Effect of soluble epoxide hydrolase polymorphism on substrate and inhibitor selectivity and dimer formation

Morisseau, Christophe; Wecksler, Aaron T.; Deng, Catherine; Dong, Hua; Yang, Jun; Lee, Kin Sing Stephen; Kodani, Sean D.; Hammock, Bruce D.

doi:10.1194/jlr.m049718

Cited by 38 publications

(53 citation statements)

References 40 publications

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“…Incubation times were optimized to ensure that the total turnover was <5% for the preferred substrate. The amount of each diol formed was quantified by LC-MS/MS as previously described (Morisseau et al, 2014). Results are mean ± SD (n = 3).…”

Section: Star Methodsmentioning

confidence: 99%

Active-Site Flexibility and Substrate Specificity in a Bacterial Virulence Factor: Crystallographic Snapshots of an Epoxide Hydrolase

et al. 2017

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Summary The CFTR inhibitory factor (Cif) is an epoxide-hydrolase virulence factor from Pseudomonas aeruginosa, with catalytic activity that perturbs essential host-defense networks. Its targets 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 α/β-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.

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Section: Star Methodsmentioning

confidence: 99%

Active-Site Flexibility and Substrate Specificity in a Bacterial Virulence Factor: Crystallographic Snapshots of an Epoxide Hydrolase

et al. 2017

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“…13 In addition, chicken sEH (chxEH) is active on EETs and the catalytic residues between chxEH and human sEH are conserved. 14 The overall selectivity of chxEH for a series of epoxy-fatty acids (Figure 1) is similar to the human sEH, 15 with a clear preference for the epoxide of DHA over the epoxides of EPA, ARA or linoleic acid. The kinetic constants for chxEH’s best substrate, 16,17-epoxy-docosapentaenoic acid, yield a K m (12 ± 3 μM) that is similar to the one of the human sEH, but a V max (728 ± 97 nmol.min −1 .mg −1 ) that is roughly 10-fold lower than the one measured for the human sEH.…”

mentioning

confidence: 74%

“…Selectivity was measured using a mixture of 14 epoxy-fatty acids each at a concentration of 1 μM, and the diols formed were quantified by LC/MS-MS. 15 Di-HOME: diols from linoleic acid epoxides; DHET: diol from arachidonic acid epoxides; Di-HETE: diols from EPA epoxides; Di-HPDE: diols from DHA epoxides.…”

Section: Figurementioning

confidence: 99%

Identification of potent inhibitors of the chicken soluble epoxide hydrolase

Shihadih

Harris

Yang

et al. 2015

Bioorganic & Medicinal Chemistry Letters

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In vertebrates, soluble epoxide hydrolase (sEH) hydrolyzes natural epoxy-fatty acids (EpFAs), which are chemical mediators modulating inflammation, pain, and angiogenesis. Chick embryos are used to study angiogenesis, particularly its role in cardiovascular biology and pathology. To find potent and bio-stable inhibitors of the chicken sEH (chxEH) a library of human sEH inhibitors was screened. Derivatives of 1(adamantan-1-yl)-3-(trans-4-phenoxycyclohexyl) urea were found to be very potent tight binding inhibitors (KI < 150 pM) of chxEH while being relatively stable in chicken liver microsomes, suggesting their usefulness to study the role of EpFAs in chickens.

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“…To our knowledge, there are no other antibodies for ELISA of native sEH. The existing polyclonal antibody is mostly used for western blot analysis, and was recently reported to have a limit of detection around 10 ng of purified human sEH loaded into a gel [39]. With a theoretical loading volume of 50 μL, and a detection limit of 15.6 ng/mL, our developed sandwich ELISA should be able to detect down to 0.8 ng of human sEH, suggesting a greater sensitivity than available polyclonal antibodies against the human sEH.…”

Section: Resultsmentioning

confidence: 99%

“…To study the utility of the sandwich ELISA, the concentration of sEH was determined with the sandwich ELISA in human tissue S9 samples (liver, kidney, and lung) (Table 3), and compared with the enzyme activity and Western blot method [39]. There are no significant variations between the enzyme activity and sandwich ELISA method.…”

Section: Resultsmentioning

confidence: 99%

Heavy chain single-domain antibodies to detect native human soluble epoxide hydrolase

Cui

Morisseau

et al. 2015

Anal Bioanal Chem

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The soluble epoxide hydrolase (sEH) is a potential pharmacological target for treating hypertension, vascular inflammation, pain, cancer and other diseases. However, there is not a simple, inexpensive and reliable method to estimate levels of active sEH in tissues. Toward developing such an assay, a polyclonal-variable domain of heavy chain antibody (VHH) sandwich immunoassay was developed. Ten VHHs, which are highly selective for native human sEH, were isolated from a phage displayed library. The ten VHHs have no significant cross-reactivity with human microsomal epoxide hydrolase, rat and mouse sEH, and denatured human sEH. There is a high correlation between protein levels of the sEH determined by the ELISA and the catalytic activity of the enzyme in S9 fractions of human tissues (liver, kidney and lung). The VHH based ELISA appears to be a new reliable method for monitoring the sEH, and may be useful as a diagnostic tool for diseases influenced by sEH. This study also demonstrates the broad utility of VHH in biochemical and pharmacological research.

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Effect of soluble epoxide hydrolase polymorphism on substrate and inhibitor selectivity and dimer formation

Cited by 38 publications

References 40 publications

Active-Site Flexibility and Substrate Specificity in a Bacterial Virulence Factor: Crystallographic Snapshots of an Epoxide Hydrolase

Active-Site Flexibility and Substrate Specificity in a Bacterial Virulence Factor: Crystallographic Snapshots of an Epoxide Hydrolase

Identification of potent inhibitors of the chicken soluble epoxide hydrolase

Heavy chain single-domain antibodies to detect native human soluble epoxide hydrolase

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