Diversity and Biocatalytic Potential of Epoxide Hydrolases Identified by Genome Analysis

Abstract

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 ␣/␤-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.Enantiopure epoxides and vicinal diols are valuable intermediates in the synthesis of a number of pharmaceutical compounds. Epoxide hydrolases (EC 3.3.2.3) catalyze the conversion of epoxides to the corresponding diols. If they are enantioselective, they can be used to produce enantiopure epoxides by means of kinetic resolution (5). In the past, when only epoxide hydrolases from mammalian sources were known (12), the use of epoxide hydrolases in biocatalysis was hampered by their poor availability and insufficient catalytic performance, such as a low turnover rate or poor enantioselectivity. The potential for biocatalytic application of epoxide hydrolases was significantly increased with the discovery of microbial epoxide hydrolases (41), which are easier to produce in large quantities. The cloning and overexpression of several enantioselective epoxide hydrolases, e.g., from Agrobacterium radiobacter (35), Aspergillus niger (3), and potato plants (40), not only facilitated large-scale production of these enzymes but also made it possible to improve their biocatalytic properties by site-directed or random mutagenesis (34,36,43).Since many microbial genome sequences are available in the public domain, it is useful to screen these databases for genes that might encode new enzymes with interesting properties.Novel epoxide hydrolases can be identified by performing a BLAST search of the genomic databases, using amino acid sequences of known epoxide hyd...

Crystal Structure of the Cystic Fibrosis Transmembrane Conductance Regulator Inhibitory Factor Cif Reveals Novel Active-Site Features of an Epoxide Hydrolase Virulence Factor

“…Conventionally, EHs have two tyrosine residues positioned above the nucleophile to assist in the initial attack step (38), and a recent phylogenetic analysis showed that all experimentally confirmed EHs utilize two tyrosines during epoxide ring opening (52). These generate an oxyanion hole for initial attack and facilitate the opening of the epoxide ring (55).…”

“…Within the ␣/␤ hydrolase family, there are three enzyme classes that utilize an aspartic acid as the nucleophile in the catalytic mechanism (10,52). These are the EHs, the HADs, and the haloalkane dehalogenases (HLDs).…”

“…strain FA1 (FAc-DEX FA1), with a Z score of 35.7 and an rmsd of 1.9 Å. FAc-DEX FA1 is an ␣/␤ hydrolase that defluorinates fluoroacetate to form glycolic acid (29), thus detoxifying this compound, which can form a potent aconitase inhibitor if ingested (24). As a family, the HADs are closely related in sequence to the EHs and also utilize an aspartic acid side chain for initial nucleophilic attack (52). Like ArEH, FAc-DEX FA1 has a catalytic triad that aligns with that of Cif.…”

“…Sequence alignment of Cif with known EHs revealed substitutions in several conserved EH motifs thought to be required for the formation of the enzyme active site. Furthermore, sequence relationships suggest that several haloacetate dehalogenases (HADs) previously had been misclassified as EHs, all of which cluster in the EH subgroup that includes Cif (52). As a basis for a detailed structure-function analysis of Cif, we have determined its crystal structure and assayed its activity against a variety of candidate substrates for EHs and related ␣/␤ hydrolases both for wild-type protein and for a mutation that abrogates Cif's host cell activity.…”

Discovery and characterization of thermophilic limonene‐1,2‐epoxide hydrolases from hot spring metagenomic libraries

“…Although the substrate specificity shown by Rv2740 is fairly different from that of Re-LEH, the available information about this enzyme is quite preliminary and no real application for its synthetic exploitation has been made. Sequence-based mining of (meta)genomes for novel EHs has been limited so far to the discovery of enzymes showing an a/b-hydrolase fold [16][17][18].…”