Molecular cloning and functional expression of rat liver cytosolic acetyl-CoA hydrolase.

A cytosolic acetyl-CoA hydrolase (CACH) was purified from rat liver to homogeneity by a new method using Triton X-100 as a stabilizer. We digested the purified enzyme with an endopeptidase and determined the N-terminal amino-acid sequences of the two proteolytic fragments. From the sequence data, we designed probes for RT-PCR, and amplified CACH cDNA from rat liver mRNA. The CACH cDNA contains a 1668-bp ORF encoding a protein of 556 amino-acid residues (62 017 Da). Recombinant expression of the cDNA in insect cells resulted in overproduction of functional acetyl-CoA hydrolase with comparable acyl-CoA chain-length specificity and Michaelis constant for acetyl-CoA to those of the native CACH. Database searching shows no homology to other known proteins, but reveals high similarities to two mouse expressed sequence tags (91% and 93% homology) and human mRNA for KIAA0707 hypothetical protein (50% homology) of unknown function.     

Human microsomal xenobiotic epoxide hydrolase. Complementary DNA sequence, complementary DNA-directed expression in COS-1 cells, and chromosomal localization

A lambda gt11 expression library constructed from human liver mRNA was screened with an antibody against human microsomal xenobiotic epoxide hydrolase. The clone pheh32 contains an insert of 1742 base pairs with an open reading frame coding for a protein of 455 amino acids with a calculated Mr of 52,956. The nucleotide sequence is 77% similar to the previously reported rat xenobiotic epoxide hydrolase cDNA sequence. The deduced amino acid sequence of the human epoxide hydrolase is 80% similar to the previously reported rabbit and 84% similar to the deduced rat protein sequence. The NH2-terminal amino acids deduced from the human xenobiotic epoxide hydrolase cDNA are identical to the published 19 NH2-terminal amino acids of the purified human xenobiotic epoxide hydrolase protein. Northern blot analysis revealed a single mRNA band of 1.8 kilobases. Southern blot analysis indicated that there is only one gene copy/haploid genome. The human xenobiotic epoxide hydrolase gene was assigned to the long arm of human chromosome 1. Several restriction fragment length polymorphisms were observed with the human epoxide hydrolase cDNA. pheh32 was expressed as enzymatically active protein in cultured monkey kidney cells (COS-1).     

Expression of human liver epoxide hydrolase in Saccharomyces pombe.

Human liver microsomal epoxide hydrolase cDNA was inserted into the yeast expression vector pEVP11. The resulting recombinant plasmid was introduced into Saccharomyces pombe. The epoxide hydrolase protein and enzymic activity was subsequently expressed and identified in the 105,000 g pellet after centrifugal fractionation of homogenized yeast cells. This method will provide a useful source of human liver epoxide hydrolase, avoiding the problems of obtaining human tissue.     

Brassica napus soluble epoxide hydrolase (BNSEH1).

Epoxide hydrolase (EC 3.3.2.3) in plants is involved in the metabolism of epoxy fatty acids and in mediating defence responses. We report the cloning of a full-length epoxide hydrolase cDNA (BNSEH1) from oilseed rape (Brassica napus) obtained by screening of a cDNA library prepared from methyl jasmonate induced leaf tissue, and the 5'-RACE technique. The cDNA encodes a soluble protein containing 318 amino acid residues. The identity on the protein level is 85% to an Arabidopsis soluble epoxide hydrolase (sEH) and 50-60% to sEHs cloned from other plants. A 5 x His tag was added to the N-terminus of the BNSEH1 and the construct was over-expressed in the yeast Pichia pastoris. The recombinant protein was recovered at high levels after Ni-agarose chromatography of lysed cell extracts, had a molecular mass of 37 kDa on SDS/PAGE and cross-reacted on Western blots with antibodies raised to a sEH from Arabidopsis thaliana. BNSEH1 was shown to be a monomer by gel filtration analysis. The activity was low towards cis-stilbene oxide but much higher using trans-stilbene oxide as substrate with Vmax of 0.47 micro mol.min.mg-1, Km of 11 micro m and kcat of 0.3 s-1. The optimum temperature of the recombinant enzyme was 55 degrees C and the optimum pH 6-7 for trans-stilbene oxide hydrolysis. The isolation of BNSEH1 will facilitate metabolic engineering of epoxy fatty acid metabolism for functional studies of resistance and seed oil modification in this important oilcrop.     

Identification and characterization of an ovary-selective isoform of epoxide hydrolase

A novel ovary-selective gene was identified by suppression subtractive hybridization (SSH) that is expressed only during the mouse periovulatory phase of a stimulated estrous cycle. Analysis of the protein encoded by the full-length cDNA revealed that the majority of it, with the exception of the first 44 amino acids, matched soluble epoxide hydrolase (Ephx2, referred to as Ephx2A). By comparing the cDNA sequence of this newly identified variant of soluble epoxide hydrolase (referred to as Ephx2B) with the mouse genome database, an exon was identified that corresponds to its unique 5' cDNA sequence. Through the use of an Ephx2A-specific probe, Northern blot analysis revealed that this mRNA was also expressed in the ovary, with the highest level of expression occurring during the luteal phase of a stimulated estrous cycle. In situ hybridization revealed that Ephx2B mRNA expression was restricted to granulosa cells of preovulatory follicles. Ephx2A mRNA expression, however, was detectable in follicles at different stages of development, as well as in the corpus luteum. Total ovarian epoxide hydrolase activity increased following the induction of follicular development, and remained elevated through the periovulatory and postovulatory stages of a stimulated estrous cycle. The change in enzyme activity paralleled the combined mRNA expression profiles for both Ephx2A and Ephx2B, thus supporting a role for epoxide metabolism in ovarian function.     

Isolation and characterization of the epoxide hydrolase-encoding gene from Xanthophyllomyces dendrorhous

The epoxide hydrolase (EH)-encoding gene (EPH1) from the basidiomycetous yeast Xanthophyllomyces dendrorhous was isolated. The genomic sequence has a 1,236-bp open reading frame which is interrupted by eight introns that encode a 411-amino-acid polypeptide with a calculated molecular mass of 46.2 kDa. The amino acid sequence is similar to that of microsomal EH and belongs to the alpha/beta hydrolase fold family. The EPH1 gene was not essential for growth of X. dendrorhous in rich medium under laboratory conditions. The Eph1-encoding cDNA was functionally expressed in Escherichia coli. A sixfold increase in specific activity was observed when we used resting cells rather than X. dendrorhous. The epoxides 1,2-epoxyhexane and 1-methylcyclohexene oxide were substrates for both native and recombinant Eph1. Isolation and characterization of the X. dendrorhous EH-encoding gene are essential steps in developing a yeast EH-based epoxide biotransformation system.     

Molecular and biochemical characterization of juvenile hormone epoxide hydrolase from the silkworm, Bombyx mori

One major route of insect juvenile hormone (JH) degradation is epoxide hydration by JH epoxide hydrolase (JHEH). A full-length cDNA (1536 bp) encoding a microsomal JHEH was isolated from the silkworm, Bombyx mori. Bommo-JHEH cDNA contains an open reading frame encoding a 461-amino acid protein (52 kDa), which reveals a high degree of similarity to the previously reported insect JHEHs. The residues Tyr298, Tyr373, and the HGWP motif corresponding to the oxyanion hole of JHEHs and the residues Asp227, His430, and Glu403 in the catalytic triad are well conserved in Bommo-JHEH. Bommo-JHEH was highly expressed in the fat body, where its mRNA expression pattern was in contrast to the pattern of hemolymph levels of JH during the larval development, suggesting that Bommo-JHEH plays an important role in JH degradation. Recombinant Bommo-JHEH (52 kDa) expressed in Sf9 insect cells was membrane-bound and had a high level of enzyme activity (300-fold over the control activity). This Bommo-JHEH study provides a better understanding of how JH levels are regulated in the domesticated silkworm.     

The effects of metyrapone, chalcone epoxide, benzil, clotrimazole and related compounds on the activity of microsomal epoxide hydrolase in situ, in purified form and in reconstituted systems towards different substrates

The influence of metyrapone, chalcone epoxide, benzil and clotrimazole on the activity of microsomal epoxide hydrolase towards styrene oxide, benzo[a]pyrene 4,5-oxide, estroxide and androstene oxide was investigated. The studies were performed using liver microsomes from rats, rabbits, mice and humans; epoxide hydrolase purified from rat liver microsomes to apparent homogeneity; and the purified enzyme incorporated into liposomes composed of egg-yolk phosphatidylcholine or total rat liver microsomal lipids. All four effectors were found to activate the hydrolysis of styrene oxide by epoxide hydrolase in situ in rat liver microsomal membranes, in agreement with earlier findings. Epoxide hydrolase activity towards styrene oxide in liver microsomes from mouse, rabbit and man was also increased by all four effectors. The most striking effect was a 680% activation by clotrimazole in rat liver microsomes. However, none of the effectors activated microsomal epoxide hydrolase more than 50% when benzo[a]pyrene 4,5-oxide, estroxide or androstene oxide was used as substrate. Indeed, clotrimazole was found to inhibit microsomal epoxide hydrolase activity towards estroxide 30-50% and towards androstene oxide 60-90%. The effects of these four compounds were found to be virtually identical in the preparations from rats, rabbits, mice and humans. The effects of metyrapone, chalcone epoxide, benzil and clotrimazole on purified epoxide hydrolase were qualitatively the same as those on epoxide hydrolase in intact microsomes, but much smaller in magnitude. These effects were increased in magnitude only slightly by incorporation of the purified enzyme into liposomes made from egg-yolk phosphatidylcholine. However, when incorporation into liposomes composed of total microsomal lipids was performed, the effects seen were essentially of the same magnitude as with intact microsomes. When the extent of activation was plotted against effector concentration, three different patterns were found with different effectors. Activation of epoxide hydrolase activity towards styrene oxide by clotrimazole was found to be uncompetitive with the substrate and highly structure specific. On the other hand, inhibition of epoxide hydrolase activity towards androstene oxide by clotrimazole was found to be competitive in microsomes. It is concluded that the marked effects of these four modulators on microsomal epoxide hydrolase activity are due to an interaction with the enzyme protein itself, but that the presence of total microsomal phospholipids allows the maximal expression leading to similar degrees of modulation as those observed in intact microsomes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Carboxyl group of residue Asp647 as possible proton donor in catalytic reaction of alpha-glucosidase from Schizosaccharomyces pombe.

cDNA encoding Schizosaccharomyces pombe alpha-glucosidase was cloned from a library constructed from mRNA of the fission yeast, and expressed in Saccharomyces cerevisiae. The cDNA, 4176 bp in length, included a single ORF composed of 2910 bp encoding a polypeptide of 969 amino-acid residues with M(r) 106 138. The deduced amino-acid sequence showed a high homology to those of alpha-glucosidases from molds, plants and mammals. Therefore, the enzyme was categorized into the alpha-glucosidase family II. By site-directed mutagenesis, Asp481, Glu484 and Asp647 residues were confirmed to be essential in the catalytic reaction. The carboxyl group (-COOH) of the Asp647 residue was for the first time shown to be the most likely proton donor acting as the acid catalyst in the alpha-glucosidase of family II. Studies with the chemical modifier conduritol B epoxide suggested that the carboxylate group (-COO-) of the Asp481 residue was the catalytic nucleophile, although the role of the Glu484 residue remains obscure.     

Cytosolic and microsomal epoxide hydrolases are immunologically distinguishable from each other in the rat and mouse

Antibodies raised to homogeneous rat liver microsomal epoxide hydrolase were used to distinguish microsomal epoxide hydrolase from epoxide hydrolase of cytosolic origin in mice and rats. Using double diffusion analysis in agarose gels, we show that anti-rat liver microsomal epoxide hydrolase forms a single precipitin line with solubilized microsomes from rat and mouse liver, but no reaction is seen with the corresponding cytosolic fractions. Rat or mouse microsomal epoxide hydrolase activity (using benzo[a]pyrene 4,5-oxide as substrate) can be completely precipitated out of solubilized preparations by the antibody, which is equipotent against rat and mouse microsomal epoxide hydrolase. No precipitation of cytosolic hydrolase activity (using trans-beta-ethyl styrene oxide as substrate) is seen with any concentration of the antibody tested. Thus, in the case of microsomal epoxide hydrolase, extensive immunological cross-reactivity exists between the two species, rat and mouse. In contrast, no cross-reactivity is detectable between cytosolic and microsomal epoxide hydrolase, even when enzymes from the same species are compared. We conclude that microsomal and cytosolic epoxide hydrolase activities represent distinct and immunologically non-cross-reactive protein species.     

Purification of hepoxilin epoxide hydrolase from rat liver

Hepoxilin epoxide hydrolase activity was demonstrated in rat liver cytosol using as substrate [1-14C] hepoxilin A3, a recently described hydroxy epoxide derivative of arachidonic acid. The enzyme was isolated and purified to apparent homogeneity using conventional chromatographic procedures resulting in 41-fold purification. The protein eluted during isoelectric focusing at a pI in the 5.3-5.4 range. The specific activity of the purified protein was 1.2 ng/microgram protein/20 min at 37 degrees C. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, under denaturing conditions, a molecular mass value of 53 kDa was observed. Using native polyacrylamide gel electrophoresis, enzyme activity corresponded to the main protein band. The purified protein used hepoxilin A3 as preferred substrate converting it to trioxilin A3. The enzyme was marginally active toward other epoxides such as leukotriene A4 and styrene oxide. The Mr, pI, and substrate specificity of the hepoxilin epoxide hydrolase indicate that this enzyme is different from the recently reported leukotriene A4 hydrolase from human erythrocytes and rat and human neutrophils and constitutes a hitherto undescribed form of epoxide hydrolase with specificity toward hepoxilin A3. Tissue screening for enzyme activity revealed that this enzyme is ubiquitous in the rat.     

Nuclear metabolism. II. Further studies on epoxide hydrolase activity

Apparent K_m- and V_max-values of nuclear styrene 7,8-oxide hydrolase were determined at different protein concentrations. In the protein concentrations range used no significant differences in the apparent K_m-values were observed. The influence of the incubation with different modifiers (i.e. SKF-525A, metyrapone, 1,2-epoxy-3,3,3 trichloropropane, cyclohexene oxide) at two different concentrations on this enzyme activity was also determined. Cyclohexene oxide and 1,2-epoxy-3,3,3-trichloropropane, two well known inhibitors of the microsomal epoxide hydrolase(s) caused a marked inhibition, metyrapone had a strong activating effect whereas SKF-525A had no effect. In vivo pretreatment with phenobarbital significantly induced the nuclear epoxide hydrolase whereas β-naphthoflavone caused a lower degree of induction. This pattern is quantitatively different but qualitatively very similar to the microsomal one. Moreover a toxifying to detoxifying enzymatic activity balance is attempted for the metabolization of the alkenic double bond of styrene, taking into account the ratio between the styrene monooxygenase (toxifying enzyme) and the styrene 7,8-oxide hydrolase (detoxifying enzyme) after the above mentioned pretreatments, both in the microsomal and nuclear fractions.     

Ingestion of soybean epoxide oil. Effects on monooxygenases, epoxide hydrolase and activities of UDP glucuronosyltransferases in hepatic microsomes of the rat

The effect of peroxidized soybean oil in the diet of male Wistar rats was studied on hepatic drug metabolizing enzymes and their phenobarbital induction and compared to that of natural soybean diet in the same conditions. No hepatomegaly or increase in serum transaminases occurred, however growth was inhibited after ingestion of peroxidized soybean oil. In addition, the protein biosynthesis of epoxide hydrase determined by immunochemistry was largely stimulated by this treatment; but the corresponding activity measured with benzo(a)pyrene 4-5 oxide as a substrate was increased in weaker proportions. This induction was limited to epoxide hydrolase only, since the enzymes of phase one were not affected and UDP glucuronosyltransferase activities toward group I substrates were randomly activated. The induction of epoxide hydrolase may affect only one or several isoforms of the membrane enzyme which are not necessarily specific to benzo(a)pyrene 4-5 oxide activity determination of the enzyme.     

Cloning,expression and enantioselective hydrolytic catalysis of a microsomal epoxide hydrolase from a marine fish, <Emphasis Type="Italic">Mugil cephalus</Emphasis>

The cDNA of a marine fish microsomal epoxide hydrolase (mEH) gene from Mugil cephalus was cloned by rapid amplification of cDNA ends (RACE) techniques. The homology model for the mEH of M. cephalus showed a characteristic structure of α/β-hydrolase-fold main domain with a lid domain over the active site. The characteristic catalytic triad, consisting of Asp(238), His(444), and Glu(417), was highly conserved. The cloned mEH gene was expressed in Escherichia coli and the recombinant mEH exhibited (R)-preferred hydrolysis activity toward racemic styrene oxide. We obtained enantiopure (S)-styrene oxide with a high enantiopurity of more than 99% enantiomeric excess and yield of 15.4% by batch kinetic resolution of 20 mM racemic styrene oxide.     

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

Endogenous, 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 lie 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 protein soluble epoxide hydrolase-SKI was strictly cytosolic, indicating that -SKI was not a temporally inefficient putative type 1 PTS. Import of soluble epoxide hydrolase-SKI into peroxisomes in plant cells revealed that the context of -SKI on soluble epoxide hydrolase was targeting permissible. These results show that the C-terminal -SKI is a non-functional putative type 1 PTS on soluble epoxide hydrolase and suggest the existence of distinct cytosolic and peroxisomal targeting variants of soluble epoxide hydrolase in mouse and rat.     

环氧化物水解酶产生菌的筛选及产酶条件研究

手性环氧化物至少含有一个手性碳,通过选择性开环和官能团转换,可以方便地合成许多有价值的手性化合物,在制药、农药、香料、精细化学品工业上有着极其重要的应用价值[1]。因此,手性环氧化物的合成一直是一个重要的研究课题。