Purification of an epoxide hydrolase from Rhodotorula glutinis

The epoxide hydrolase from Rhodotorula glutinis was isolated and initially characterized. The enzyme was membrane associated and could be solubilized by Triton X-100. Purification yielded an enzyme with sp. act. of 66 mol 1,2-epoxyhexane hydrolyzed min^–1 mg^–1 protein. The enzyme was not completely purified to homogeneity but, nevertheless, a major protein was isolated by SDS-PAGE for subsequential amino acid determination of peptide fragments. From sequence alignments to related enzymes, a high homology towards the active site sequences of other microsomal epoxide hydrolases was found. Molecular mass determinations indicated that the native enzyme exists as a homodimer, with a subunit molecular mass of about 45 kDa. Based upon these, this epoxide hydrolase is structurally related to other microsomal epoxide hydrolases.     

Attenuation of cisplatin nephrotoxicity by inhibition of soluble epoxide hydrolase

Cisplatin is a highly effective chemotherapeutic agent against many tumors; however, it is also a potent nephrotoxicant. Given that there have been no significant advances in our ability to clinically manage acute renal failure since the advent of dialysis, the development of novel strategies to ablate nephrotoxicity would represent a significant development. In this study, we investigated the ability of an inhibitor of soluble epoxide hydrolase (sEH), n-butyl ester of 12-(3-adamantan-1-yl-ureiido)-dodecanoic acid (nbAUDA), to attenuate cisplatin-induced nephrotoxicity. nbAUDA is quickly converted to AUDA and results in maintenance of high AUDA levels in vivo. Subcutaneous administration of 40 mg/kg of nbAUDA to C3H mice every 24 h resulted in elevated blood levels of AUDA; this protocol was also associated with attenuation of nephrotoxicity induced by cisplatin (intraperitoneal injection) as assessed by BUN levels and histological evaluation of kidneys. This is the first report of the use of sEH inhibitors to protect against acute nephrotoxicity and suggests a therapeutic potential of these compounds.     

Fluorometric assay for determining epoxide hydrolase activity

We have developed a rapid screening procedure that enables the screening of hundreds of enzyme samples or variants for epoxide hydrolase activity towards any substrate. The procedure detects the products of the enzymatic reaction via periodate cleavage and remaining fluorescence of carboxyfluorescein.     

Screening of epoxide hydrolase producing microorganisms for biotransformation of deoxynivalenol

Deoxynivalenol (DON) transformation products from selected time course experiments were analyzed by thin-layer chromatography. With the strainAlternaria alternata f. sp.lycopersici AS27-3, one major metabolite of DON in ethyl acetate was observed. This unidentified metabolite was more polar than DON and has a R_f value of 0.71. Derivatization indicated that this metabolite was probably an unidentified trichothecene. Screening of 29 other microbial isolates (bacteria, yeast, filamentous fungi) for DON transformation did not result in any active organism. Presented at the 26^th Mykotoxin-Workshop in Herrching, Germany, May 17–19, 2004     

Affinity purification and characterization of a yeast epoxide hydrolase

Purification of the membrane-associated epoxide hydrolase from the yeast Rhodosporidium toruloides CBS 0349 to electrophoretic homogeneity was achieved in a single chromatographic step employing the affinity ligand adsorbent Mimetic Green. More than 68% of the total epoxide hydrolase activity present in the whole cells was recovered from the membrane fraction. The enzyme was purified 26-fold with respect to the solubilized membrane proteins and was obtained in a 90% yield. The purified epoxide hydrolase has an apparent monomeric molecular weight of 54 kDa, and a pI of 7.3. The enzyme was optimally active at 30–40 °C, and pH 7.3–8.5. The enzyme is highly glycosylated with a carbohydrate content >42%. The specific activity of the purified enzyme for (±)-1,2-epoxyoctane is 172 mol min^–1 mg protein^–1. The amino acid composition of the protein was determined. This is the first report of a yeast epoxide hydrolase purified to homogeneity in milligram amounts.     

Molecular engineering of epoxide hydrolase and its application to asymmetric and enantioconvergent hydrolysis

Safety and regulatory issues favor increasing use of enantiopure compounds in pharmaceuticals. Enantiopure epoxides and diols are valuable intermediates in organic synthesis for the production of optically active pharmaceuticals. Enantiopure epoxide can be prepared using epoxide hydrolase (EH)-catalyzed asymmetric hydrolysis of its racemate. Enantioconvergent hydrolysis of racemic epoxides by EHs possessing complementary enantioselectivity and regioselectivity can lead to the formation of enantiopure vicinal diols with high yield. EHs are cofactor-independent and easy-to-use catalysts. EHs will attract much attention as commercial biocatalysts for the preparation of enantiopure epoxides and diols. In this paper, recent progress in molecular engineering of EHs is reviewed. Some examples and prospects of asymmetric and enantioconvergent hydrolysis reactions are discussed as supplements to molecular engineering to improve EH performance.     

Adamantyl thioureas as soluble epoxide hydrolase inhibitors

A series of inhibitors of the soluble epoxide hydrolase (sEH) containing one or two thiourea groups has been developed. Inhibition potency of the described compounds ranges from 50?μM to 7.2?nM. 1,7-(Heptamethylene)bis[(adamant-1-yl)thiourea] (6f) was found to be the most potent sEH inhibitor, among the thioureas tested. The inhibitory activity of the thioureas against the human sEH is closer to the value of activity against rat sEH rather than murine sEH. While being less active, thioureas are up to 7-fold more soluble than ureas, which makes them more bioavailable and thus promising as sEH inhibitors.     

Symmetric adamantyl-diureas as soluble epoxide hydrolase inhibitors

A series of inhibitors of the soluble epoxide hydrolase (sEH) containing two urea groups has been developed. Inhibition potency of the described compounds ranges from 2.0 μM to 0.4 nM. 1,6-(Hexamethylene)bis[(adamant-1-yl)urea] (3b) was found to be a potent slow tight binding inhibitor (IC₅₀ = 0.5 nM) with a strong binding to sEH (K_i = 3.1 nM) and a moderately long residence time on the enzyme (k_off = 1.05 × 10⁻³ s⁻¹; t_1/2 = 11 min).     

Novel aliphatic epoxide hydrolase activities from dematiaceous fungi

Abstract Epoxide hydrolases were found to be constitutively expressed in dematiaceous fungi coincident with secondary metabolite pigment production in stationary or idiophase. Washed-cell preparations of two fungi, Ulodadium atrum CMC 3280 and Zopfiella karachiensis CMC 3284, exhibited affinity for 2,2-dialkylated oxiranes, for which contrasting enantioselectivities were observed, but not for aromatic styrene oxide or alicyclic cyclohexene oxide type substrates. Lyophilised preparations of soluble epoxide hydrolase activities proved to be effective catalysts for the mild hydrolysis of aliphatic epoxides.     

Effects of human soluble epoxide hydrolase polymorphisms on isoprenoid phosphate hydrolysis

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.     

Development of fluorescent substrates for microsomal epoxide hydrolase and application to inhibition studies

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.     

Physico-chemical properties of the epoxide hydrolase from Rhodosporidium toruloides

Residue-specific chemical modification of amino acid residues of the microsomal epoxide hydrolase (mEH) from Rhodosporidium toruloides UOFS Y-0471 revealed that the enzyme is inactivated through modification of Asp/Glu and His residues, as well as through modification of Ser. Since Asp acts as the nucleophile, and Asp/Glu and His serve as charge relay partners in the catalytic triad of microsomal and soluble epoxide hydrolases during epoxide hydrolysis, inactivation of the enzyme by modification of the Asp/Glu and His residues agrees with the established reaction mechanism of these enzymes. However, the inactivation of the enzyme through modification of Ser residues is unexpected, suggesting that a Ser in the catalytic site is indispensable for substrate binding by analogy of the role of Ser residues in the related L-2-haloacid dehalogenases, as well as the ATPase and phosphatase enzymes. Co²⁺, Hg²⁺, Ag⁺, Mg²⁺ and Ca²⁺ inhibited enzyme activity and EDTA increased enzyme activity. The activation energy for inactivation of the enzyme was 167 kJ mol^–1. Kinetic constants for the enzyme could not be determined since unusual behaviour was displayed during hydrolysis of 1,2-epoxyoctane by the purified enzyme. Enantioselectivity w as strongly dependent on substrate concentration. When the substrate was added in concentrations ensuring two-phase conditions, the enantioselectivity was greatly enhanced. On the basis of these results, it is proposed that this enzyme acts at an interface, analogous to lipases.     

Cytosolic epoxide hydrolase from liver of control and clofibrate-treated mice. Structural comparison by HPLC peptide mapping

Cytosolic epoxide hydrolases purified from livers of control and clofibrate-induced male C57B1/6 mice were compared. The proteins were reduced, alkylated and cleaved with trypsin and chymotrypsin. The digests were analyzed by HPLC and no qualitative differences were observed in the peptide mapping profiles of the two types of epoxide hydrolase preparation. The amino acid compositions and N-terminal residues of selected tryptic peptides also gave identical results for the control and clofibrate-induced mice. Both intact proteins have e-amino-blocked N-termini. The two enzyme forms are concluded to have highly similar, if not identical, primary structures.Abbreviations HPLC high-performance liquid chromatography - DABITC dimethylaminoazobenzene isothiocyanate     

Fluorescent substrates for soluble epoxide hydrolase and application to inhibition studies

Inhibition of the mammalian soluble epoxide hydrolase (sEH) is a promising new therapy in the treatment of disorders resulting from hypertension and vascular inflammation. A spectrophotometric assay (4-nitrophenyl-trans-2,3-epoxy-3-phenylpropyl carbonate, NEPC) is currently used to screen libraries of chemicals; however this assay lacks the required sensitivity to differentiate the most potent inhibitors. A series of fluorescent alpha-cyanoester and alpha-cyanocarbonate epoxides that produce a strong fluorescent signal on epoxide hydrolysis by both human and murine sEH were designed as potential substrates for an in vitro inhibition assay. The murine enzyme showed a broad range of specificities, whereas the human enzyme showed the highest specificity for cyano(6-methoxy-naphthalen-2-yl)methyl trans-[(3-phenyloxiran-2-yl)methyl] carbonate. An in vitro inhibition assay was developed using this substrate and recombinant enzyme. The utility of the fluorescent assay was confirmed by determining the IC(50) values for a series of known inhibitors. The new IC(50) values were compared with those determined by spectrophotometric NEPC and radioactive tDPPO assays. The fluorescent assay ranked these inhibitors on the basis of IC(50) values, whereas the NEPC assay did not. The ranking of inhibitor potency generally agreed with that determined using the tDPPO assay. These results show that the fluorescence-based assay is a valuable tool in the development of sEH inhibitors by revealing structure-activity relationships that previously were seen only by using the costly and labor-intensive radioactive tDPPO assay.     

Inhibition of soluble epoxide hydrolase by fulvestrant and sulfoxides

The soluble epoxide hydrolase (sEH) is a key enzyme in the metabolism of epoxy-fatty acids, signaling molecules involved in numerous biologies. Toward finding novel inhibitors of sEH, a library of known drugs was tested for inhibition of sEH. We found that fulvestrant, an anticancer agent, is a potent (K_I = 26 nM) competitive inhibitor of sEH. From this observation, we found that alkyl-sulfoxides represent a new kind of pharmacophore for the inhibition of sEH.     

Discovery of 3,3-disubstituted piperidine-derived trisubstituted ureas as highly potent soluble epoxide hydrolase inhibitors

3,3-Disubstituted piperidine-derived trisubstituted urea entA-2b was discovered as a highly potent and selective soluble epoxide hydrolase (sEH) inhibitor. Despite the good compound oral exposure, excellent sEH inhibition in whole blood, and remarkable selectivity, compound entA-2b failed to lower blood pressure acutely in spontaneously hypertensive rats (SHRs). This observation further challenges the premise that sEH inhibition can provide a viable approach to the treatment of hypertensive patients.     

Exploring the size of the lipophilic unit of the soluble epoxide hydrolase inhibitors

Soluble epoxide hydrolase (sEH) inhibitors are potential drugs for several diseases. Adamantyl ureas are excellent sEH inhibitors but have limited metabolic stability. Herein, we report the effect of replacing the adamantane group by alternative polycyclic hydrocarbons on sEH inhibition, solubility, permeability and metabolic stability. Compounds bearing smaller or larger polycyclic hydrocarbons than adamantane yielded all good inhibition potency of the human sEH (0.4 ≤ IC₅₀ ≤ 21.7 nM), indicating that sEH is able to accommodate inhibitors of very different size. Human liver microsomal stability of diamantane containing inhibitors is lower than that of their corresponding adamantane counterparts.     

Immobilization of the epoxide hydrolase from Aspergillus niger

Three methods for the immobilization of the epoxide hydrolase from the fungus Aspergillus niger were tested. The highest immobilization yield (90%) and retention of activity (65%) were obtained by adsorption onto DEAE-cellulose compared to adsorption onto hydrophobic porous polypropylene and covalent linkage using Eupergit resin. The enzymatic properties of the immobilized enzyme were similar to those of the free enzyme with respect to the effect of temperature and pH on both activity and stability as well as the effect of solvent (DMF) on activity. The kinetic parameters were affected leading to lower K _M(app) and higher Vm _(app).     

Human soluble epoxide hydrolase: structural basis of inhibition by 4-(3-cyclohexylureido)-carboxylic acids

X-ray crystal structures of human soluble epoxide hydrolase (sEH) complexed with four different dialkylurea inhibitors bearing pendant carboxylate "tails" of varying length have been determined at 2.3-3.0 A resolution. Similarities among inhibitor binding modes reinforce the proposed roles of Y381 and/or Y465 as general acids that protonate the epoxide ring of the substrate in concert with nucleophilic attack of D333 at the electrophilic epoxide carbon. Additionally, the binding of these inhibitors allows us to model the binding mode of the endogenous substrate 14,15-epoxyeicosatrienoic acid. Contrasts among inhibitor binding modes include opposite orientations of inhibitor binding in the active-site hydrophobic tunnel. Alternative binding orientations observed for this series of inhibitors to human sEH, as well as the binding of certain dialkylurea inhibitors to human sEH and murine sEH, complicate the structure-based design of human sEH inhibitors with potential pharmaceutical applications in the treatment of hypertension. Thus, with regard to the optimization of inhibitor designs targeting human sEH, it is critical that human sEH and not murine sEH be utilized for inhibitor screening, and it is critical that structures of human sEH-inhibitor complexes be determined to verify inhibitor binding orientations that correlate with measured affinities.     

Isolation and characterization of Xenopus soluble epoxide hydrolase

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.