Direct comparison of the ammo acid sequences of mlcrosomal and soluble epoxlde hydrolase superficially mdlcates that these enzymes are unrelated Both protems, however, share slgmficant sequence slmllanty to a bacterial haloalkane dehalogenase that has earlier been shown to belong to the a@ hydrolase fold family of enzymes The catalytic mechanism for the dehalogenase has been elucidated m detail [Verschueren et al (1993) Nature 363, 693-6981 and proceeds via an ester mtermedlate where the substrate IS covalently bound to the enzyme From these observations we conclude (I) that mlcrosomal and soluble epoxlde hydrolase are distantly related enzymes that have evolved from a common ancestral protein together with the haloalkane dehalogenase and a variety of other proteins specified m the present paper, (u) that these enzymes most likely belong to the a//3 hydrolase fold family of enzymes and (III) that the enzymatic epoxlde hydrolysis proceeds via a hydroxy ester mtermedlate, m contrast to the presently favoured base-catalyzed direct attack of the epoxlde by an activated water

We have analyzed amino acid sequence relationships among soluble and microsomal epoxide hydrolases, haloacid dehalogenases, and a haloalkane dehalogenase. The amino-terminal residues (1-229) of mammalian soluble epoxide hydrolase are homologous to a haloacid dehalogenase. The carboxy-terminal residues (230-554) of mammalian soluble epoxide hydrolase are homologous to haloalkane dehalogenase, to plant soluble epoxide hydrolase, and to microsomal epoxide hydrolase. The shared identity between the haloacid and haloalkane dehalogenases does not indicate relatedness between these two types of dehalogenases. The amino-terminal and carboxy-terminal homologies of mammalian soluble epoxide hydrolase to the respective dehalogenases suggests that this epoxide hydrolase, but not the soluble epoxide hydrolase of plant or the microsomal epoxide hydrolase, derives from a gene fusion. The homology of microsomal to soluble epoxide hydrolase suggests they derive from a gene duplication, probably of an ancestral bacterial (epoxide) hydrolase gene. Based on homology to haloalkane dehalogenase, the catalytic residues for the soluble and microsomal epoxide hydrolases are predicted. A nomenclature system based on divergent molecular evolution is proposed for these epoxide hydrolases.

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SummaryFive cDNAs encoding a putative soluble epoxide hydrolase (sEH) from potato were isolated and characterized. The cDNAs contained open reading frames encoding 36 kDa polypeptides which were highly homologous to the carboxy terminal region of mammalian sEH. When one of the cDNAs was expressed in a baculovirus system a soluble 38 kDa protein with epoxide hydrolase activity was produced. The recombinant enzyme hydrolyzed a commonly used dlagnostic substrata for the soluble form of mammalian EH. Inhibitor profiles of the recombinant potato and mammalian sEH were also similar. The expression of sEH in potato was found to be regulated by both developmental and environmental signals. Levels of mRNA for sEH were higher in meristematic tissue than in mature leaves. This mRNA was also observed to accumulate on wounding and application of exogenous methyl jasmonate.

In order to investigate the involvement of amino acids in the catalytic mechanism of the soluble epoxide hydrolase, different mutants of the murine enzyme were produced using the baculovirus expression system. Our results are consistent with the involvement of Asp-333 and His-523 in a catalytic mechanism similar to that of other alpha/beta hydrolase fold enzymes. Mutation of His-263 to asparagine led to the loss of approximately half the specific activity compared to wild-type enzyme. When His-332 was replaced by asparagine, 96.7% of the specific activity was lost and mutation of the conserved His-523 to glutamine led to a more dramatic loss of 99.9% of the specific activity. No activity was detectable after the replacement of Asp-333 by serine. However, more than 20% of the wild-type activity was retained in an Asp-333-->Asn mutant produced in Spodoptera frugiperda cells. We purified, by affinity chromatography, the wild-type and the Asp-333-->Asn mutant enzymes produced in Trichoplusia ni cells. We labeled these enzymes by incubating them with the epoxide containing radiolabeled substrate juvenile hormone III (JH III). The purified Asp-333-->Asn mutant bound 6% of the substrate compared to the wild-type soluble epoxide hydrolase. The mutant also showed 8% of the specific activity of the wild-type. Preincubation of the purified Asp-333-->Asn mutant at 37 degrees C (pH 8), however, led to a complete recovery of activity and to a change of isoelectric point (pI), both of which are consistent with hydrolysis of Asn-333 to aspartic acid. This intramolecular hydrolysis of asparagine to aspartic acid may explain the activity observed in this mutant. Wild-type enzyme that had been radiolabeled with the substrate was digested with trypsin. Using reverse phase-high pressure liquid chromatography, we isolated four radiolabeled peptides of similar polarity. These peptides were not radiolabeled if the enzyme was preincubated with a selective competitive inhibitor of soluble epoxide hydrolase 4-fluorochalcone oxide. This strongly suggested that these peptides contained a catalytic amino acid. Each peptide was characterized with N-terminal amino acid sequencing and electrospray mass spectrometry. All four radiolabeled peptides contained overlapping sequences. The only aspartic acid present in all four peptides and conserved in all epoxide hydrolases was Asp-333. These peptides resulted from cleavage at different trypsin sites and the mass of each was consistent with the covalent linkage of Asp-333 to the substrate.

SummaryA cDNA (1122 bp) was isolated from a cDNA library prepared from Arabidopais thaliana L. that had been subjected to drought stress for I h. The sequencing of a genomic clone corresponding to the cDNA and Sl mepplng analysis revealed that the cDNA lacked the first 6 bp from its translational start (ATG). The resulting open reading frame encodes a polypaptlde of 321 amino acids, and the calculated molecular weight of this polypeptide is 36 423 De. The deduced amino acid sequence shows a high degree of similarity to C terminal halves of those of soluble epoxide hydrolases (sEHs) of human, mouse and rat, 35.5%, 34.1% and 33.1%, respectively. The cDNA was expressed In Escherichla coil cells, and the expressed protein migrates at 40 kDa when analyzed by SDS-PAGE. The recombinant protein at 40 kDa Is much smaller than the mammalian sEH (58 kDa) but has characteristics of activity and inhibition slmller to the mammalian sEHs when assayed with the substrate trans-stilbene oxide and the inhibitors 4-fluorochalcone oxide (4FCO), (2R,3R)-3-(4-nitrophenyl) glycidol (RRNPG), and (2S,3S)-3-(4-nitrophenyl)glycidol (SSNPG), which indicates that the cDNA did encode a soluble epoxide hydrolese of A. thallana (AtsEH). Drought stress, but not heat or cold stress, slightly Increased the accu-

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