RESUMO
CYP20A1 is a well-conserved member of the human cytochrome P450 enzyme family for which no endogenous or xenobiotic substrate is known. We have recently shown that this enzyme has moderate activity towards two proluciferin probe substrates. In order to facilitate the search for physiological substrates we have tested nine additional proluciferins in this study and identified three such probe substrates that give much higher product yields. Using one of these probes, we demonstrate inhibition of CYP20A1 activity by 1-benzylimidazole, ketoconazole and letrozole. Finally, we show that the combination of two common single nucleotide polymorphisms (SNPs) of CYP20A1 leads to an enzyme (CYP20A1Leu97Phe346) with reduced activity.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Imidazóis/farmacologia , Cetoconazol/farmacologia , Letrozol/farmacologia , Sistema Enzimático do Citocromo P-450/genética , Humanos , Estrutura Molecular , Especificidade por SubstratoRESUMO
The human cytochrome P450 (CYP) enzyme CYP4A22 is an orphan CYP with unknown function. Here, through functional expression in fission yeast, we show that CYP4A22 catalyzes fatty acid hydroxylation as well as aliphatic or aromatic hydroxylations of luciferin-based probe substrates. Mechanistic molecular modeling of CYP4A22 suggests that its ω-hydroxylation activity is hampered by a more spacious active site compared to CYP4B1. Substrate recognition via side-chains R96 and R233 is indicated by dynamic three-dimensional pharmacophores (dynophores) derived from molecular dynamics simulations. CYP4A22 activity is inhibited by three unspecific CYP inhibitors. A comparison of CYP4A22*1 (the reference standard sequence) with CYP4A22-WT (the most common allele) revealed that for the four substrates tested the WT-enzyme always had lower activity.
Assuntos
Citocromo P-450 CYP4A/química , Citocromo P-450 CYP4A/metabolismo , Ácidos Láuricos/metabolismo , Ácidos Mirísticos/metabolismo , Arginina/metabolismo , Domínio Catalítico , Citocromo P-450 CYP4A/genética , Inibidores das Enzimas do Citocromo P-450/química , Inibidores das Enzimas do Citocromo P-450/farmacologia , Humanos , Hidroxilação , Modelos Moleculares , Simulação de Dinâmica Molecular , Estrutura Secundária de Proteína , Schizosaccharomyces/genética , Schizosaccharomyces/crescimento & desenvolvimento , Especificidade por SubstratoRESUMO
Here, a complete set of recombinant fission yeast strains that coexpress each of the 57 human cytochrome P450 (CYP) enzymes together with their natural human electron transfer partner(s) was cloned. This strain collection was tested with two luminogenic probe substrates, and 31 human CYPs (including the orphan enzymes CYP2A7, CYP4A22 and CYP20A1) were found to metabolize at least one of these. Since other substrates are known for the remaining enzymes, all human CYPs are now shown to be active. Interestingly, CYP5A1 was found for the first time to work on a substrate other than prostaglandin H2 , and, moreover, to catalyze an aliphatic hydroxylation reaction that consumes molecular oxygen. Also, the ability of CYP11A1 to catalyze an aryl hydroxylation is another unexpected result.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Schizosaccharomyces/enzimologia , Catálise , Sistema Enzimático do Citocromo P-450/genética , Humanos , Schizosaccharomyces/genética , Especificidade por SubstratoRESUMO
The human cytochrome P450 enzyme CYP8B1 is a crucial regulator of the balance of cholic acid (CA) and chenodeoxycholic acid (CDCA) in the liver. It was previously shown to catalyze the conversion of 7α-hydroxycholest-4-en-3-one, a CDCA precursor, to 7α,12α-dihydroxycholest-4-en-3-one, which is an intermediate of CA biosynthesis. In this study we demonstrate that CYP8B1 can also convert CDCA itself to CA. We also show that five derivatives of luciferin are metabolized by CYP8B1 and established a rapid and convenient inhibitor test system. In this way we were able to identify four new CYP8B1 inhibitors, which are aminobenzotriazole, exemestane, ketoconazole and letrozole.