure recombinant proteins each have a distinct substrate specificity profile against a range of azo substrates. Using the structure of P. aeruginosa azoreductase (paAzoR) 1 and the homology models of paAzoR2 and paAzoR3, we have identified residues important for substrate specificity. We have defined a novel flavin mononucleotide binding cradle, which is a recurrent motif in many flavodoxin-like proteins. A novel structure of paAzoR1 with the azo pro-drug balsalazide bound within the active site was determined by X-ray crystallography and demonstrates that the substrate is present in a hydrazone tautomer conformation. We propose that the structure with balsalazide bound represents an enzyme intermediate and, together with the flavin mononucleotide binding cradle, we propose a novel catalytic mechanism.     

4-Nitrotryptophan is a substrate for the non-ribosomal peptide synthetase TxtB in the thaxtomin A biosynthetic pathway

Thaxtomin A, a cyclic dipeptide with a nitrated tryptophan moiety, is a phytotoxic pathogenicity determinant in scab-causing Streptomyces species that inhibits cellulose synthesis by an unknown mechanism. Thaxtomin A is produced by the action of two non-ribosomal peptide synthetase modules (TxtA and TxtB) and a complement of modifying enzymes, although the order of biosynthesis has not yet been determined. Analysis of a thaxtomin dual module knockout mutant and single module knockout mutants revealed that 4-nitrotryptophan is an intermediate in thaxtomin A biosynthesis prior to backbone assembly. The 4-nitrotryptophan represents a novel substrate for non-ribosomal peptide synthetases. Through identification of N -methyl-4-nitrotryptophan in a single module knockout and the use of adenylation domain specificity prediction software, TxtB was identified as the non-ribosomal peptide synthetase module specific for 4-nitrotryptophan.     

Characterization and Fungal Inhibition Activity of Siderophore from Wheat Rhizosphere Associated Acinetobacter calcoaceticus Strain HIRFA32

Acinetobacter calcoaceticus HIRFA32 from wheat rhizosphere produced catecholate type of siderophore with optimum siderophore (ca. 92 % siderophore units) in succinic acid medium without FeSO₄ at 28 °C and 24 h of incubation. HPLC purified siderophore appeared as pale yellow crystals with molecular weight [M⁺¹] m/z 347.18 estimated by LCMS. The structure elucidated by ¹H NMR, ¹³C NMR, HMQC, HMBC, NOESY and decoupling studies, revealed that siderophore composed of 2,3-dihydroxybenzoic acid with hydroxyhistamine and threonine as amino acid subunits. In vitro study demonstrated siderophore mediated mycelium growth inhibition (ca. 46.87 ± 0.5 %) of Fusarium oxysporum. This study accounts to first report on biosynthesis of acinetobactin-like siderophore by the rhizospheric strain of A. calcoaceticus and its significance in inhibition of F. oxysporum.     

Functional and structural analysis of the siderophore synthetase AsbB through reconstitution of the petrobactin biosynthetic pathway from Bacillus anthracis

Petrobactin, a mixed catechol-carboxylate siderophore, is required for full virulence of Bacillus anthracis, the causative agent of anthrax. The asbABCDEF operon encodes the biosynthetic machinery for this secondary metabolite. Here, we show that the function of five gene products encoded by the asb operon is necessary and sufficient for conversion of endogenous precursors to petrobactin using an in vitro system. In this pathway, the siderophore synthetase AsbB catalyzes formation of amide bonds crucial for petrobactin assembly through use of biosynthetic intermediates, as opposed to primary metabolites, as carboxylate donors. In solving the crystal structure of the B. anthracis siderophore biosynthesis protein B (AsbB), we disclose a three-dimensional model of a nonribosomal peptide synthetase-independent siderophore (NIS) synthetase. Structural characteristics provide new insight into how this bifunctional condensing enzyme can bind and adenylate multiple citrate-containing substrates followed by incorporation of both natural and unnatural polyamine nucleophiles. This activity enables formation of multiple end-stage products leading to final assembly of petrobactin. Subsequent enzymatic assays with the nonribosomal peptide synthetase-like AsbC, AsbD, and AsbE polypeptides show that the alternative products of AsbB are further converted to petrobactin, verifying previously proposed convergent routes to formation of this siderophore. These studies identify potential therapeutic targets to halt deadly infections caused by B. anthracis and other pathogenic bacteria and suggest new avenues for the chemoenzymatic synthesis of novel compounds.     

Insights into Substrate Specificity of Geranylgeranyl Reductases Revealed by the Structure of Digeranylgeranylglycerophospholipid Reductase, an Essential Enzyme in the Biosynthesis of Archaeal Membrane Lipids

Archaeal membrane lipids consist of branched, saturated hydrocarbons distinct from those found in bacteria and eukaryotes. Digeranylgeranylglycerophospholipid reductase (DGGR) catalyzes the hydrogenation process that converts unsaturated 2,3-di-O-geranylgeranylglyceryl phosphate to saturated 2,3-di-O-phytanylglyceryl phosphate as a critical step in the biosynthesis of archaeal membrane lipids. The saturation of hydrocarbon chains confers the ability to resist hydrolysis and oxidation and helps archaea withstand extreme conditions. DGGR is a member of the geranylgeranyl reductase family that is also widely distributed in bacteria and plants, where the family members are involved in the biosynthesis of photosynthetic pigments. We have determined the crystal structure of DGGR from the thermophilic heterotrophic archaea Thermoplasma acidophilum at 1.6 Å resolution, in complex with flavin adenine dinucleotide (FAD) and a bacterial lipid. The DGGR structure can be assigned to the well-studied, p-hydroxybenzoate hydroxylase (PHBH) SCOP superfamily of flavoproteins that include many aromatic hydroxylases and other enzymes with diverse functions. In the DGGR complex, FAD adopts the IN conformation (closed) previously observed in other PHBH flavoproteins. DGGR contains a large substrate-binding site that extends across the entire ligand-binding domain. Electron density corresponding to a bacterial lipid was found within this cavity. The cavity consists of a large opening that tapers down to two, narrow, curved tunnels that closely mimic the shape of the preferred substrate. We identified a sequence motif, PxxYxWxFP, that defines a specificity pocket in the enzyme and precisely aligns the double bond of the geranyl group with respect to the FAD cofactor, thus providing a structural basis for the substrate specificity of geranylgeranyl reductases. DGGR is likely to share a common mechanism with other PHBH enzymes in which FAD switches between two conformations that correspond to the reductive and oxidative half cycles. The structure provides evidence that substrate binding likely involves conformational changes, which are coupled to the two conformational states of the FAD.     

Biosynthetic analysis of the petrobactin siderophore pathway from Bacillus anthracis

The asbABCDEF gene cluster from Bacillus anthracis is responsible for biosynthesis of petrobactin, a catecholate siderophore that functions in both iron acquisition and virulence in a murine model of anthrax. We initiated studies to determine the biosynthetic details of petrobactin assembly based on mutational analysis of the asb operon, identification of accumulated intermediates, and addition of exogenous siderophores to asb mutant strains. As a starting point, in-frame deletions of each of the genes in the asb locus (asbABCDEF) were constructed. The individual mutations resulted in complete abrogation of petrobactin biosynthesis when strains were grown on iron-depleted medium. However, in vitro analysis showed that each asb mutant grew to a very limited extent as vegetative cells in iron-depleted medium. In contrast, none of the B. anthracis asb mutant strains were able to outgrow from spores under the same culture conditions. Provision of exogenous petrobactin was able to rescue the growth defect in each asb mutant strain. Taken together, these data provide compelling evidence that AsbA performs the penultimate step in the biosynthesis of petrobactin, involving condensation of 3,4-dihydroxybenzoyl spermidine with citrate to form 3,4-dihydroxybenzoyl spermidinyl citrate. As a final step, the data reveal that AsbB catalyzes condensation of a second molecule of 3,4-dihydroxybenzoyl spermidine with 3,4-dihydroxybenzoyl spermidinyl citrate to form the mature siderophore. This work sets the stage for detailed biochemical studies with this unique acyl carrier protein-dependent, nonribosomal peptide synthetase-independent biosynthetic system.     

Acute effects of insulin on the activity of mitochondrial GPAT1 in primary adipocytes

The mitochondrial enzyme 1-acyl-sn-glycerol-3-phosphate acyltransferase (mtGPAT1) catalyzes a rate-limiting step in triacylglycerol and glycerophospholipid biosynthesis, which can be modulated by protein kinases in cell free analyses. We report that treatment of primary rat adipocytes with insulin acutely affects the activity of mtGPAT1 by increasing V_MAX and K_M for the substrates glycerol-3-phosphate and palmitoyl-CoA. Proteolytic cleavage of isolated mitochondrial membranes and mass spectrometric peptide sequencing identify in vivo phosphorylation of serine 632 and serine 639 in mtGPAT1. These phosphorylation sites correspond to casein kinase-2 consensus sequences and are highly conserved in chordate animal, but not fly, fungal or plant, mtGPAT1.     

The structural basis for the narrow substrate specificity of an acetyl esterase from Thermotoga maritima

Acetyl esterases from carbohydrate esterase family 7 exhibit unusual substrate specificity. These proteins catalyze the cleavage of disparate acetate esters with high efficiency, but are unreactive to larger acyl groups. The structural basis for this distinct selectivity profile is unknown. Here, we investigate a thermostable acetyl esterase (TM0077) from Thermotoga maritima using evolutionary relationships, structural information, fluorescent kinetic measurements, and site directed mutagenesis. We measured the kinetic and structural determinants for this specificity using a diverse series of small molecule enzyme substrates, including novel fluorogenic esters. These experiments identified two hydrophobic plasticity residues (Pro228, and Ile276) surrounding the nucleophilic serine that impart this specificity of TM0077 for small, straight-chain esters. Substitution of these residues with alanine imparts broader specificity to TM0077 for the hydrolysis of longer and bulkier esters. Our results suggest the specificity of acetyl esterases have been finely tuned by evolution to catalyze the removal of acetate groups from diverse substrates, but can be modified by focused amino acid substitutions to yield enzymes capable of cleaving larger ester functionalities.     

Global substrate specificity profiling of post‐translational modifying enzymes

Enzymes that modify the proteome, referred to as post‐translational modifying (PTM) enzymes, are central regulators of cellular signaling. Determining the substrate specificity of PTM enzymes is a critical step in unraveling their biological functions both in normal physiological processes and in disease states. Advances in peptide chemistry over the last century have enabled the rapid generation of peptide libraries for querying substrate recognition by PTM enzymes. In this article, we highlight various peptide‐based approaches for analysis of PTM enzyme substrate specificity. We focus on the application of these technologies to proteases and also discuss specific examples in which they have been used to uncover the substrate specificity of other types of PTM enzymes, such as kinases. In particular, we highlight our multiplex substrate profiling by mass spectrometry (MSP‐MS) assay, which uses a rationally designed, physicochemically diverse library of tetradecapeptides. We show how this method has been applied to PTM enzymes to uncover biological function, and guide substrate and inhibitor design. We also briefly discuss how this technique can be combined with other methods to gain a systems‐level understanding of PTM enzyme regulation and function.     

Ironing out siderophore biosynthesis: a review of non-ribosomal peptide synthetase (NRPS)-independent siderophore synthetases

Iron is required for microbial growth and proliferation. To survive in low-iron environments, some microorganisms secrete ferric iron chelators called siderophores. Siderophore biosynthesis occurs via two pathways: the non-ribosomal peptide synthetase (NRPS) pathway and the NRPS-independent siderophore (NIS) synthetase pathway. NIS enzymes function by adenylating a carboxylic acid substrate, typically citrate, or a derivative, followed by nucleophilic capture of an amine or alcohol and displacement of a citryl intermediate. In this review, we summarize recent advances in NIS biochemistry with a particular focus on structural biology and confirm the classification of NIS enzymes into Types A, A’, B, and C based on substrate specificity. Based on a phylogenetic analysis, we also propose a new subclass of NIS enzymes, Type C’, responsible for dimerization and macrocyclization of complex and substituted amine or amide intermediates. Finally, we describe the role of NIS enzymes in virulence of pathogenic microbes and discuss NIS inhibitors as potential anti-microbial agents.     

Purification and characterisation of a novel enantioselective epoxide hydrolase from Aspergillus niger M200

Purification of a novel enantioselective epoxide hydrolase from Aspergillus niger M200 has been achieved using ammonium sulphate precipitation, ionic exchange, hydrophobic interaction, and size-exclusion chromatography, in conjunction with two additional chromatographic steps employing hydroxylapatite, and Mimetic Green. The enzyme was purified 186-fold with a yield of 15%. The apparent molecular mass of the enzyme was determined to be 77 kDa under native conditions and 40 kDa under denaturing conditions, implying a dimeric structure of the native enzyme. The isoelectric point of the enzyme was estimated to be 4.0 by isoelectric focusing electrophoresis. The enzyme has a broad substrate specificity with highest specificities towards tert-butyl glycidyl ether, para-nitrostyrene oxide, benzyl glycidyl ether, and styrene oxide. Enantiomeric ratios of 30 to more than 100 were determined for the hydrolysis reactions of 4 epoxidic substrates using the purified enzyme at a reaction temperature of 10 °C. Product inhibition studies suggest that the enzyme is able to differentiate to a high degree between the (R)-diol and (S)-diol product of the hydrolysis reaction with tert-butyl glycidyl ether as the substrate. The highest activity of the enzyme was at 42 °C and a pH of 6.8. Six peptide sequences, which were obtained by cleavage of the purified enzyme with trypsin and mass spectrometry analysis of the tryptic peptides, show high similarity with corresponding sequences originated from the epoxide hydrolase from Aspergillus niger LCP 521.