Sequence similarity of mammalian epoxide hydrolases to the bacterial haloalkane dehalogenase and other related proteins Implication for the potential…

1994

Direct comparison of the amino acid sequences of microsomal and soluble epoxide hydrolase superficially indicates that these enzymes are unrelated. Both proteins, however, share significant sequence similarity to a bacterial haloalkane dehalogenase that has earlier been shown to belong to the alpha/beta hydrolase fold family of enzymes. The catalytic mechanism for the dehalogenase has been elucidated in detail [Verschueren et al. (1993) Nature 363, 693-698] and proceeds via an ester intermediate where the substrate is covalently bound to the enzyme. From these observations we conclude (i) that microsomal and soluble epoxide hydrolase are distantly related enzymes that have evolved from a co…

Differential subcellular localization of endogenous and transfected soluble epoxide hydrolase in mammalian cells: evidence for isozyme variants

1999

AbstractEndogenous, constitutive soluble epoxide hydrolase in mice 3T3 cells was localized via immunofluorescence microscopy exclusively in peroxisomes, whereas transiently expressed mouse soluble epoxide hydrolase (from clofibrate-treated liver) accumulated only in the cytosol of 3T3 and HeLa cells. When the C-terminal Ile of mouse soluble epoxide hydrolase was mutated to generate a prototypic putative type 1 PTS (-SKI to -SKL), the enzyme targeted to peroxisomes. The possibility that soluble epoxide hydrolase-SKI was sorted slowly to peroxiosmes from the cytosol was examined by stably expressing rat soluble epoxide hydrolase-SKI appended to the green fluorescent protein. Green fluorescent…

The enzymatic mechanism of epoxide hydrolysis

1995

Isolation of a Putative Hydroxyacyl Enzyme Intermediate of an Epoxide Hydrolase

1994

A putative covalent, alpha-hydroxyacyl intermediate was isolated by the brief exposure of murine soluble epoxide hydrolase to its substrate. The reaction was reversed by time and blocked by competitive inhibitors. The formation of the intermediate was dependent upon the concentration of the enzyme and was increased by incubation under acidic conditions. The structure of the intermediate was supported by microchemical methods.

Visualization of a covalent intermediate between microsomal epoxide hydrolase, but not cholesterol epoxide hydrolase, and their substrates

1997

Mammalian soluble and microsomal epoxide hydrolases have been proposed to belong to the family of alpha/beta-hydrolase-fold enzymes. These enzymes hydrolyse their substrates by a catalytic triad, with the first step of the enzymatic reaction being the formation of a covalent enzyme-substrate ester. In the present paper, we describe the direct visualization of the ester formation between rat microsomal epoxide hydrolase and its substrate. Microsomal epoxide hydrolase was precipitated with acetone after brief incubation with [1-(14)C]epoxystearic acid. After denaturing SDS gel electrophoresis the protein-bound radioactivity was detected by fluorography. Pure epoxide hydrolase and crude micros…

Expression and characterization of the recombinant juvenile hormone epoxide hydrolase (JHEH) from Manduca sexta.

1998

The cDNA of the microsomal Juvenile Hormone Epoxide Hydrolase (JHEH) from Manduca sexta was expressed in vitro in the baculovirus system. In insect cell culture, the recombinant enzyme (Ms-JHEH) was produced at a high level (100 fold over background EH catalytic activity). As expected, Ms-JHEH was localized in the microsomal fraction with a molecular mass of approximately 50 kDa. Ms-JHEH showed a substrate and inhibitor spectrum similar to the wild type JHEH isolated from eggs of M. sexta. Its enzymatic activity was the highest for Juvenile Hormone III. Ms-JHEH hydrolyzed several trans-epoxides faster than cis-epoxides. A putative hydroxyl-acyl enzyme intermediate was isolated suggesting a …