RESUMO
Epoxide hydrolases are a small superfamily of enzymes important for the detoxification of chemically reactive xenobiotic epoxides and for the processing of endogenous epoxides that act as signaling molecules. Here, we report the identification of two human epoxide hydrolases: EH3 and EH4. They share 45% sequence identity, thus representing a new family of mammalian epoxide hydrolases. Quantitative RT-PCR from mouse tissue indicates strongest EH3 expression in lung, skin, and upper gastrointestinal tract. The recombinant enzyme shows a high turnover number with 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acid (EET), as well as 9,10-epoxyoctadec-11-enoic acid (leukotoxin). It is inhibited by a subclass of N,N'-disubstituted urea derivatives, including 12-(3-adamantan-1-yl-ureido)-dodecanoic acid, 1-cyclohexyl-3-dodecylurea, and 1-(1-acetylpiperidin-4-yl)-3-(4-(trifluoromethoxy)phenyl)urea, compounds so far believed to be selective inhibitors of mammalian soluble epoxide hydrolase (sEH). Its sensitivity to this subset of sEH inhibitors may have implications on the pharmacologic profile of these compounds. This is particularly relevant because sEH is a potential drug target, and clinical trials are under way exploring the value of sEH inhibitors in the treatment of hypertension and diabetes type II.
Assuntos
Epóxido Hidrolases/metabolismo , Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Ácido 8,11,14-Eicosatrienoico/metabolismo , Animais , Epóxido Hidrolases/antagonistas & inibidores , Epóxido Hidrolases/química , Compostos de Epóxi/metabolismo , Humanos , Inativação Metabólica , Camundongos , Camundongos Endogâmicos C57BL , Filogenia , Ácidos Esteáricos/metabolismo , Xenobióticos/metabolismoRESUMO
Inhibition of fungal lanosterol-14 alpha-demethylase (CYP51) is the working principle of the antifungal activity of azoles used in agriculture and medicine. Inhibition of human CYP51 may result in endocrine disruption since follicular fluid-meiosis activating steroid (FF-MAS), the direct product of lanosterol demethylation, is involved in the control of meiosis. To investigate the specificity of antifungal agents for the fungal enzyme, assays to determine inhibitory potencies of 13 agricultural fungicides and 6 antimycotic drugs were established. FF-MAS product formation was measured by LC-MS/MS analysis in the incubations using lanosterol as substrate. Recombinant human enzyme (hCYP51) was available from BD Gentest. CYP51 of Candida albicans (cCYP51) was co-expressed with Candida tropicalis oxidoreductase in the baculovirus system. IC(50) values of 13 fungicides for cCYP51 ranged about six-fold (0.059-0.35 microM); for hCYP51 the range was about 30-fold (1.3-37.2 microM). The most favourable IC(50) ratio human to Candida was observed for imazalil (440-fold), while the specificity of epoxiconazole and tebuconazole for cCYP51 was only by a factor of 10. For the antimycotic drugs, the range of IC(50) values for cCYP51 was similar to those of fungicides (0.039-0.30 microM). For the inhibition of hCYP51, IC(50) values split into two classes: the newer drugs fluconazole and itraconazole showed little inhibition (> or = 30 microM) while the older drugs were even more potent than the agricultural fungicides, with miconazole being the most potent (0.057 microM). No correlation was seen between the IC(50) values determined for the two enzymes, indicating that a housekeeping gene can show significant diversity if inhibition is concerned. Our data indicate that fungicide residues in food are unlikely to exert a relevant inhibition of CYP51 in humans whereas systemic use of some antimycotic drugs, e.g. ketoconazole or miconazole, should be carefully considered regarding disturbance of human steroid biosynthesis.
Assuntos
Antifúngicos/farmacologia , Azóis/farmacologia , Candida albicans/enzimologia , Inibidores das Enzimas do Citocromo P-450 , Inibidores Enzimáticos/farmacologia , Proteínas Fúngicas/antagonistas & inibidores , Oxirredutases/antagonistas & inibidores , Azóis/química , Azóis/classificação , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/classificação , Contaminação de Alimentos , Humanos , Concentração Inibidora 50 , Resíduos de Praguicidas , Medição de Risco , Especificidade da Espécie , Esterol 14-Desmetilase , Relação Estrutura-AtividadeRESUMO
Epoxide hydrolases are a class of enzymes important in the detoxification of genotoxic compounds, as well as in the control of physiological signaling molecules. This chapter gives an overview on the function, structure, and enzymatic mechanism of structurally characterized epoxide hydrolases and describes selected assays for the quantification of epoxide hydrolase activity.
Assuntos
Bioensaio/métodos , Epóxido Hidrolases/química , Epóxido Hidrolases/metabolismo , Animais , Epóxido Hidrolases/genética , Humanos , Inativação Metabólica , Estrutura Molecular , Dobramento de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ácidos Esteáricos/análise , Ácidos Esteáricos/metabolismo , Xenobióticos/metabolismoRESUMO
We have recently reported that human soluble epoxide hydrolase (sEH) is a bifunctional enzyme with a novel phosphatase enzymatic activity. Based on a structural relationship with other members of the haloacid dehalogenase superfamily, the sEH N-terminal phosphatase domain revealed four conserved sequence motifs, including the proposed catalytic nucleophile D9, and several other residues potentially implicated in substrate turnover and/or Mg(2+) binding. To enlighten the catalytic mechanism of dephosphorylation, we constructed sEH phosphatase active-site mutants by site-directed mutagenesis. A total of 18 mutants were constructed and recombinantly expressed in Escherichia coli as soluble proteins, purified to homogeneity and subsequently analysed for their kinetic parameters. A replacement of residues D9, K160, D184 or N189 resulted in a complete loss of phosphatase activity, consistent with an essential function for catalysis. In contrast, a substitution of D11, T123, N124 and D185 leads to sEH mutant proteins with altered kinetic properties. We further provide evidence of the formation of an acylphosphate intermediate on D9 by liquid chromatography-tandem mass spectrometry based on the detection of homoserine after NaBH(4) reduction of the phosphorylated enzyme, which identifies D9 as the catalytic nucleophile. Surprisingly, we could only show such homoserine formation using the D11N mutant, which strongly suggests D11 to be involved in the acylphosphate hydrolysis. In the D11 mutant, the second catalytic step becomes rate limiting, which then allows trapping of the labile intermediate. Substrate turnover in the presence of (18)H(2)O revealed that the nucleophilic attack during the second reaction step occurs at the acylphosphate phosphorous. Based on these findings, we propose a two-step catalytic mechanism of dephosphorylation that involves the phosphate substrate hydrolysis by nucleophilic attack by the catalytic nucleophile D9 followed by hydrolysis of the acylphosphate enzyme intermediate supported by D11.