Gene_locus Report for: ratno-hyep

The microsomal epoxide hydrolase (mEH) plays a significant role in the metabolism of xenobiotics such as polyaromatic toxicants. Additionally, polymorphism studies have underlined a potential role of this enzyme in relation to a number of diseases, such as emphysema, spontaneous abortion, eclampsia, and several forms of cancer. We recently demonstrated that fatty amides, such as elaidamide, represent a new class of potent inhibitors of mEH. While these compounds are very active on recombinant mEH in vitro, they are quickly inactivated in liver extracts reducing their value in vivo. We investigated the effect of structural changes on mEH inhibition potency and microsomal stability. Results obtained indicate that the presence of a small alkyl group alpha to the terminal amide function and a thio-ether beta to this function increased mEH inhibition by an order of magnitude while significantly reducing microsomal inactivation. The addition of a hydroxyl group 9-10 carbons from the terminal amide function resulted in better inhibition potency without improving microsomal stability. The best compound obtained, 2-nonylsulfanyl-propionamide, is a competitive inhibitor of mEH with a K I of 72 nM. Furthermore, this new inhibitor significantly reduces mEH diol production in ex vivo lungs exposed to naphthalene, underlying the usefulness of the inhibitors described herein. These novel inhibitors could be valuable tools to investigate the physiological and biological roles of mEH.

The gene for the microsomal xenobiotic rat liver epoxide hydrolase has been isolated and characterized. Clones were obtained from a Wistar Furth Charon 35 genomic library by hybridization with a full-length epoxide hydrolase cDNA. The gene for the xenobiotic epoxide hydrolase is approximately 16 kilobases in length and consists of 9 exons ranging in size from 109 to 420 base pairs and 8 intervening sequences, the largest of which is 3.2 kilobases. S1-nuclease mapping, primer extension studies, and sequence analysis were used to determine the 5' cap site and the size of the first exon (170 base pairs). Regulatory sequences analogous to TATA, CCAAT, and core enhancer sequences were noted in the 5'-flanking region of the gene. The cDNA and gene for epoxide hydrolase displayed nucleotide sequence identity although they were isolated from different rat strains. Also, Southern blot analysis of restricted liver DNA from inbred Fischer 344 and Wistar Furth rat strains, and outbred Sprague-Dawley rats indicated a high degree of structural similarity for the epoxide hydrolase gene within these three strains. Only a single functional epoxide hydrolase gene was identified and no evidence of hybridization to the genes for the microsomal cholesterol epoxide hydrolase or the cytosolic epoxide hydrolase was observed. However, a pseudogene for the microsomal xenobiotic epoxide hydrolase was isolated and characterized from the genomic library.

The coding nucleotide sequence for rat liver microsomal, xenobiotic epoxide hydrolase was determined from two overlapping cDNA clones, which together contain 1750 nucleotides complementary to epoxide hydrolase mRNA. The single open reading frame of 1365 nucleotides codes for a 455 amino acid polypeptide with a molecular weight of 52,581. The deduced amino acid composition agrees well with those determined by direct amino acid analysis of the rat protein, and the amino acid sequence is 81% identical to that of rabbit epoxide hydrolase. Analysis of codon usage for epoxide hydrolase, and that of rabbit epoxide hydrolase. Analysis of codon usage for epoxide hydrolase, and comparison to codon usage for NADPH-cytochrome P-450 oxidoreductase and cytochromes P-450b, P-450d, and P-450PCN, suggest that epoxide hydrolase is more conserved than cytochromes P-450b and P-450PCN; comparison of the extent of sequence conservation for 12 homologous proteins between the rat and rabbit, including cytochrome P-450b, supports this hypothesis, and indicates that much of epoxide hydrolase is constrained to maintain its hydrophobic character, consistent with its intramembranous location. The predicted membrane topology of epoxide hydrolase delineates 6 membrane-spanning segments, less than the 8 or 10 predicted for two cytochrome P-450 isozymes; the lower number of membrane-spanning segments predicted for epoxide hydrolase correlates with its lesser dependence on the membrane for maintenance of its tertiary structure and catalytic activity.

The microsomal epoxide hydrolase (mEH) plays a significant role in the metabolism of numerous xenobiotics. In addition, it has a potential role in sexual development and bile acid transport, and it is associated with a number of diseases such as emphysema, spontaneous abortion, eclampsia, and several forms of cancer. Toward developing chemical tools to study the biological role of mEH, we designed and synthesized a series of absorbent and fluorescent substrates. The highest activity for both rat and human mEH was obtained with the fluorescent substrate cyano(6-methoxy-naphthalen-2-yl)methyl glycidyl carbonate (11). An in vitro inhibition assay using this substrate ranked a series of known inhibitors similarly to the assay that used radioactive cis-stilbene oxide but with a greater discrimination between inhibitors. These results demonstrate that the new fluorescence-based assay is a useful tool for the discovery of structure-activity relationships among mEH inhibitors. Furthermore, this substrate could also be used for the screening chemical library with high accuracy and with a Z' value of approximately 0.7. This new assay permits a significant decrease in labor and cost and also offers the advantage of a continuous readout. However, it should not be used with crude enzyme preparations due to interfering reactions.

The microsomal epoxide hydrolase (mEH) plays a significant role in the metabolism of xenobiotics such as polyaromatic toxicants. Additionally, polymorphism studies have underlined a potential role of this enzyme in relation to a number of diseases, such as emphysema, spontaneous abortion, eclampsia, and several forms of cancer. We recently demonstrated that fatty amides, such as elaidamide, represent a new class of potent inhibitors of mEH. While these compounds are very active on recombinant mEH in vitro, they are quickly inactivated in liver extracts reducing their value in vivo. We investigated the effect of structural changes on mEH inhibition potency and microsomal stability. Results obtained indicate that the presence of a small alkyl group alpha to the terminal amide function and a thio-ether beta to this function increased mEH inhibition by an order of magnitude while significantly reducing microsomal inactivation. The addition of a hydroxyl group 9-10 carbons from the terminal amide function resulted in better inhibition potency without improving microsomal stability. The best compound obtained, 2-nonylsulfanyl-propionamide, is a competitive inhibitor of mEH with a K I of 72 nM. Furthermore, this new inhibitor significantly reduces mEH diol production in ex vivo lungs exposed to naphthalene, underlying the usefulness of the inhibitors described herein. These novel inhibitors could be valuable tools to investigate the physiological and biological roles of mEH.

The laboratory rat (Rattus norvegicus) is an indispensable tool in experimental medicine and drug development, having made inestimable contributions to human health. We report here the genome sequence of the Brown Norway (BN) rat strain. The sequence represents a high-quality 'draft' covering over 90% of the genome. The BN rat sequence is the third complete mammalian genome to be deciphered, and three-way comparisons with the human and mouse genomes resolve details of mammalian evolution. This first comprehensive analysis includes genes and proteins and their relation to human disease, repeated sequences, comparative genome-wide studies of mammalian orthologous chromosomal regions and rearrangement breakpoints, reconstruction of ancestral karyotypes and the events leading to existing species, rates of variation, and lineage-specific and lineage-independent evolutionary events such as expansion of gene families, orthology relations and protein evolution.

The Golgi complex functions to posttranslationally modify newly synthesized proteins and lipids and to sort them to their sites of function. In this study, a stacked Golgi fraction was isolated by classical cell fractionation, and the protein complement (the Golgi proteome) was characterized using multidimensional protein identification technology. Many of the proteins identified are known residents of the Golgi, and 64% of these are predicted transmembrane proteins. Proteins localized to other organelles also were identified, strengthening reports of functional interfacing between the Golgi and the endoplasmic reticulum and cytoskeleton. Importantly, 41 proteins of unknown function were identified. Two were selected for further analysis, and Golgi localization was confirmed. One of these, a putative methyltransferase, was shown to be arginine dimethylated, and upon further proteomic analysis, arginine dimethylation was identified on 18 total proteins in the Golgi proteome. This survey illustrates the utility of proteomics in the discovery of novel organellar functions and resulted in 1) a protein profile of an enriched Golgi fraction; 2) identification of 41 previously uncharacterized proteins, two with confirmed Golgi localization; 3) the identification of arginine dimethylated residues in Golgi proteins; and 4) a confirmation of methyltransferase activity within the Golgi fraction.

Microsomal epoxide hydrolase (mEH) belongs to the superfamily of alpha/beta-hydrolase fold enzymes. A catalytic triad in the active centre of the enzyme hydrolyses the substrate molecules in a two-step reaction via the intermediate formation of an enzyme-substrate ester. Here we show that the mEH catalytic triad is composed of Asp226, Glu404 and His431. Replacing either of these residues with non-functional amino acids results in a complete loss of activity of the enzyme recombinantly expressed in Saccharomyces cerevisiae. For Glu404 and His431 mutants, their structural integrity was demonstrated by their retained ability to form the substrate ester intermediate, indicating that the lack of enzymic activity is due to an indispensable function of either residue in the hydrolytic step of the enzymic reaction. The role of Asp226 as the catalytic nucleophile driving the formation of the ester intermediate was substantiated by the isolation of a peptide fraction carrying the 14C-labelled substrate after cleavage of the ester intermediate with cyanogen bromide. Sequence analysis revealed that one of the two peptides within this sample harboured Asp226. Surprisingly, the replacement of Glu404 with Asp greatly increased the Vmax of the enzyme with styrene 7,8-oxide (23-fold) and 9, 10-epoxystearic acid (39-fold). The increase in Vmax was paralleled by an increase in Km with both substrates, in line with a selective enhancement of the second, rate-limiting step of the enzymic reaction. Owing to its enhanced catalytic properties, the Glu404-->Asp mutant might represent a versatile tool for the enantioselective bio-organic synthesis of chiral fine chemicals. The question of why all native mEHs analysed so far have a Glu in place of the acidic charge relay residue is discussed.

Microsomal epoxide hydrolase (MEH) catalyzes the addition of water to epoxides in a two-step reaction involving initial attack of an active site carboxylate on the oxirane to give an ester intermediate followed by hydrolysis of the ester. An efficient bacterial expression system for the enzyme from rat that facilitates the production of native and mutant enzymes for mechanistic analysis is described. Pre-steady-state kinetics of the native enzyme toward glycidyl-4-nitrobenzoates, 1, indicate the rate-limiting step in the reaction is hydrolysis of the alkyl-enzyme intermediate. The enzyme is enantioselective, turning over (2R)-1 about 10-fold more efficiently than (2S)-1, and regiospecific toward both substrates with exclusive attack at the least hindered oxirane carbon. Facile isomerization of the monoglyceride product is observed and complicates the regiochemical analysis. The D226E and D226N mutants of the protein are catalytically inactive, behavior that is consistent with the role of D226 as the active-site nucleophile as suggested by sequence alignments with other alpha/beta-hydrolase fold enzymes. The D226N mutant undergoes hydrolytic autoactivation with a half-life of 9.3 days at 37 degreesC, suggesting that the mutant is still capable of catalyzing the hydrolytic half-reaction (in this instance an amidase reaction) and confirming that D226 is in the active site. The indoylyl side chain of W227, which is in or near the active site, is not required for efficient alkylation of the enzyme or for hydrolysis of the intermediate. However, the W227F mutant does exhibit altered stereoselectivity toward (2R)-1, (2S)-1, and phenanthrene-9,10-oxide, suggesting that modifications at this position might be used to manipulate the stereo- and regioselectivity of the enzyme.

A tritiated photoaffinity labelling analogue of tamoxifen, [(2-azido-4-benzyl)-phenoxy]-N-ethylmorpholine (azido-MBPE), was used to identify the anti-oestrogen-binding site (AEBS) in rat liver tissue [Poirot, Chailleux, Fargin, Bayard and Faye (1990) J. Biol. Chem. 265, 17039-17043]. UV irradiation of rat liver microsomal proteins incubated with tritiated azido-MBPE led to the characterization of two photolabelled proteins of molecular masses 40 and 50 kDa. The amino acid sequences of proteolytic products from the 50 kDa protein were identical with those from rat microsomal epoxide hydrolase (mEH). Treatment of hepatocytes with anti-sense mRNA directed against mEH abolished AEBS in these cells. In addition we found that tamoxifen and N-morpholino-2-[4-(phenylmethyl)phenoxy]ethanamine, a selective ligand of AEBS, were potent inhibitors of the catalytic hydration of styrene oxide by mEH. However, functional overexpression of the human mEH did not significantly modify the binding capacity of [3H]tamoxifen. Taken together, these results suggest that the 50 kDa protein, mEH, is necessary but not sufficient to reconstitute AEBS.

Microsomal epoxide hydrolase (MEH) is a member of the alpha/beta-hydrolase fold family of enzymes, each of which has a catalytic triad consisting of a nucleophile involved in the formation of a covalent intermediate and a general base and charge relay carboxylate that catalyze the hydrolysis of the intermediate. The rate-limiting step in the catalytic mechanism of MEH is hydrolysis of the ester intermediate. An efficient bacterial expression system for a C-terminal hexahistidine tagged version of the native enzyme, which facilitates the isolation of mutant enzymes in which residues involved in the hydrolytic half-reaction have been altered, is described. The H431S mutant of this enzyme is efficiently alkylated by substrate to form the ester intermediate but is unable to hydrolyze the ester to complete the catalytic cycle, a fact that confirms that H431 acts as the base in the hydrolytic half-reaction. The charge relay carboxylate, which is not apparent in paired sequence alignments with other alpha/beta-hydrolase fold enzymes, is thought to be located between residues 340 and 405. A mutagenic survey of all eight Asp and Glu residues in this region reveals that only two (E376 and E404) influence the catalytic mechanism. Steady-state and pre-steady-state kinetic analyses of these residues suggest that both E404 and E376 may serve the charge relay function in the hydrolysis half-reaction. Finally, the tryptophan residue (W150), which resides in the oxyanion hole sequence HGWP, is demonstrated to contribute to the large change in intrinsic protein fluorescence observed when the enzyme is alkylated.

The microsomal epoxide hydrolase (mEH) and cytochrome P450s catalyze the sequential formation of carcinogenic metabolites. According to one algorithm for predicting the membrane topology of proteins, the human, the rabbit, and the rat mEH should adopt a type II topology. The type II topology is also predicted by a recently established neuronal network which is trained to recognize signal peptides with very high accuracy. In contrast to these predictions we find, based on N-glycosylation analysis in a cell-free and in a cellular system, that the membrane anchor of human, rat, and rabbit mEH displays a type I topology. This result is correctly predicted by the positive inside rule in which negatively charged residues, the distribution of which differs in the mEH membrane anchor of these species, have only a modulating role for the membrane topology of proteins. However, our results demonstrate that this role is not strong enough to force the mEHs into a type II topology, not even in the case of the rabbit mEH, in which the only positively charged residue in the C-terminal part of the topogenic sequence is flanked by five negatively charged residues.

In order to investigate the role of the microsomal epoxide hydrolase (mEH) in the detoxification of arene oxides in the presence of a high endogenous glutathione S-transferase (GST) activity-a situation found in several organs--we expressed the rat mEH cDNA in BHK21 Syrian hamster cells. These cells have high GST activities but contain an extremely low endogenous mEH enzyme activity. We obtained several cell clones which expressed the mEH heterologously, as determined by immunoblotting. The cell clone BHK21-mEH/Mz1 had the highest level of mEH protein. Immunofluorescence showed that the level of expression was almost homogeneous throughout the cell population. Total protein isolated from the cell line BHK21-mEH/Mz1 had a specific mEH activity of 123 pmol/min/mg protein, as determined with benzo[a]pyrene 4,5-oxide (B[a]P 4,5-oxide), which was 60 times higher than the activity in the parental cell line and eight times lower than the activity found in rat hepatocytes. However, BHK21-mEH/Mz1 cell homogenates were found to catalyze the conjugation of B[a]P 4,5-oxide to glutathione extremely well. The ratio of the GST enzyme activity to the mEH enzyme activity towards this substrate was 23 in the BHK21-mEH/Mz1 cell line. For hepatocytes this ratio was only six. Despite their already high potential to inactivate B[a]P 4,5-oxide by conjugation to glutathione, BHK21-mEH/Mz1 cells were better protected against the toxic and mutagenic effects of B[a]P 4,5-oxide than the parental cell line due to the expression of the mEH. The mEH, however, failed to protect the cells from the toxic and mutagenic effects of the bay region epoxide anti-7-methylbenz[a]anthracene-3,4-diol 1,2-oxide.

The cDNA containing the complete coding region for rat microsomal epoxide hydrolase (EC 3.3.2.3) was cloned into the expression/secretion vector pIN-III-OmpA3 and expressed in Escherichia coli strain TG1. Recombinant epoxide hydrolase was found to represent 4-9% of total bacterial protein and catalyzed the hydrolysis of styrene oxide and benzo[a]pyrene 4,5-oxide with specific activities of 421 and 734 nmol min-1 mg of epoxide hydrolase-1, respectively. Previous work implicated a histidyl residue at or near the active site of the enzyme (DuBois, G. C., Appella, E., Levin, W., Lu, A. Y. H., and Jerina, D. M. (1978) J. Biol. Chem. 253, 2932-2939). Comparison of the amino acid sequences of rat, human, and rabbit epoxide hydrolases revealed the presence of 14 conserved histidyl residues. To investigate the role of these residues in epoxide hydrolysis, site-specific mutants were generated and expressed in E. coli. Mutants H64L, H82L, H115N, H126N, H129L, H148N, H170L, H176L, H242L, H247L, H301L, H385L, K386M-H387L, delta 385-391, and H407L catalyzed the hydrolysis of benzo[a]pyrene 4,5-oxide with specific activities between 115 and 830 nmol min-1 mg-1. Mutants H431L, H431N, and H431R were all found to have activities of < 5 nmol min-1 mg-1, which is at least 150-fold less than the activity of the wild type enzyme. A Vm versus pH profile for the recombinant wild type epoxide hydrolase revealed a broad pH optimum of 6.5 to 8.5 and the presence of three ionizable groups with pKa values of 5.8 +/- 0.2, 9.2 +/- 0.1, and 9.7 +/- 0.4. The group with a pKa of 5.8 is preferentially unprotonated, while the other two groups are preferentially protonated for catalysis. We propose that histidine 431 corresponds to the group with a pKa of 5.8, while the others, with pKa values of 9.2 and 9.7 likely represent lysyl, cysteinyl, or tyrosyl residues. Thus, the data are consistent with a model where His-431 acts as a general base, abstracting a proton from water, while another residue(s), perhaps lysine, act as a general acid protonating the alkoxide anion that forms upon cleavage of the carbon-oxygen bond.

The gene for the microsomal xenobiotic rat liver epoxide hydrolase has been isolated and characterized. Clones were obtained from a Wistar Furth Charon 35 genomic library by hybridization with a full-length epoxide hydrolase cDNA. The gene for the xenobiotic epoxide hydrolase is approximately 16 kilobases in length and consists of 9 exons ranging in size from 109 to 420 base pairs and 8 intervening sequences, the largest of which is 3.2 kilobases. S1-nuclease mapping, primer extension studies, and sequence analysis were used to determine the 5' cap site and the size of the first exon (170 base pairs). Regulatory sequences analogous to TATA, CCAAT, and core enhancer sequences were noted in the 5'-flanking region of the gene. The cDNA and gene for epoxide hydrolase displayed nucleotide sequence identity although they were isolated from different rat strains. Also, Southern blot analysis of restricted liver DNA from inbred Fischer 344 and Wistar Furth rat strains, and outbred Sprague-Dawley rats indicated a high degree of structural similarity for the epoxide hydrolase gene within these three strains. Only a single functional epoxide hydrolase gene was identified and no evidence of hybridization to the genes for the microsomal cholesterol epoxide hydrolase or the cytosolic epoxide hydrolase was observed. However, a pseudogene for the microsomal xenobiotic epoxide hydrolase was isolated and characterized from the genomic library.

The coding nucleotide sequence for rat liver microsomal, xenobiotic epoxide hydrolase was determined from two overlapping cDNA clones, which together contain 1750 nucleotides complementary to epoxide hydrolase mRNA. The single open reading frame of 1365 nucleotides codes for a 455 amino acid polypeptide with a molecular weight of 52,581. The deduced amino acid composition agrees well with those determined by direct amino acid analysis of the rat protein, and the amino acid sequence is 81% identical to that of rabbit epoxide hydrolase. Analysis of codon usage for epoxide hydrolase, and that of rabbit epoxide hydrolase. Analysis of codon usage for epoxide hydrolase, and comparison to codon usage for NADPH-cytochrome P-450 oxidoreductase and cytochromes P-450b, P-450d, and P-450PCN, suggest that epoxide hydrolase is more conserved than cytochromes P-450b and P-450PCN; comparison of the extent of sequence conservation for 12 homologous proteins between the rat and rabbit, including cytochrome P-450b, supports this hypothesis, and indicates that much of epoxide hydrolase is constrained to maintain its hydrophobic character, consistent with its intramembranous location. The predicted membrane topology of epoxide hydrolase delineates 6 membrane-spanning segments, less than the 8 or 10 predicted for two cytochrome P-450 isozymes; the lower number of membrane-spanning segments predicted for epoxide hydrolase correlates with its lesser dependence on the membrane for maintenance of its tertiary structure and catalytic activity.