Characterisation of a catabolic epoxide hydrolase from a Corynebacterium sp.

Abstract

The epoxide hydrolase (EH) from Corynebacterium sp. C12, which grows on cyclohexene oxide as sole carbon source, has been purified to homogeneity in two steps, involving anion exchange followed by hydrophobic-interaction chromatography. The purified enzyme is multimeric (probably tetrameric) with a subunit size of 32 140 Da. The gene encoding Corynebacterium EH was located on a 3.5-kb BamHI fragment of C12 chromosomal DNA using a DNA probe generated by PCR using degenerate primers based on the N-terminal and an internal amino acid sequence. Sequencing and database comparison of the predicted amino acid sequence of Corynebacterium EH shows that it is similar to mammalian and plant soluble EH, and the recently published sequence of epichlorohydrin EH from Agrobacterium radiobacter AD1 [Rink, R., Fennema, M., Smids, M., Dehmel, U. & Janssen, D. B. (1997) J. Biol. Chem. 272, 14 650Ϫ14 657), particularly around the catalytic site. All of these proteins belong to the A/β-hydrolase-fold family of enzymes. Similarity to the mammalian microsomal EH is weaker.Keywords : epoxide hydrolase; Corynebacterium C12 ; cyclohexene oxide ; A/β hydrolase fold.Extensive studies on the mammalian soluble (cytosolic) and chemical studies of soluble EH have demonstrated the formation microsomal epoxide hydrolases (EH), enzymes with a central of intermediate aspartate adducts , and role in detoxification (Oesch, 1973;Hammock et al., 1997), single-turnover experiments with microsomal EH (Lacourciere have established that they belong to a family of proteins that and Armstrong, 1993) have confirmed that the hydroxyl group share regions of amino acid sequence similarity with the active transferred to the product derives from the enzyme and not disite of bacterial haloalkane dehalogenase . rectly from water. These experiments also demonstrated the laThe three-dimensional structure of the latter was characterised belling of a specific aspartate residue during turnover, by H 2 18 O. by Verschueren et al. and coworkers (1993) who demonstrated Site-directed-mutagenesis studies with soluble EH (Pinot et al., that the enzyme belongs to the A/β-hydrolase-fold class of en-1995; Arand et al., 1996) have also confirmed the essential role zymes and employs a two-step mechanism involving initial for-of a histidine and aspartate pair, predicted by sequence alignmation of an aspartate ester, which is hydrolysed subsequently ment with haloalkane dehalogenase and other A/β hydrolase fold by a water molecule activated by a histidine-aspartate pair. enzymes, to be involved in promoting the hydrolytic step. While the core structure of the A/β hydrolase family, formed by Genes encoding the soluble EH have been cloned from a sheet of parallel β strands sandwiched by A helices (Ollis et mammals and plants (Beetham et al., 1995), and microsomal EH al., 1992), supports the catalytic triads of hydrolytic enzymes genes have been cloned from mammals and an insect source such as lipase and serine carboxypeptidase, a distinguishing fea- (Wojtasek and Prestwich, 1996). W...

A novel cold active esterase derived from Colombian high Andean forest soil metagenome

“…3). Nonheme peroxidases, epoxide hydrolases, dehalogenases and a haloperoxidases have the typical structure of the a/bhydrolases, lipase catalytic triad and approximately 25% identity with family V (Misawa et al 1998;Tirawongsaroj et al 2008). However, site-directed mutagenesis would be required to confirm the amino acids involved in the catalytic triad.…”

Diversity and Biocatalytic Potential of Epoxide Hydrolases Identified by Genome Analysis

“…Upon bacterial expression of these constructs, epoxide hydrolases were obtained as fusion proteins with a maltose binding protein (MalE) domain linked to the N terminus of the epoxide hydrolase domain via a polyasparagine linker and a small decapeptide. The following DNA sources served as templates: whole-cell material from an overnight culture (Bsueh, Bfueh1, Npueh1, Npueh2, and Ppueh), genomic DNA added up to 0.05 ng l Ϫ1 (Draeh, Rpaeh2, Scoeh6, and Tfueh), and plasmid DNA (AraEchA [43], Coreh [29], and MtuEphF). Primers were used at 0.4 nM in a reaction mixture with a 0.2 mM concentration of each deoxynucleoside triphosphate and 0.025 U l Ϫ1 Pwo DNA polymerase or, in the case of DNAs with high GϩC contents (see the supplemental material), 0.05 U l Ϫ1 Pfu polymerase.…”

Pseudomonas aeruginosa Cif Defines a Distinct Class of α/β Epoxide Hydrolases Utilizing a His/Tyr Ring-Opening Pair

“…The sequence of Cif is aligned with previously identified HAD-like EH homologues (O52866 [33] and BD8877 [34]), as well as putative homologues from opportunistic human pathogens, identified by NCBI accession number. Residues flanking Glu153 and His177 are shown.…”