Substrate Report for: Cis-stilbene-oxide

Pseudomonas aeruginosa secretes an epoxide hydrolase with catalytic activity that triggers degradation of the cystic fibrosis transmembrane conductance regulator (CFTR) and perturbs other host defense networks. Targets of this CFTR inhibitory factor (Cif) are largely unknown, but include an epoxy-fatty acid. In this class of signaling molecules, chirality can be an important determinant of physiological output and potency. Here we explore the active-site chemistry of this two-step alpha/beta-hydrolase and its implications for an emerging class of virulence enzymes. In combination with hydrolysis data, crystal structures of 15 trapped hydroxyalkyl-enzyme intermediates reveal the stereochemical basis of Cif's substrate specificity, as well as its regioisomeric and enantiomeric preferences. The structures also reveal distinct sets of conformational changes that enable the active site to expand dramatically in two directions, accommodating a surprising array of potential physiological epoxide targets. These new substrates may contribute to Cif's diverse effects in vivo, and thus to the success of P. aeruginosa and other pathogens during infection.

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 open reading frame YNR064c in Saccharomyces cerevisiae encodes a protein tentatively assigned as similar to a bacterial dehalogenase. In this study we conclude that the YNR064c protein displays characteristics of an epoxide hydrolase belonging to the alpha/beta-hydrolase fold family of enzymes. Endogenous expression of the protein in S. cerevisiae was confirmed and a His-tagged variant of the protein was heterologously expressed in both Escherichia coli and Pichia pastoris for isolation and characterization. The YNR064c protein displayed low but reproducible epoxide hydrolase activity with racemic phenanthrene 9,10-oxide and trans- or cis-stilbene oxide. Phylogenetic analysis of related gene products found in various microorganisms suggested that the YNR064c protein is a member of a new subclass of alpha/beta-hydrolase fold enzymes.

Pseudomonas aeruginosa secretes an epoxide hydrolase with catalytic activity that triggers degradation of the cystic fibrosis transmembrane conductance regulator (CFTR) and perturbs other host defense networks. Targets of this CFTR inhibitory factor (Cif) are largely unknown, but include an epoxy-fatty acid. In this class of signaling molecules, chirality can be an important determinant of physiological output and potency. Here we explore the active-site chemistry of this two-step alpha/beta-hydrolase and its implications for an emerging class of virulence enzymes. In combination with hydrolysis data, crystal structures of 15 trapped hydroxyalkyl-enzyme intermediates reveal the stereochemical basis of Cif's substrate specificity, as well as its regioisomeric and enantiomeric preferences. The structures also reveal distinct sets of conformational changes that enable the active site to expand dramatically in two directions, accommodating a surprising array of potential physiological epoxide targets. These new substrates may contribute to Cif's diverse effects in vivo, and thus to the success of P. aeruginosa and other pathogens during infection.

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.

JH III esterase and JH III epoxide hydrolase (EH) in vitro activity was compared in whole body Trichoplusia ni homogenates at each stage of development (egg, larva, pupa and adult). While activity of both enzymes was detected at all ages tested, JH esterase was significantly higher than EH activity except for day three of the fifth (last) stadium (L5D3). For both enzymes, activity was highest in eggs. Adult virgin females had 4.6- and 4.0-fold higher JH esterase and EH activities, respectively, than adult virgin males. JH III metabolic activity also was measured in whole body homogenates of fifth stadium T. ni that were fed a nutritive diet (control) or starved on a non-nutritive diet of alphacel, agar and water. With larvae that were starved for 6, 28 and 52 h, EH activity per insect equivalent was 48%, 5% and 1%, respectively, of the control insects. At the same time points, JH esterase activity levels in starved T. ni were 29%, 4% and 3% of that of insects fed the nutritive diet. Selected insect hormones and xenobiotics were administered topically or orally to fifth stadium larvae for up to 52 h, and the effects on whole body EH and JH esterase activity analyzed. JH III increased the JH III esterase activity as high as 2.2-fold, but not the JH III EH activity. The JH analog, methoprene, increased both JH esterase and EH activity as high as 2.5-fold. The JH esterase inhibitor, 3-octylthio-1,1,1-trifluoropropan-2-one (OTFP), had no impact on EH activity. The epoxides trans- and cis-stilbene oxide (TSO and CSO) in separate experiments increased the EH activity approximately 2.0-fold. TSO did not alter JH esterase levels when topically applied, but oral administration reduced activity to 70% of the control at 28 h, and then increased the activity 1.8-fold at 52 h after the beginning of treatment. CSO had no effect on JH esterase activity. Phenobarbital increased EH activity by 1.9-fold, but did not change JH esterase levels. Clofibrate and cholesterol 5alpha,6alpha-epoxide had no effect on EH. JH esterase activity also was not affected by clofibrate, but cholesterol 5alpha,6alpha-epoxide reduced the JH esterase activity to 60-80% of the control. The biological significance of these results is discussed.

The open reading frame YNR064c in Saccharomyces cerevisiae encodes a protein tentatively assigned as similar to a bacterial dehalogenase. In this study we conclude that the YNR064c protein displays characteristics of an epoxide hydrolase belonging to the alpha/beta-hydrolase fold family of enzymes. Endogenous expression of the protein in S. cerevisiae was confirmed and a His-tagged variant of the protein was heterologously expressed in both Escherichia coli and Pichia pastoris for isolation and characterization. The YNR064c protein displayed low but reproducible epoxide hydrolase activity with racemic phenanthrene 9,10-oxide and trans- or cis-stilbene oxide. Phylogenetic analysis of related gene products found in various microorganisms suggested that the YNR064c protein is a member of a new subclass of alpha/beta-hydrolase fold enzymes.

Human microsomal epoxide hydrolase (EPHX1) is active in the metabolism of many potentially carcinogenic or otherwise genotoxic epoxides, such as those derived from the oxidation of polyaromatic hydrocarbons. EPHX1 is polymorphic and encodes allelic variation at least two amino acid positions, Y113H and H139R. In a number of recent molecular epidemiological investigations, EPHX1 polymorphism has been suggested as a susceptibility factor for several human diseases. To better evaluate the functional contribution of EPHX1 genetic polymorphism, we characterized the enzymatic properties associated with each of the respective variant proteins. Enzymatic profiles were evaluated with cis-stilbene oxide (cSO) and benzo[a]pyrene-4,5-epoxide (BaPO), two prototypical substrates for the hydrolase. In one series of experiments, activities of recombinant EPHX1 proteins were analyzed subsequent to their expression using the pFastbac baculovirus vector in Spodoptera frugiperda-9 (Sf9) insect cells, and purification by column chromatography. In parallel studies, EPHX1 activities were evaluated with human liver microsomes derived from individuals of known EPHX1 genotype. Using the purified protein preparations, rates of cSO and BaPO hydrolysis for the reference protein, Y113/H139, were approximately 2-fold greater than those measured with the other EPHX1 allelic variants. However, when activities were analyzed using human liver microsomal fractions, no major differences were evident in the reaction rates generated among preparations representing the different EPHX1 alleles. Collectively, these results suggest that the structural differences encoded by the Y113H and H139R variant alleles exert only modest impact on EPHX1-specific enzymatic activities in vivo.

In order to understand the roles of the epoxide hydrolases (EHs) in xenobiotic biotransformation in insects, we examined the induction of EHs by exogenous compounds in Drosophila melanogaster third instar larvae. Among the chemicals tested, clofibrate, a phenoxyacetate hypolipidermics drug, increased EH activity towards cis-stilbene oxide approximately twofold in larval whole-body homogenates. The same dose of clofibrate also induced glutathione S-transferase activity. The effect of clofibrate on EH induction was dose-dependent and the highest activity occurred with a 10% clofibrate application. Three other substrates conventionally used in EH assays (trans-stilbene oxide, trans-diphenylpropene oxide and juvenile hormone III) were poorly hydrolysed by larval homogenates, with or without clofibrate administration. Because the increased EH activity was localized predominantly in the microsomal fraction, we synthesized degenerate oligonucleotide primers with sequences corresponding to conserved regions of known microsome EHs from mammals and insects in order to isolate the gene. The 1597 bp putative cDNA of D. melanogaster microsomal EH (DmEH) obtained from a larval cDNA library encoded 463 amino acids in an open reading frame. Northern blot analysis showed that the transcription of DmEH was increased in larvae within 5 h of clofibrate treatment. Recombinant DmEH expressed in baculovirus hydrolysed cis-stilbene oxide (23 nmol.min-1.mg protein-1) and was located mainly in the microsomal fraction of virus-infected Sf9 cells. There was no detectable EH activity toward juvenile hormone III. These observations suggest that DmEH is involved in xenobiotic biotransformation, but not in juvenile hormone metabolism, in D. melanogaster.

Epoxide hydrolases are enzymes involved in metabolism and defense of plants. Genome scanning suggested the presence of several genes encoding epoxide hydrolase in Arabidopsis thaliana. To assure that the predicted genes are functional and the translated products have epoxide hydrolase activity analysis at the protein level is needed. We have started to clone the cDNAs and overexpress them for catalytic and physico-chemical analysis. We here report that Pichia pastoris serves as an efficient system for overexpression of soluble epoxide hydrolase 1 (AtsEH1) from A. thaliana. A tag containing six histidine residues was added to the N-terminus to enable efficient one-step purification on nickel-agarose. The enzyme was expressed at levels >18 mg.L(-1) of culture and a French Press was found to be effective to achieve cell lysis. The recombinant enzyme had a molecular mass of 37 or 38 kDa based on SDS-PAGE or MALDI-TOF analysis, respectively. The enzyme was highly active towards the substrate trans-stilbene oxide (TSO) and had a pH optimum at 7 and a temperature optimum at 54 degrees C. Using TSO as substrate the K(m) and V(max) values were determined to 5 micro M and 2 micromol min(-1) mg protein(-1), respectively. The activity was 50-fold lower towards cis-stilbene oxide. The stability over time was tested from 20 to 54 degrees C and the enzyme lost activity at varying degrees at the temperatures tested but was stable for several months at 4 degrees C.

Microsomal epoxide hydrolase (mEH) is involved in the detoxification of xenobiotics that are or can form epoxide metabolites, including the ovotoxicant, 4-vinylcyclohexene (VCH). This industrial chemical is bioactivated by hepatic CYP450 to the diepoxide metabolite, VCD, which destroys mouse small preantral follicles (F1). Since ovarian mEH may play a role in VCD detoxification, these studies investigated the expression and activity of mEH in isolated ovarian fractions. Mice were given 1 or 15 daily doses (ip) of VCH (7.4 mmol/kg/day) or VCD (0.57 mmol/kg/day); 4 h following the final dose, ovaries were removed, distinct populations of intact follicles (F1, 25-100 microm; F2, 100-250 microm; F3, > 250 microm) and interstitial cells (Int) were isolated, and total RNA and protein were extracted. Real-time polymerase chain reaction and the substrate cis-stilbene oxide (CSO; 12.5 microM) were used to evaluate expression and specific activity of mEH, respectively. Confocal microscopy evaluated ovarian distribution of mEH protein. Expression of mRNA encoding mEH was increased in F1 (410 +/- 5% VCH; 292 +/- 5% VCD) and F2 (1379 +/- 4% VCH; 381 +/- 11% VCD) follicles following repeated dosing with VCH or VCD. Catalytic activity of mEH increased in F1 follicles following repeated dosing with VCH/VCD (381 +/- 11% VCH; 384 +/- 27% VCD). Visualized by confocal microscopy, mEH protein was distributed throughout the ovary with the greatest staining intensity in the interstitial cells and staining in the theca cells that was increased by dosing (56 +/- 0.8% VCH; 29 +/- 0.9% VCD). We conclude that mEH is expressed and is functional in mouse ovarian follicles. Additionally,in vivo dosing with VCH and VCD affects these parameters.

Microsomal epoxide hydrolase (HYL1) is a single-gene enzyme responsible for the hydrolysis of epoxides derived from the oxidative metabolism of xenobiotics. Variation in HYL1, therefore, may be an important determinant of drug toxicity. We have investigated HYL1 enzyme kinetics in six different species including man, for which a liver bank genotyped for polymorphisms in exons 3 and 4 of the HYL1 gene was used. Activity was measured by radiochromatography with high specific activity radiolabeled substrates, cis-stilbene oxide (CSO) and carbamazepine 10,11-epoxide (CBZ-E). In addition, naphthalene was used to investigate the hydrolysis of an epoxide (naphthalene 1,2-epoxide [N-E] generated in situ. There was marked species variation in enzyme activity that was substrate dependent. CSO was rapidly hydrolyzed by microsomes from all species, the rank order of specific activity being human > rabbit > dog > rat > hamster > mouse. In contrast, hydrolysis of CBZ-E was only observed with human liver microsomes. CBZ-E was only a weak (IC50 = 1 mM) inhibitor of CSO hydrolysis. The hydrolysis of N-E, determined as the diol-to-total metabolite ratio, was human > rabbit > dog > hamster > mouse > rat. Intraspecies variation in man was 4-fold, 7-fold and 2-fold for CSO, CBZ-E and N-E, respectively: none of this variation could be directly accounted for by the HYL1 polymorphisms in exons 3 and 4. These data emphasize the need for careful toxicokinetic evaluation of species used in the safety evaluation of compounds likely to form epoxide intermediates in vivo.

Administration of tridiphane (Tandem, DOWCO 356, 2-(3,5-dichlorophenyl)-2-(2,2,2-trichloroethyl)oxirane) to male Swiss-Webster mice for 3 days at 100, 250, and 500 mg/kg (ip) resulted in increases in liver weight accompanied by an increase in mitotic index and increases in large particle and microsomal protein. Epoxide hydrolase (EH) activity towards cis-stilbene oxide (CSO, microsomal EH) was elevated in microsomes and cytosol, a decrease in microsomal cholesterol EH was found, and hydrolysis of trans-stilbene oxide (TSO, cytosolic EH) was elevated in the cytosol but not in the microsomes. Glutathione S-transferase (GST) activity was elevated in cytosol for CSO, TSO, and 1,2-dichloro-4-nitrobenzene (DCNB), with inconsistent responses found with 1-chloro-2,4-dinitrobenzene (CDNB) and 1,2-epoxy-3-(p-nitrophenoxy)propane (ENPP). Microsomal GST was not consistently effected by tridiphane. Clofibrate (500 mg/kg, 3 daily ip injections) treatment resulted in similar responses in liver size, microsomal protein, and the EHs. The increase in cytosolic EH activity previously has been noted only in animals treated with peroxisome proliferators. Examination of livers from mice treated with 250 mg/kg tridiphane revealed that an increase in hepatic peroxisomes was apparent after 3 days of treatment. This was accompanied by decreases in serum cholesterol and triglyceride levels and increases in liver carnitine acetyl transferase and cyanide-insensitive oxidation of palmitoyl-CoA. This study demonstrates that tridiphane does have in vivo effects on mammalian epoxide-metabolizing enzymes and extends the association of increased cytosolic epoxide hydrolase activity with peroxisome proliferation.