Polymorphisms in Human Soluble Epoxide Hydrolase

Abstract

Human soluble epoxide hydrolase (hsEH) metabolizes a variety of epoxides to the corresponding vicinal diols. Arachidonic and linoleic acid epoxides are thought to be endogenous substrates for hsEH. Enzyme activity in humans shows high interindividual variation (e.g., 500-fold in liver) suggesting the existence of regulatory and/or structural gene polymorphisms. We resequenced each of the 19 exons of the hsEH gene (EPHX2) from 72 persons representing black, Asian, and white populations. A variety of polymorphisms was found, six of which result in amino acid substitutions. Amino acid variants were localized on the crystal structure of the mouse sEH, resulting in the prediction that at least two of these (Arg287Gln and Arg103Cys) might significantly affect enzyme function. The six variants of the hsEH cDNA corresponding to each single polymorphism and one corresponding to a double polymorphism were then constructed by site-directed mutagenesis and expressed in insect cells. As predicted, Arg287Gln and the double mutant Arg287Gln/Arg103Cys showed decreased enzyme activity using trans-stilbene oxide, trans-diphenylpropene oxide, and 14,15-epoxyeicosatrienoic acid as substrates. Lys55Arg and Cys154Tyr mutants had elevated activity for all three substrates. Detailed kinetic studies revealed that the double mutant Arg287Gln/Arg103Cys showed significant differences in K m and V max . In addition, stability studies showed that the double mutant was less stable than wild-type protein when incubated at 37°C. These results suggest that at least six hsEH variants exist in the human population and that at least four of these may influence hsEH-mediated metabolism of exogenous and endogenous epoxide substrates in vivo.Epoxide hydrolases (EC 3.3.2.3) metabolize exogenous and endogenous epoxides by hydrolyzing them to vicinal diols, which are usually less reactive and less mutagenic because of their higher hydrophilicity. sEH is one of five epoxide hydrolases (the others are hepoxilin EH, leukotriene A 4 hydrolase, cholesterol EH, and microsomal EH), which differ in molecular weight, subcellular localization, pI and substrate specificity.Previous work suggests the existence of one hsEH gene localized to chromosomal region 8p21-p12 (Larsson et al., 1995). The human sEH gene (EPHX2) consists of 19 exons encoding 555 amino acids (Sandberg and Meijer, 1996). Because the human and mouse proteins are 73% identical (Beetham et al., 1995) with 100% identity in residues forming the catalytic triad, the crystal structure of murine sEH (Argiriadi et al., 1999) is a good model for predicting structurefunction correlations of the hsEH. Each monomer of the homodimeric mouse sEH has two domains: an N-terminal domain and a C-terminal catalytic domain connected by a proline rich linker (Argiriadi et al., 1999). The catalytic mechanism involves formation of a covalent alkylenzyme ester intermediate as a result of nucleophilic attack by Asp333. This is subsequently hydrolyzed with assistance of the general base His523 in a charge relay with...

Role of epoxyeicosatrienoic acids in protecting the myocardium following ischemia/reperfusion injury

“…There is little known about the importance of this endogenous system within the heart even though the heart contains significant levels of functionally active CYP. Finally, and most importantly, recent human epidemiological evidence has identified associations between CYP2J2 polymorphisms and CAD, and EPHX2 polymorphisms and CHD [43,45,46,84]. These findings provide strong evidence supporting the notion that EETs play an important role in postischemic functional recovery and perhaps infarct size reduction [31,33,36,83].…”

“…These studies highlight the complexity of the CYP enzyme system, emphasize the role of different CYP metabolites in cardioprotection and suggest caution in the interpretation of results when using non-selective CYP inhibitors. Recent epidemiologic data suggests an association between single nucleotide polymorphisms in the genes encoding CYP2J2, CYP2C8, CYP2C9 and EPHX2 and cardiovascular disease risk in humans supporting the functional relevance of the CYP epoxygenase pathway in the heart [43][44][45][46][47].…”

Protein Quaternary Structure and Expression Levels Contribute to Peroxisomal-Targeting-Sequence-1-Mediated Peroxisomal Import of Human Soluble Epoxide Hydrolase

“…47 The fragments were then subcloned into BglII/SalI sites of pAcGFP-C1 vector (BD Bioscience) to produce green fluorescent protein (GFP) fusion constructs. When expressed in mammalian cells alone, GFP is a monomeric protein (BD Bioscience), and so does not affect dimerization of hsEH.…”

Soluble Epoxide Inhibition Is Protective Against Cerebral Ischemia via Vascular and Neural Protection

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