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Over the last decade oxylipins have become more recognized for their involvement in several diseases. Non-steroidal anti-inflammatory drugs (NSAIDs) such as ibuprofen are known to inhibit cyclooxygenase (COX) enzymes, but how NSAIDs affect oxylipins, in addition to COX products, in animal tissues is not well understood. Oxylipins in livers from male and female mice treated with 100 mg/kg/day of ibuprofen for 7 days were investigated. The results showed that ibuprofen treated male livers contained 7 times more altered oxylipins than ibuprofen treated female livers. In male and female livers some prostaglandins were altered, while diols, hydroxy fatty acids and epoxides were significantly altered in male livers. Some soluble epoxide hydrolase (sEH) products, such as 9,10-DiHODE were found to be decreased, while sEH substrates (such as 9(10)-EpODE and 5(6)-EpETrE) were found to be increased in male livers treated with ibuprofen, but not in ibuprofen treated female livers. The enzymatic activities of sEH and microsomal epoxide hydrolase (mEH) were elevated by ibuprofen in both males and females. Analyzing the influence of sex on the effect of ibuprofen on oxylipins and COX products showed that approximately 27% of oxylipins detected were influenced by sex. The results reveal that ibuprofen disturbs not only the COX pathway, but also the CYP450 and lipoxygenase pathways in male mice, suggesting that ibuprofen is likely to generate sex related differences in biologically active oxylipins. Increased sEH activity after ibuprofen treatment is likely to be one of the mechanisms by which the liver reduces the higher levels of EpODEs and EpETrEs.

Background: COVID19 is a heterogeneous disease;while some patients experience mild or asymptomatic disease, others develop respiratory failure and death. Although there is increased understanding of clinical factors associated with severe disease, studies investigating biomarkers to predict severity and uncover treatment targets are still needed. Polyunsaturated fatty acid-derived lipid mediators (PUFA-LM) can promote inflammation or resolution of inflammation. Previous research showed differences in PUFA-LM expression in septic patients that developed ARDS. Here we evaluate the plasma PUFA-LM profile at admission in 20 patients with COVID-19 and correlate our findings with disease severity. Methods: Blood was collected at admission from 41 patients with COVID-19 at Virginia Commonwealth University between April and July, 2020. Platelet poor plasma from 20 patients were selected;10 with nonsevere COVID19 (NSC) defined by

The genome of the human intracellular pathogen Mycobacterium tuberculosis encodes an unusually large number of epoxide hydrolases, which are thought to be involved in lipid metabolism and detoxification reactions needed to endure the hostile environment of host macrophages. These enzymes therefore represent suitable targets for compounds such as urea derivatives, which are known inhibitors of soluble epoxide hydrolases. In this work, we studied in vitro the effect of the thiourea drug isoxyl on six epoxide hydrolases of M. tuberculosis using a fatty acid substrate. We show that one of the proteins inhibited by isoxyl is EphD, an enzyme involved in the metabolism of mycolic acids, key components of the mycobacterial cell wall. By analyzing mycolic acid profiles, we demonstrate the inhibition of EphD epoxide hydrolase activity by isoxyl and two other urea-based inhibitors, thiacetazone and AU1235, inside the mycobacterial cell.

Sphingorhabdus sp. YGSMI21, a novel microbial strain with an enantioselective epoxide hydrolase activity, was isolated from tidal samples contaminated by accidental oil spills subjected to enriched culture with polycyclic aromatic hydrocarbon. This strain was able to optically decompose (R)-styrene oxide (SO) and showed 100% optical purity. In addition, it showed a good enantioselectivity for the derivatives of (S)-SO, (S)-2-chlorostyrene oxide (CSO), (S)-3-CSO and (S)-4-CSO. For (S)-2-CSO, (S)-3-CSO and (S)-4-CSO, 99.9%ee was obtained with the yield of 26.2%, 24.8%, and 11.0%, respectively, when using 10 mg cells of Sphingorhabdus sp. YGSMI21 at pH 8.0 with 4 mM racemic substrates at pH 8.0 and 25°C. The values obtained in this study for (S)-2-CSO, particularly the yield of 26.2%, is noteworthy, considering that obtaining an enantiomerically pure form is difficult. Taken together, Sphingorhabdus sp. YGSMI21 can be regarded as a whole-cell biocatalyst in the production of various (S)-CSO with the chlorine group at a different position.

Leukotriene B4 (LTB4) is a potent, proinflammatory lipid mediator implicated in the pathologies of an array of inflammatory diseases and cancer. The biosynthesis of LTB4 is regulated by the leukotriene A4 hydrolase (LTA4H). Compounds capable of limiting the formation of LTB4, through selective inhibition of LTA4H, are expected to provide potent anti-inflammatory and anti-cancer agents. The aim of the current study is to obtain potential LTA4H inhibitors using computer-aided drug design. A hybrid 3D structure-based pharmacophore model was generated based on the crystal structure of LTA4H in complex with bestatin. The generated pharmacophore was used in a virtual screen of the Maybridge database. The retrieved hits were extensively filtered, then docked into the active site of the enzyme. Finally, they were consensually scored to yield five hits as potential LTA4H inhibitors. Consequently, the selected hits were purchased and their biological activity assessed in vitro against the epoxide hydrolase activity of LTA4H. The results were very promising, with the most active compound showing 73.6% inhibition of the basal epoxide hydrolase activity of LTA4H. The results from this exploratory study provide valuable information for the design and development of more potent and selective inhibitors.

The symbiotic relationship between native microbiota and their hosts probably is a key factor in animal survival. In this study, the relationships of Pseudomonas spp. strains with specific biomarkers of exposure to polycyclic aromatic hydrocarbons (PAHs) in wild fish Chirostoma jordani, as well as the capacity of these bacteria to biotransform PAHs were evaluated. The activity of the naphthalene dioxygenase system of the bacteria exposed to 0.1, 1.0 and 10 µg/L of PAHs was higher than the mean isoform 1A1 (CYP1A1) of the wild fish, particularly bacterial species related to Pseudomonas spp. However, the epoxide hydrolase activity of the strains was lower in all cases compared to the fish. Glutathione S-transferase (GST) activity of the bacterial strains was lower than in the liver, but higher than in viscera of C. jordani. Using redundancy analysis, two differential patterns were found: (i) CYP1A1 activity of fish was induced by naphthalene and anthracene water levels independently of sampling season and lakes monitored, and (ii) the unidentified strain of the Pseudomonas genus biotransforms the endogenous levels of benzo[a]pyrene, benzo[k]fluoranthene and indeno[1,2,3-cd]pyrene in the fish allowing the hydroxylated metabolites to conjugate with glutathione through GST activity of the fish.

LTA4H is a monozinc bifunctional enzyme which exhibits both aminopeptidase and epoxide hydrolase activities. Its dual functions in anti- and pro-inflammatory roles have attracted wide attention to the inhibitor design. In this work, we tried to construct Michaelis complexes of LTA4H with both a native peptide substrate and LTA4 molecule using combined quantum mechanics and molecular mechanics molecular dynamics simulations. First of all, the zinc ion is coordinated by H295, H299, and E318. For its aminopeptidase activity, similar to conventional peptidases, the fourth ligand to the zinc ion is suggested to be an active site water, which is further hydrogen bonded with a downstream glutamic acid, E296. For the epoxide hydrolase activity, the fourth ligand to the zinc ion is found to be an epoxy oxygen atom. The potential of mean force calculation indicates about an 8.5 kcal/mol activation barrier height for the ring-opening reaction, which will generate a metastable carbenium intermediate. Subsequent frontier molecular orbital analyses suggest that the next step would be the nucleophilic attacking reaction at the C12 atom by a water molecule activated by D375. Our simulations also analyzed functions of several important residues like R563, K565, E271, Y383, and Y378 in the binding of peptide and LTA4.

Soluble epoxide hydrolase (sEH) is a bifunctional enzyme that exhibits lipid epoxide hydrolase (sEH-H) and lipid phosphatase activity (sEH-P), with each being located in its own distinct domain. While the epoxide hydrolase activity is well-investigated, the role of the phosphatase domain remains unclear. This article briefly summarizes the evolution, structure and SNPs of the human sEH, with a special focus on the function and postulated role of the N-terminal domain of sEH. Furthermore, the article provides an overview of tools to study sEH-P.

1. Carbamazepine is an antiepileptic drug which is metabolized by CYP3A4 into carbamazepine-10,11-epoxide. This metabolite is then detoxified by epoxide hydrolase. As carbamazepine-10,11-epoxide has been associated with neurotoxicity, it is critical to identify whether a new antiepileptic drug has the potential to inhibit epoxide hydrolase and therefore increase carbamazepine-10,11-epoxide plasma levels.2. In this study, an in vitro assay was developed to evaluate epoxide hydrolase activity by using carbamazepine-10,11-epoxide as probe substrate. The ability of this assay to predict drug–drug interactions (DDI) at the epoxide hydrolase level was also investigated.3. To this aim, known inhibitors of epoxide hydrolase for which in vivo data are available were used. Firstly, carbamazepine-10,11-epoxide hydrolase activity was determined in liver microsomes, cytosol and hepatocytes. Thereafter, the IC50 of epoxide hydrolase inhibitors (progabide, valproic acid, valpromide and valnoctamide) was determin...

Batatasin IV is a dihydrostilbenoid isolated from Chinese yams which was shown to have inhibitory activities against plant growth. Later studies showed that this compound may exhibit anti-inflammatory properties by inhibiting the epoxide hydrolase activity of the leukotriene A4 hydrolase enzyme. To access the dihydrostilbenoid skeleton, a copper-free SPhos-mediated Sonogashira reaction was conceived and the substrate scope was explored. Our results indicate that the reaction can tolerate the presence of free alcohols, aldehydes, nitro groups, and anilinyl groups. However, a substituent with an acidic phenol or carboxylic acid group gave significantly lower yields. Next, a total synthesis of batatasin IV was accomplished in 16% overall yield incorporating the reported copper-free Sonogashira reaction. Finally, we show that batatasin IV inhibits the hydrolysis of alanine p-nitroanilide by leukotriene A4 hydrolase with an IC50 of 91.4 µM.

Sphingorhabdus sp. YGSMI21 is a novel strain exhibiting high enantioselective hydrolysis activity for styrene oxide. Here, we present its complete genome sequence, consisting of one circular chromosome (3.86 Mb) and one plasmid (0.196 Mb).

Marine bacteria possessing epoxide hydrolase (EH) activity were isolated from the oil-spilled foreshore of South Korea. While the isolated Rhodococcus sp. YSMI04 and YSNA32 showed different enantioselectivities to racemic styrene oxide (SO), two strains exhibited same enantiopreference to 3-chlorostyrene oxide (3-CSO) and 4-CSO. In the case of 3-CSO, Rhodococcus sp. YSMI04 and YSNA32 exhibited (R)-3-CSO preferred hydrolysis activity, whereas Roseobacter sp. TSBP12 showed a preference for (S)-3-CSO. In the case of 4-CSO, Rhodococcus sp. YSMI04 and YSNA32 showed enantiopreference to (S)-4-CSO, Roseobacter sp. TSBP12 exhibited (R)-4-CSO preferred activity. (S)-4-CSO was obtained with 35% yield (theoretically 50%) using Roseobacter sp. TSBP12.

Lysophosphatidic acids (LPAs) are phospholipids which have many physiological and pathophysiological functions. The human soluble epoxide hydrolase (sEH) plays a role in the metabolism of xenobiotics through its metabolism of aromatic hydrocarbon epoxides such as styrene oxide. sEH also has a phosphatase activity, and metabolizes LPAs. In this study, we investigated a purified wild-type (WT) and six allelic variants of sEH to evaluate differences in their activities toward LPAs. We found that the R103C and R287Q showed significantly lower activity than the WT sEH. We also found that the R103C and R287Q had significantly lower activity even when applied to only the N-terminal or C-terminal domain. The kinetic study determined that the R103C and R287Q had a lower Vmax/Km ratio toward stearoyl-LPA than the other variants. In a previous study, we found that WT sEH suppressed vascular endothelial growth factor (VEGF) mRNA in Hep3B cells; in the present experiments, all sEH variants except V442A suppressed VEGF mRNA levels in Hep3B cells. These results suggest that the R103C and R287Q have lower phosphatase activity, but that all the allelic variants have similar effects on VEGF suppression.

The antihyperlipidemic and antioxidant activities of dietary supplementation of sea tangle from Goseong and the alginate-free residue of sea tangle were investigated in Sprague Dawley rats treated with a high-fat diet, streptozotocin, poloxamer 407, and bromobenzene. The alginate-free residue of Goseong sea tangle induced a significant reduction in triglycerides and total cholesterol levels, as well as a significant increase in high-density lipoprotein cholesterol levels. Alginate-free Goseong sea tangle residue reduced the activities of the phase I enzymes aminopyrine N-demethylase and aniline hydroxylase, which had been increased by intraperitoneal injection of bromobenzene. Pretreatment with Goseong sea tangle residue prevented a bromobenzene-induced decrease in epoxide hydrolase activity. Bromobenzene reduced hepatic glutathione content and increased hepatic lipid peroxide levels. Pretreatment with alginate-free Goseong sea tangle residue prevented lipid peroxidation induced by bromobenzene, but pretreatment with Goseong sea tangle did not. These results suggest that Goseong sea tangle residue exerted antihyperlipidemic and antioxidant activities that were higher than those induced by alginate-containing sea tangle. Therefore, the alginate-free residue may contain physiologically unknown active components, other than alginic acid, which may potentially be used to prevent hyperlipidemic atherosclerosis.

Recalcitrant environmental pollutants, like bromoorganics and epoxides are hydrolysed with limited substrate specificities by microbial oxygenases, reductases, hydrolases and dehalogenases. Here, we report the identification and characterisation of a protein (XP_504164) from the tropical marine yeast Yarrowia lipolytica NCIM 3589, known to degrade bromoorganics and epoxides. Multiple sequence alignment suggests it belongs to α/β superfamily with conservation of catalytic triad and oxyanion hole motifs. The corresponding gene cloned and protein (Ylehd) expressed in E. coli BL21AI exhibited epoxide hydrolase activity (24 ± 0.7 nmol s−1 mg−1 protein) at pH 8.0 and promiscuous haloalkane dehalogenase (1.5 ± 0.2 nmol s−1 mg−1 protein) at pH 4.5. Recombinant Ylehd catalyses structurally diverse epoxides and bromoorganics with maximum catalytic efficiency (kcat/Km) of 96.56 and 10.1 mM−1 s−1 towards 1,2-Epoxyoctane (EO) and 1-Bromodecane (BD). The expression of Ylehd was highly induced in presence of BD and EO but not in glucose grown cells as studied by immunoblot analyses, q-PCR and activity levels. Immunoelectron microscopy confirmed higher expression in presence of xenobiotics and located it to cytosol. Such inducible nature of Ylehd suggests its physiological role in xenobiotic stress mitigation. This study represents the first functional characterisation of a bifunctional EH/HLD in eukaryotic microbes with broad substrate specificity making it a potential biocatalyst for bioremediation/biosensing of mixed pollutants.

Aim: mesT of Mycobacterium tuberculosis, a hypothetical/putative epoxide hydrolase, is predicted to convert toxic epoxides to the more water-soluble and less toxic diols. Detailed characterization of the protein was carried out. Results: mesT demonstrated esterase as well as epoxide hydrolase activity. It was membrane bound and was upregulated under hypoxic conditions. The enzyme was able to degrade styrene oxide. The presence of antisense against this gene resulted in the inhibition of in vitro bacterial growth/survival in the presence of styrene oxide. Conclusion & future perspective: We demonstrated that mesT possessed epoxide hydrolase activity and styrene oxide might be its physiological substrate. Inhibition of mesT reduced the growth of the bacteria in presence of styrene oxide and its expression under hypoxic condition suggested its role in intracellular survival of bacteria.

The by-products of olive oil production can be used as sources of microbial strains. Penicillium sp., Aspergillus terreus, Penicillium aurantiogriseum, Aspergillus tubingensis and Aspergillus niger were selected on the basis of their epoxide-hydrolyzing activity towards racemic rac-glycidyl phenyl ether. We studied the effect on enzymatic activity of adding styrene oxide to the growth medium. It induced the biosynthesis of epoxide hydrolases and reduced cell growth. The resolution capacity of the five fungi was tested on rac-glycidyl phenyl ether, rac-benzyl glycidyl ether, rac-1,2-epoxyhexane and rac-1,2-epoxyoctane. The resolution of rac-glycidyl phenyl ether by A. niger, rac-benzyl glycidyl ether by P. aurantiogriseum and A. terreus, rac-1,2-epoxyhexane by A. tubingensis and rac-1,2-epoxyoctane by A. terreus provided (S)-3-phenoxy-1,2-propanediol (45.1% yield, 51.4% ee), (R)-3-benzyloxy-1,2-propanediol (40.8% yield, 43.3% ee), (S)-3-benzyloxy-1,2-propanediol (45.4% yield, 45.6% ee), (R)-1,2-hexanediol (70.4% yield, 24.4% ee) and (R)-1,2-octanediol (21.4% yield, 27.5% ee), respectively. The (R)-enantiopreference of the epoxide hydrolases from P. aurantiogriseum is unprecedented.

Cytochrome P450 (CYP) epoxygenases metabolize arachidonic acid into epoxyeicosatrienoic acids (EETs), which play an important role in blood pressure regulation, protection against ischemia-reperfusion injury, angiogenesis, and inflammation. Epoxide hydrolases metabolize EETs to their corresponding diols (dihydroxyeicosatrienoic acids; DHETs) which are biologically less active. Microsomal epoxide hydrolase (EPHX1, mEH) and soluble epoxide hydrolase (EPHX2, sEH) were identified >30 years ago and are capable of hydrolyzing EETs to DHETs. A novel epoxide hydrolase, EPHX3, was recently identified by sequence homology and also exhibits epoxide hydrolase activity in vitro with a substrate preference for 9,10-epoxyoctadecamonoenoic acid (EpOME) and 11,12-EET. EPHX3 is highly expressed in the skin, lung, stomach, esophagus, and tongue; however, its endogenous function is unknown. Therefore, we investigated the impact of genetic disruption of Ephx3 on fatty acid epoxide hydrolysis and EET-related physiology in mice. Ephx3-/- mice were generated by excising the promoter and first four exons of the Ephx3 gene using Cre-LoxP methodology. LC-MS/MS analysis of Ephx3-/- heart, lung, and skin lysates revealed no differences in endogenous epoxide:diol ratios compared to wild type (WT). Ephx3-/- mice also exhibited no change in plasma levels of fatty acid epoxides and diols relative to WT. Incubations of cytosolic and microsomal fractions prepared from Ephx3-/- and WT stomach, lung, and skin with synthetic 8,9-EET, 11,12-EET, and 9,10-EpOME revealed no significant differences in rates of fatty acid diol formation between the genotypes. Ephx3-/- hearts had similar functional recovery compared to WT hearts following ischemia/reperfusion injury. Following intranasal lipopolysaccharide (LPS) exposure, Ephx3-/- mice were not different from WT in terms of lung histology, bronchoalveolar lavage fluid cell counts, or fatty acid epoxide and diol levels. We conclude that genetic disruption of Ephx3 does not result in an overt phenotype and has no significant effects on the metabolism of EETs or EpOMEs in vivo.

Soluble epoxide hydrolase (sEH) contributes to cell growth, but the contribution of sEH to embryonic development is not well understood. In this study, Xenopus sEH cDNA was isolated from embryos of Xenopus laevis. The Xenopus sEH was expressed in Escherichia coli and was purified. The epoxide hydrolase and phosphatase activities of purified sEH were investigated. The Xenopus sEH did not show phosphatase activity toward 4-methylumbelliferyl phosphate or several lysophosphatidic acids although it had EH activity. The amino acid sequence of Xenopus sEH was compared with that reported previously. We found amino acid substitutions of the 29th Thr to Asn and the 146th Arg to His and prepared a sEH mutant (N29T/H146R), designed as mutant 1. Neither wild-type sEH nor mutant 1 had phosphatase activity. Additional substitution of the 11th Gly with Asp was found by comparison with human sEH which has phosphatase activity, but the Xenopus sEH mutant G11D prepared as mutant 2 did not have phosphatase activity. The epoxide hydrolase activity of sEH seemed to be similar to that of human sEH, while Xenopus sEH did not have phosphatase activity toward several substrates that human sEH metabolizes.

Endocytic recycling of the cystic fibrosis transmembrane conductance regulator (CFTR) is blocked by the CFTR inhibitory factor (Cif). Originally discovered in Pseudomonas aeruginosa, Cif is a secreted epoxide hydrolase that is transcriptionally regulated by CifR, an epoxide-sensitive repressor. In this report, we investigate a homologous protein found in strains of the emerging nosocomial pathogens Acinetobacter nosocomialis and Acinetobacter baumannii ("aCif"). Like Cif, aCif is an epoxide hydrolase that carries an N-terminal secretion signal and can be purified from culture supernatants. When applied directly to polarized airway epithelial cells, mature aCif triggers a reduction in CFTR abundance at the apical membrane. Biochemical and crystallographic studies reveal a dimeric assembly with a stereochemically conserved active site, confirming our motif-based identification of candidate Cif-like pathogenic EH sequences. Furthermore, cif expression is transcriptionally repressed by a CifR homolog ("aCifR") and is induced in the presence of epoxides. Overall, this Acinetobacter protein recapitulates the essential attributes of the Pseudomonas Cif system and thus may facilitate airway colonization in nosocomial lung infections.

The biocatalytic resolution of (R,S)-epichlorohydrin to (R)-epichlorohydrin by whole cells from a recombinant Escherichia coli BL21(ED3) expressing epoxide hydrolase activity in a series of organic solvent/buffer biphasic systems was studied. Isooctane was found to be the best organic solvent. The optimum volume ratio of the organic phase to the aqueous phase, reaction temperature, pH, substrate concentration, cell concentration, and reaction time were 7:3, 30 °C, 8.0, 574 mmol/L, 0.07 g/ml wet cells, and 45 min, respectively. Under the optimized conditions, a yield of 37.5% with 99.3% ee and an average productivity of 0.286 mol/(L·h) were obtained in a stirred reactor (1-L scale). Thus, the bioconversion was more efficient in the isooctane/buffer biphasic system than that in a monophasic aqueous system, where the substrate concentration and the average productivity of (R)-epichlorohydrin were improved by 55.2% and 98.6%, respectively.

A novel epoxide hydrolase (EHase) from polycyclic aromatic hydrocarbon (PAH)-degrading bacteria was identified and characterized. EHase activity was identified in four strains of PAH-degrading bacteria isolated from commercial gasoline and oil-contaminated sediment based on their growth on styrene oxide and its derivatives, such as 2,3- and 4-chlorostyrene oxides, as a sole carbon source. Gordonia sp. H37 exhibited high enantioselective hydrolysis activity for 4-chlorostyrene oxide with an enantiomeric ratio of 27. Gordonia sp. H37 preferentially hydrolyzed the (R)-enantiomer of styrene oxide derivatives resulting in the preparation of a (S)-enantiomer with enantiomeric excess greater than 99.9 %. The enantioselective EHase activity was identified and characterized in various PAH-degrading bacteria, and whole cell Gordonia sp. H37 was employed as a biocatalyst for preparing enantiopure (S)-styrene oxide derivatives.

The objectives of the present study were two-fold: (a) to evaluate the role of molecular shape on the interaction of polycyclic aromatic hydrocarbons (PAHs) with the Ah receptor and CYP1A1 upregulation, and (b) to evaluate the potential of PAHs to induce epoxide hydrolase and glutathione S-transferase, two major enzymes involved in their metabolism. In order to achieve these objectives, precision-cut rat liver slices were incubated with a range of concentrations of seven 5-ring isomeric PAHs, namely benzo[c]chrysene, benzo[b]chrysene, benzo[g]chrysene, dibenzo[a,j]anthracene, dibenzo[a,c]anthracene, picene and pentacene, for 24h. All compounds, with the exception of pentacene, elevated the O-deethylation of ethoxyresorufin, an activity associated with CYP1A1; induction of this enzyme by the various PAHs correlated with their avidity for the Ah receptor. None of the PAHs studied increased epoxide hydrolase activity, monitored using benzo[a]pyrene 4,5-oxide. Of the seven PAHs, only benzo[g]chrysene elevated glutathione S-transferase activity, measured using 1-chloro-2,4-dinitrobenzene or 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole as substrates. No relationship could be established between length or length/width and interaction with the Ah receptor and CYP1A1 up-regulation indicating that other structural or electronic factors are likely to be more important. Finally, 5-ring PAHs are poor inducers of the epoxide hydrolase and glutathione S-transferase enzyme systems.

The data presented in this article are related to the research article entitled “Epoxide hydrolase of Trichoderma reesei: Biochemical properties and conformational characterization” [1]. Epoxide hydrolases (EHs) are enzymes that catalyze the hydrolysis of epoxides to the corresponding vicinal diols. This article describes the optimal parameters for the colorimetric red assay to determine the enzymatic activity, with an emphasis on the characterization of the kinetic parameters, pH optimum and thermal stability of this enzyme. The effects of reagents that are not resistant to oxidation by sodium periodate on the reactions can generate false positives and interfere with the final results of the red assay. Keywords: Colorimetric red assay, Epoxide hydrolase, Kinetic parameters

Epoxide hydrolases have been detected in prokaryotes and eukaryotes ranging from plants to mammals.1,2, 3 In mammals these include the soluble epoxide hydrolase (sEH), microsomal epoxide hydrolase (mEH), cholesterol epoxide hydrolase, and leukotriene A4 (LTA4) hydrolase. These enzymes mediate the addition of water to both exogenous and endogenous epoxides, leading to the corresponding vicinal diols except for LTA4 hydrolase, and they display different substrate selectivity. For example, the mammalian sEH is selective for aliphatic epoxides and particularly fatty acid epoxides whereas mEH is more selective for cyclic and arene epoxides. Studies on the mEH have focused on its role in xenobiotic metabolism, but its distribution, particularly in the brain and adrenal gland suggests a possible endogenous role.4 Although its catalytic activity on fatty acid epoxides is low, the high level of the mEH in some brain regions may contributeto their hydrolysis. The catalytic activity of the sEH on arene oxides and other cyclic epoxides is so low that its contribution appears insignificant compared to the mEH as well as for chemical and glutathione S-transferase-catalyzed conjugation of reactive epoxides. Although the sEH can metabolize some aliphatic natural products, the sEH is thought to be involved largely in the metabolism of regulatory epoxylipids, particularly those of the arachidonic acid cascade (Figure 1). Titers of free arachidonic acid are very low, but when it is released it is converted to a wide variety of biologically active metabolites. Most research has focused on the cyclooxygenase and lipoxygenase pathways, but increasing attention is being paid to the cytochrome P450 branch of the cascade. One set of P450 enzymes carry out allylic and ω and ω-1 oxidation. Another set of P450 enzymes form regioisomeric epoxides of arachidonic acid and other unsaturated lipids. In the arachidonate series these epoxides are called epoxyeicosatrienoic acids (EETs). The EETs are metabolized by incorporation into phospholipids, chain shortening, chain elongation, hydroxylation and other pathways.5 However, the dominant pathway is hydration of the epoxides to the corresponding 1,2-diols by sEH. It should be noted that multiple drugs have already been discovered to act on the cyclooxygenase and lipoxygenase branches of the arachidonic acid metabolic cascade. For example, numerous non-steroidal anti-inflammatory drugs (NSAIDs) are inhibitors ofcyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2).6 In addition, montelukast is a leukotriene (LT) receptor antagonist blocking the action of LTD4 and secondary ligands LTC4 and LTE4.7 Zileuton inhibits 5-lipoxygenase, an enzyme of the eicosanoid synthesis pathway for the production of LTs.8 Both montelukast and zileuton are effective therapies for treatment of asthma. Lastly, laropiprant, an antagonist of the DP1 receptor of prostaglandin D2, is used in combination with niacin to suppress the niacin-induced vasodilation.9 Figure 1 Major pathways of arachidonic acid metabolism. As shown in Figure2, sEH in human (hsEH, EPHX2, EC 3.3.2.10) is a bifunctional homodimeric enzyme located in both cytosol and peroxisomes with both epoxide hydrolase and phosphatase activity.1 Specifically, the C-terminus epoxide hydrolase motif of sEH transforms four regioisomers of EETs, namely 5,6-, 8,9-, 11,12- and 14,15-EETs which are the endogenous chemical mediators derived from arachidonic acid by cytochrome P450 epoxygenases, to the corresponding dihydroxyeicosatrienoic acids (DHETs), whereby the biological effects of EETs are diminished, eliminated or altered.10 Recent work has shown that some fatty acid diols have unique biological activities, but the diols are far more polar than the epoxides and thus quickly move out of cells and often are conjugated.11, 12 The sEH hydrates all fatty acids so far tested. For example, sEH converts linoleic acid epoxides to proinflammatory linoleic acid diols which are proposed endogenous chemical mediators as well. Regarding the enzyme activity in tissues, it was found that human liver possesses the highest sEH specific activity followed by the kidney.13,14 Of note, specific cell types in the heart, vasculature, brain, lung, and kidney have quite high levels of enzyme. However much less is known about the N-terminal phosphatase regarding its endogenous substrates and physiological roles. Figure 2 Left: X-ray cocrystal structure of murine sEH (PDB code: 1CR6). One of the antiparallel monomers is shown in blue and the other in yellow. Right: X-ray cocrystal structure of a single subunit of the human sEH (PDB code: 1ZD3). The catalytic mechanism of epoxide hydrolases was worked out by a series of biochemical studies in several laboratories based on the homology of the mEH and sEH to haloalkane dehalogenase. Similarly the hypothetical arrangement of binding sites in the enzyme was predicted by three-dimensionalquantitative structure-activity relationship (3D QSAR).15 The X-ray crystal structure of human sEH complexed with an sEH inhibitor (PDB code: 1ZD3) and later structures revealed the catalytic pocket and the key structural features required to inhibit the epoxide hydrolase activity of this enzyme in great detail (Figure 3).16 The epoxide hydrolase catalytic pocket consists of two tyrosine residues (Tyr381 and Tyr465) which activate the epoxide ring opening by Asp333. The resulting ester is then rapidly hydrolyzed into DHETs. It has been recognized that amide, carbamate and urea groups fit well in the hydrolase catalytic pocket. Specifically, the carbonyl oxygen of amide or urea is involved in a hydrogen bonding interaction with Tyr381 and Tyr465, and the N-H of ureas or amides acts as a hydrogen bond donor to Asp333. Therefore, various ureas and amides (1 and 2) have been developed as competitive, reversible and often tight binding sEH inhibitors. Several of these inhibitors bind to the sEH in the picomolar range. The X-ray structure also showed that the two domains of the sEH were joined by a proline rich bridge, the enzyme in mammals is an anti parallel homodimer, and the putative catalytic site of the N-terminal domain suggested an active phosphatase of unknown role. The W334 niche and F265 binding pocket, as depicted in Figure 3, can each accommodate a variety of functional groups of sEH inhibitors. These structures not only have assisted with optimization of sEH inhibitors but have proven valuable to evaluate the biology associated with single nucleotide polymorphisms (SNPs) in the enzyme. Figure 3 X-ray cocrystal structure of human sEH C domain showing the binding site which exists as an “L” shape hydrophobic tunnel. (PDB code: 1ZD3) In mammals the mEH (EPHX1, EC 3.3.2.9) is membrane bound and largely in the endoplasmic reticulum.17,18 In some pathological states the mEH dissociates from the membrane and appears in the blood where it is known as the preneoplatic antigen and is a marker for tissue damage including cancer.19 Activity and polymorphisms of the enzyme have been associated with a variety of diseases, and this is a quite active area of research.20 With regard to this review it is important to note that although the mEH and sEH have similar catalytic mechanisms and are members of the α/β-hydrolase fold family of proteins, their evolutionary paths diverged at the level of prokaryotes. Thus it is possible to differentially inhibit the mEH and sEH with high selectivity.21 Evidence is excellent that the enzyme referred to as hepoxilin epoxide hydrolase is in fact the sEH. The cholesterol 5,6-epoxide hydrolase and leukotriene A4 hydrolase are in different enzyme families working by different catalytic mechanisms and will not be discussed further. In studying the evolution of the sEH, related genes were further found. These hypothetical products of these genes are known as EH3 and EH4. Their expression in vertebrates, particularly in man is under investigation and their biological activity is so far not clear.18 In light of the recent progress in this field, this perspective will provide biological rationales, medicinal chemistry approaches, and possible paths for sEH inhibitors to enter clinical trials.

Zinc metallopeptidases are ubiquitous enzymes with diverse cellular functions that can be found in most organisms. Leukotriene A4 hydrolase (LTA4H; E.C. 3.3.2.6) is an unusual zinc metallopeptidase of the M1 family that also possesses an epoxide hydrolase activity; however, the role of its peptidase activity remains unknown. To further characterize the peptidase activity of LTA4H and other closely related metallopeptidases, a multiple sequence alignment and predicted structure were used to target three amino acid residues of yeast LTA4H for mutagenesis: Asn362, Trp365, and Asp399. Although mutating Trp365 and Asp399 had little effect on catalysis, altering Asn362 had varying effects on catalysis, depending on the replacement residue. Mutation of Asn362 to glutamine (N362Q) caused minor catalytic defects, while mutation to leucine (N362L) or glutamate (N362E) caused large reductions in activity. Both N362L and N362E also exhibited an altered pH dependence of catalysis, reduced chloride activation, and reduced zinc affinity and content, indicating that Asn362 may interact with the nearby zinc coordinating residue His344, and possibly with Glu363 as well, to polarize and/or orient these residues.

We studied the effects of a single, oral dose of methotrexate (MTX) on arachidonic acid metabolism in neutrophils from 6 patients with rheumatoid arthritis, which were obtained 1 day before and 1 day after their usual weekly MTX dose. The 6 patients had received a mean weekly MTX dose of 9.6 mg (range 5-15) for a mean of 61.7 months (range 58-64), and none received concomitant corticosteroids. Total generation of leukotriene B4 (LTB4) in neutrophils stimulated ex vivo with 10 microM calcium ionophore A23187 for 20 minutes was significantly suppressed, by a mean of 53%, after the MTX dose compared with the predose levels (mean +/- SEM 13.0 +/- 1.4 ng/10(6) cells versus 6.0 +/- 0.9 ng/10(6) cells; P = 0.0019), reflecting a comparable suppression of both released and cell-retained LTB4. A 49% decrease in omega-oxidation products of LTB4 demonstrates that decreased LTB4 synthesis, rather than increased degradation, is responsible for the decrease in LTB4 generation. The absence of a significant change in either 3H-labeled arachidonic acid release or platelet-activating factor generation indicates that the observed decrease in LTB4 synthesis was apparently not caused by diminished phospholipase A2 activity. A 28% decrease in the total formation of the 5-lipoxygenase products 5-hydroxyeicosatetraenoic acid and the 6-trans-LTB4 diastereoisomers, and a 48% suppression of production of LTB4 plus its omega-oxidation metabolites after the MTX dose suggest inhibition of 5-lipoxygenase activity and possible suppression of leukotriene A4 epoxide hydrolase activity.

A newly isolated Bacillus megaterium with epoxide hydrolase activity resolved racemic glycidyl (o, m, p)-methylphenyl ethers to give enantiopure epoxides in 84-99% enantiomeric excess and with 21-73 enantiomeric ratios. The (S)-enantiomer was obtained from rac-glycidyl (o or m)-methylphenyl ether while the (R)-epoxides was obtained from glycidyl p-methylphenyl ether. The observations are explained at the level by enzyme-substrate docking studies.

Inhibition of the soluble epoxide hydrolase (sEH) has beneficial effects on vascular inflammation and hypertension indicating that the enzyme may be a promising target for drug development. As the enzymatic core of the hydrolase domain of the human sEH contains two tyrosine residues (Tyr(383) and Tyr(466)) that are theoretically crucial for enzymatic activity, we addressed the hypothesis that the activity of the sEH may be affected by nitrosative stress. Epoxide hydrolase activity was detected in human and murine endothelial cells as well in HEK293 cells and could be inhibited by either authentic peroxynitrite (ONOO(-)) or the ONOO(-) generator 3-morpholino-sydnonimine (SIN-1). Protection of the enzymatic core with 1-adamantyl-3-cyclohexylurea in vitro decreased sensitivity to SIN-1. Both ONOO(-) and SIN-1 elicited the tyrosine nitration of the sEH protein and mass spectrometry analysis of tryptic fragments revealed nitration on several tyrosine residues including Tyr(383) and Tyr(466). Mutation of the latter residues to phenylalanine was sufficient to abrogate epoxide hydrolase activity. In vivo, streptozotocin-induced diabetes resulted in the tyrosine nitration of the sEH in murine lungs and a significant decrease in its activity. Taken together, these data indicate that the activity of the sEH can be regulated by the tyrosine nitration of the protein. Moreover, nitrosative stress would be expected to potentiate the physiological actions of arachidonic acid epoxides by preventing their metabolism to the corresponding diols.

Entering the fold: A common structural motif in hydrolytic enzymes is the alpha,beta-hydrolase fold. The interconversion of one enzyme into another by introduction of mechanistically important residues is not enough; only substitution of a loop allows epoxide hydrolase activity in the esterase scaffold to be formed (see picture; structure comparison of epoxide hydrolases (green) with the esterase (orange)). The result is an enantioselective chimeric enzyme.

The effects of phenoxyacid herbicides 2,4-D (2,4-dichlorophenoxyacetic acid) and MCPA (4-chloro-2-methylphenoxyacetic acid), clofibrate, and glyphosate on hepatic and intestinal drug metabolizing enzyme activities were studied in rats intragastrically exposed for 2 weeks. The hepatic ethoxycoumarin O-deethylase activity increased about 2-fold with MCPA. Both 2,4-D and MCPA increased the hepatic epoxide hydrolase activity and decreased the hepatic glutathione S-transferase activity. MCPA also increased the intestinal activities of ethoxycoumarin O-deethylase and epoxide hydrolase. Glyphosate decreased the hepatic level of cytochrome P-450 and monooxygenase activities and the intestinal activity of aryl hydrocarbon hydroxylase. Clofibrate decreased the hepatic activities of UDPglucuronosyltransferase with p-nitrophenol or methylumbelliferone as the substrate. Also 2,4-D decreased the hepatic activity of UDPglucuronosyltransferase with p-nitrophenol as the substrate. MCPA decreased the intestinal activities of UDPglucuronosyltransferase with either p-nitrophenol or methylumbelliferone as the substrate. The results indicate that phenoxyacetic acids, especially MCPA, may have potent effects on the metabolism of xenobiotics. Glyphosate, not chemically related to phenoxyacids, seems to inhibit monooxygenases. Whether these changes are related to the toxicity of these xenobiotics remains to be clarified in further experiments.

Epoxide hydrolase plays an important role in the detoxification of genotoxic compounds and in the control of physiological signaling molecules. Altered levels of epoxide hydrolase activity are associated with many diseases, such as emphysema, lung cancer, ovarian cancer, and laryngeal carcinoma. We designed and synthesized a resorufin-based fluorogenic probe, 7-(2-(oxiran-2-yl)ethoxy) resorufin, which was hydrolyzed by microsomal epoxide hydrolase to form the corresponding diol, which upon further treatment with sodium periodate released the strongly fluorescent resorufin. The probe exhibits good biological compatibility and photophysical properties, such as long wavelength excitation (571 nm) and emission (585 nm) and a wide working pH range (from 6.0 to 10.0), and thus facilitates the determination of the activity of microsomal epoxide hydrolase.

A crude extract of Mung bean meal ( Phaseolus radiatus ) possessing epoxide hydrolase activity immobilized in gelatin gel (gelozyme) is employed in the stereoselective epoxide ring opening of glycidate esters. Thus, ethyl trans -(±)-3-phenyl glycidate 1a and methyl trans -(±)-3-(4-methoxyphenyl) glycidate 1b gave (2 S ,3 R )-glycidate esters (ee >99% and 45% yield) with gelatin immobilized enzyme in diisopropyl ether. The corresponding (2 R ,3 S )-enantiomer of 1a was hydrolyzed by an epoxide hydrolase to predominantly give the anti -product, ethyl (2 R ,3 R )-2,3-dihydroxy-3-phenylpropanoate, with a diastereomeric excess of 78% and ee 94% (40%). A small amount (5%) of racemic syn -product was also obtained as a result of the spontaneous hydrolysis. In the case of 1b , the hydrolysis product was racemic due to high reactivity of the glycidate toward water.

Erythrocytes serve as reservoirs for cis- and trans-epoxyeicosatrienoic acids (EETs). Incubation of rat red blood cells (RBCs) with cis- and trans-EETs produces threo- and erythro-dihydroxyeicosatrienoic acids, respectively. The V(max) of EET hydrolysis by rat intact RBCs (2.35 +/- 0.24 pmol/min/10(8) RBCs for 14,15-trans-EET) decreased by approximately 2 to 3-fold sequentially from 14,15-, 11,12- to 8,9-EETs for both cis- and trans-isomers. The V(max) of trans-EET hydrolysis by RBCs is approximately 2 to 3 times that of the corresponding cis-EETs. Incubation of EETs with recombinant murine soluble epoxide hydrolase (sEH) yielded the same geometric and regio preferences of EET hydrolysis as with rat intact RBCs. The principal epoxide hydrolase activity for EET hydrolysis (approximately 90%) is present in the erythrocyte cytosol. Western blots of sEH suggested a concentration of sEH protein to be approximately 2 microg/mg protein or 0.4 microg/10(9) RBCs. The apparent K(m) values of EETs were between 1 and 2 microM, close to the K(m) for purified sEH as reported. Erythrocyte hydration of cis- and trans-EETs was blocked by sEH inhibitors, 1,3-dicyclohexylurea and 4-[4-(3-adamantan-1-ylureido)cyclohexyloxy]benzoic acid. Erythrocyte sEH activity was inhibited more than 80% by 0.2% bovine serum albumin in the buffer. Preferred hydrolysis of 14,15-EETs and trans-epoxides characterizes sEH activity in RBCs that regulates the hydrolysis and release of cis- and trans-EETs in the circulation. Inhibition of sEH has produced antihypertensive and antiinflammatory effects. Because plasma trans-EETs would increase more than cis-EETs with sEH inhibition, the potential roles of trans-EETs and erythrocyte sEH in terms of circulatory regulation deserve attention.

The filamentous fungus Botryosphaeria rhodina (ATCC 9055) was investigated related to its ability for epoxide hydrolase (EH) production. Epoxide hydrolase activity is located at two different sites of the cells. The larger part is present in the cytosol (70%), while the smaller part is associated to membranes (30%). In media optimization experiments, an activity of 3.5 U/gDW for aromatic epoxide hydrolysis of para-nitro-styrene oxide (pNSO) could be obtained. Activity increased by 30% when pNSO was added to the culture during exponential growth. An increase of enzyme activity up to 6 U/gDW was achieved during batch-fermentations in a bioreactor with 2.7 l working volume. Evaluation of fermentations with 30 l working volume revealed a relation of oxygen uptake rate to EH expression. Oxygen limitation resulted in a decreased EH activity. Parameter estimation by the linearization method of Hanes yielded Km values of 2.54 and 1.00 mM for the substrates S-pNSO and R-pNSO, respectively. vmax was 3.4 times higher when using R-pNSO. A protein purification strategy leading to a 47-fold increase in specific activity (940 U/mgProtein) was developed as a first step to investigate molecular and structural characteristics of the EH.

We previously reported that Pseudomonas aeruginosa PA14 secretes a protein that can reduce the apical membrane expression of the c ystic f ibrosis t ransmembrane conductance r egulator (CFTR) protein. Here we report that we have used a proteomic approach to identify this secreted protein as PA2394, and we have named the gene cif , for C FTR i nhibitory f actor. We demonstrate that Cif is a secreted protein and is found associated with outer membrane-derived vesicles. Expression of Cif in Escherichia coli and purification of the C-terminal six-His-tagged Cif protein showed that Cif is necessary and sufficient to mediate the reduction in apical membrane expression of CFTR and a concomitant reduction in CFTR-mediated Cl − ion secretion. Cif demonstrates epoxide hydrolase activity in vitro and requires a highly conserved histidine residue identified in α/β hydrolase family enzymes to catalyze this reaction. Mutating this histidine residue also abolishes the ability of Cif to reduce apical membrane CFTR expression. Finally, we demonstrate that the cif gene is expressed in the cystic fibrosis (CF) lung and that nonmucoid isolates of P. aeruginosa show greater expression of the gene than do mucoid isolates. We propose a model in which the Cif-mediated decrease in apical membrane expression of CFTR by environmental isolates of P. aeruginosa facilitates the colonization of the CF lung by this microbe.

Rhodococcus sp. ML-0004, a novel strain for producing epoxide hydrolase, was isolated from soil in this study. The epoxide hydrolase can catalyze the stereo-specific hydrolysis of cis -epoxysuccinic acid to generate l (+)-tartaric acid. By examining physiological, biochemical characteristics and comparing its 16S rDNA gene sequence, it was identified as Rhodococcus opacus , and named R. opacus ML-0004. The optimal conditions for epoxide hydrolase production from R. opacus ML-0004 were also investigated. Propanediol and (NH 4 ) 2 SO 4 were selected as carbon source and nitrogen source, respectively, for the production of R. opacus ML-0004 epoxide hydrolase. The optimal conditions for epoxide hydrolase production were fermentation temperature = 28 °C, pH 7.0, and cultivation time = 26 h. Under these conditions, the maximum epoxide hydrolase activity reached 10.5 U mL −1 .

Leukotriene A4 hydrolase is a bifunctional zinc metalloenzyme with an epoxide hydrolase activity as well as an arginyl tri-peptidase activity. Detailed enzymological and mechanistic investigations of the latter activity have been hampered by the lack of a rapid and convenient enzyme assay. Here we have developed a new method allowing direct spectrophotometric assessment of the tri-peptide cleaving activity of leukotriene A4 hydrolase, as well as other peptidases. The method utilizes two competing substrates, one chromogenic reference substrate together with the tri-peptide substrate of interest, and relies on computer-assisted analysis of progress curves. The chromogenic reference substrate serves to disclose the "invisible" tri-peptide substrate for kinetic analysis. The method is fast and simple and will allow detailed kinetic studies and screening for natural peptide substrates of leukotriene A4 hydrolase as well as other members of the M1 family of aminopeptidases.

Soluble epoxide hydrolase (sEH) is a bifunctional enzyme with a C-terminal epoxide hydrolase activity and an N-terminal phosphatase activity. Arachidonic acid epoxides, previously suggested to be involved in apoptosis, oncogenesis and cell proliferation, are generated by cytochrome P450 epoxygenases and are good substrates of the sEH C-terminal domain. In addition, the N-terminal phosphatase domain hydrolyzes isoprenoid mono- and pyrophosphates, which are involved in cell signaling and apoptosis. Here we provide a comprehensive analysis of the distribution of sEH, CYP2C8, 2C9 and 2J2 in human neoplastic tissues using tissue micro-arrays. The human neoplastic tissue micro-arrays provide a well-controlled side by side analysis of a wide array of neoplastic tissues and their surrounding normal tissue controls. Many of the neoplastic tissues showed altered expression of these enzymes as compared to normal tissues. Altered expression was not limited to the neoplastic tissues but also found in the surrounding non-neoplastic tissues. For example, sEH expression in renal and hepatic malignant neoplasms and surrounding non-neoplastic tissues was found to be significantly decreased, whereas expression was found to be increased in seminoma as compared to normal tissues. Our study warrants further investigation of the role of altered expression of these enzymes in neoplastic tissues.

A marine microorganism, Sphingomonas echinoides EH-983, which possesses epoxide hydrolase (EH) activity was isolated from seawater and characterized. The EH of S. echinoides EH-983 preferentially metabolized (R)-enantiomer when the racemic styrene oxides were supplied as substrates. The optimal pH and temperature for the enantioselective hydrolysis by whole-cells of S. echinoides EH-983 were 7.0 and 20 °C, respectively. When kinetic resolution was conducted with a racemic mixture of styrene oxides at an initial concentration of 40 mM, enantiopure (S)-styrene oxide was obtained in 180 min with a yield of 21.3%. To our best knowledge, S. echinoides EH-983 is the first marine microorganism that is reported to have EH activity.

Soluble epoxide hydrolase (sEH) is highly expressed in human liver and contains a C-terminal epoxide hydrolase activity and an N-terminal phosphatase activity. Endogenous C-terminal hydrolase substrates include arachidonic acid epoxides, however, data are limited regarding possible endogenous substrates for the N-terminal phosphatase. Possible sEH N-terminal substrates include isoprenoid phosphate precursors of cholesterol biosynthesis and protein isoprenylation. Here, we report the kinetic analysis for a range of sEH isoprenoid substrates. We also provide an analysis of the effects of human sEH polymorphisms on isoprenoid hydrolysis. Interestingly, the Arg287Gln polymorphism recently suggested to be involved in hypercholesterolemia was found to possess a higher isoprenoid phosphatase activity than the wild type sEH. Consistent with the finding of isoprenoid phosphates as substrates for sEH, we identified isoprenoid-derived N-terminal inhibitors with IC50 values ranging from 0.84 (+/-0.9) to 55.1 (+/-30.7) microM. Finally, we evaluated the effects of the different isoprenoid compounds on the C-terminal hydrolase activity.