Epoxide Hydrolases: Structure, Function, Mechanism, and Assay
Introduction
Epoxide hydrolases (EH) are a class of enzymes that cleave oxiran derivatives to yield the corresponding diols. If viewed from the perspective of the classical phase concept of xenobiotic metabolism, EH lead a life at the interface of phase I and phase II (Fig. 1), thus representing a kind of chimera between a functionalizing and a conjugating enzyme: typical hydrolases, such as the esterases, belong to the phase I (functionalization) of xenobiotic metabolism, yet epoxide hydrolysis may as well be regarded as a conjugation with water because water is added to the molecule without splitting it into fragments.
The first EH to be characterized was the membrane‐bound mammalian microsomal epoxide hydrolase (Oesch, 1973), which plays a major role in the control of chemically reactive and thus potentially cytotoxic/genotoxic epoxides. These arise as intermediates in the metabolism of numerous xenobiotics, and their metabolic detoxification is therefore of primary importance (Guengerich, 1982). A few years later, a soluble mammalian epoxide hydrolase (sEH) was identified that was able to convert the insect signaling molecule juvenile hormone III, an oxiran derivative, to the corresponding diol (Hammock et al., 1976). In addition, this enzyme turned out to complement the substrate profile of mEH in that it hydrolyzed 1,2‐trans‐substituted epoxides efficiently, a class of compounds that is only poorly, if at all, processed by mEH (Ota and Hammock, 1980). mEH and sEH turned out to be distantly related by structure and phylogeny (Arand 1994, Lacourciere 1994, Pries 1994), and most other EH identified to date belong to this family of enzymes. Three more families of epoxide hydrolyzing enzymes of separate evolutionary origin have been identified, as will be described in more detail later. Thus, EH has expanded to a large heterogeneous group of enzymes, and these are found in all phylae of life. It appears that all complex organisms that have been analyzed so far possess actually more than one EH gene (M. Arand, unpublished observation), indicating the essential function(s) of this class of enzymes.
Section snippets
Detoxification
The first function of EH to be well understood was its role in the detoxification of genotoxic epoxides (Jerina and Daly, 1974). Many epoxides are sufficiently reactive electrophiles that can chemically attack electron‐rich structures in nucleic acids, thus leading to the formation of DNA adducts (Kim et al., 1984). Depending on the miscoding potential of those adducts, gene mutations can occur during DNA replication and, if a protooncogene or a tumor suppressor gene was the target, may
α/β Hydrolase Fold EHs
The vast majority of known EH belong to the family of α/β hydrolase fold enzymes (Barth et al., 2004). The common three‐dimensional structure of these enzymes is composed of the α/β hydrolase fold domain with a lid domain on top and an optional N‐terminal domain (Argiriadi 1999, Nardini 1999, Zou 2000). The substrate‐binding pocket is situated between the α/β hydrolase fold domain and the lid domain. Three residues of the α/β hydrolase fold domain at the interface to the lid domain constitute a
EH as Tools in Biocatalysis
Epoxides and, to some extent, vicinal diols represent interesting building blocks in the synthesis of a variety of drugs and fine chemicals (Archelas and Furstoss, 1998). Typically, the resulting molecules are optically active and exist in two enantiomeric forms. In the case of drugs, the desired therapeutic effect is usually associated with one of the two enantiomers, whereas the other one only contributes to unwanted side effects. Thus, enantioselective synthesis is highly desirable for
Recombinant Expression of EH
A variety of different expression systems have been employed for the recombinant production of EH. The first EH to be produced this way was the mammalian mEH. Although expression in Escherichia coli is possible (Bell and Kasper, 1993), Saccharomyces cerevisiae seems to be a somewhat superior expression host for this enzyme (Arand 1999, Eugster 1991, Gautier 1993), probably because it has an endoplasmic reticulum. Another, yet more cost‐intense alternative turned out to be baculovirus‐mediated
Assay of EH
Numerous assays for the analysis of specific epoxide hydrolase activities are available, and the methodologies employed cover a broad range of analytical techniques. The following section describes three representative examples.
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2018, Journal of Biological ChemistryCitation Excerpt :The ephD gene from M. smegmatis mc2155, orthologous gene from M. tuberculosis H37Rv and the EH- and SDR-encoding segments of ephDtb were individually expressed as C-terminal histidine-tagged proteins in E. coli BL21(DE3) using the expression system pET29a (Fig. 1B). Cell-free extracts from the resulting strains BL21(DE3) pET29a-ephDtb, BL21(DE3) pET29a-ephDtb-EH, BL21(DE3) pET29a-ephDtb-SDR, and BL21(DE3) pET29a-ephDsmeg together with the control strain BL21(DE3) pET29a were used as enzyme sources in an EH assay where each recombinant protein was tested for its ability to hydrolyze the generic substrate [14C]9,10-cis-epoxystearic acid (Fig. 1C) (25). TLC analyses of organic solvent-extracted lipids clearly demonstrated that cell-free extracts from the EphDtb- and EphDsmeg-producing strains, but not those from the control strain, were capable of converting 9,10-epoxystearic acid into the corresponding diol.
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