Dataset Information

Coumarin Derivatives as Substrate Probes of Mammalian Cytochromes P450 2B4 and 2B6: Assessing the Importance of 7-Alkoxy Chain Length, Halogen Substitution, and Non-Active Site Mutations.

ABSTRACT: Using a combined structural and biochemical approach, the functional importance of a recently described peripheral pocket bounded by the E-, F-, G-, and I-helices in CYP2B4 and 2B6 was probed. Three series of 4-substituted-7-alkoxycoumarin derivatives with -H, -CH3, or -CF3 at the 4 position of the coumarin core were used initially to monitor functional differences between CYP2B4 and 2B6. 7-Ethoxy-4-(trifluoromethyl)coumarin (7-EFC) displayed the highest catalytic efficiency among these substrates. Mutants were made to alter side-chain polarity (V/E194Q) or bulk (F/Y244W) to alter access to the peripheral pocket. Modest increases in catalytic efficiency of 7-EFC O-deethylation by the mutants were magnified considerably by chlorination or bromination of the substrate ethoxy chain. A structure of CYP2B6 Y244W in complex with (+)-?-pinene was solved at 2.2 Å and showed no CYMAL-5 in the peripheral pocket. A ligand free structure of CYP2B4 F244W was solved at 3.0 Å with CYMAL-5 in the peripheral pocket. In both instances, comparison of the respective wild-type and mutant CYP2B enzymes revealed that CYMAL-5 occupancy of the peripheral pocket had little effect on the topology of active site residue side-chains, despite the fact that the peripheral pocket and active site are located on opposite sides of the I-helix. Analysis of available CYP2B structures suggest that the effect of the amino acid substitutions within the peripheral pocket derive from altered interactions between the F and G helices.

SUBMITTER: Liu J

PROVIDER: S-EPMC4864431 | biostudies-literature | 2016 Apr

REPOSITORIES: biostudies-literature

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Coumarin Derivatives as Substrate Probes of Mammalian Cytochromes P450 2B4 and 2B6: Assessing the Importance of 7-Alkoxy Chain Length, Halogen Substitution, and Non-Active Site Mutations.

Liu Jingbao J Shah Manish B MB Zhang Qinghai Q Stout C David CD Halpert James R JR Wilderman P Ross PR

Biochemistry 20160324 13

Using a combined structural and biochemical approach, the functional importance of a recently described peripheral pocket bounded by the E-, F-, G-, and I-helices in CYP2B4 and 2B6 was probed. Three series of 4-substituted-7-alkoxycoumarin derivatives with -H, -CH3, or -CF3 at the 4 position of the coumarin core were used initially to monitor functional differences between CYP2B4 and 2B6. 7-Ethoxy-4-(trifluoromethyl)coumarin (7-EFC) displayed the highest catalytic efficiency among these substrat ...[more]

PMID: 26982502

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Project description:Xanthates (alkyl or aryl derivatives of dithiocarbonic acid) have been shown to be selective mechanism-based inactivators of cytochromes P450 2B1/2B6 and 2E1 due to covalent binding of a reactive intermediate to apoprotein after double hydrogen abstraction at α-carbon atom, suggesting interaction of the xanthate dithiocarbonic head with the enzyme heme. The structures of xanthates with a long alkyl chain are similar to the fatty acids. Saturated fatty acids (FA) such as lauric acid (LA), are metabolized by different cytochrome P450 isoforms to ω- and (ω-1)-hydroxy products, in humans done by CYP4A11 and CYP2E1, respectively. In the present study we aimed at elucidating the possible interactions of xanthates with two cytochrome P450 isoforms CYP4A11 and CYP2E1 involved in the metabolism of the FA. Our experiments showed that LA-ω-hydroxylation by CYP4A11 is inhibited in a competitive manner by xanthates with long alkyl chain (C12-xanthate being the most potent inhibitor). On the other hand LA-(ω-1)-hydroxylation reaction by purified CYP2E1 is inactivated by a mechanism-based type. The suggested differences in the interactions of C12-xanthate with the two cytochrome P450 isoforms were investigated by molecular modeling using docking approach. The results suggested that in CYP2E1 active site C12-xanthate coordinates to the heme with its most vulnerable dithiocarbonic head leading to a mechanism-based inactivation. In CYP4A11 xanthate alkyl chain is exposed to the heme, thus, a potenial ω-hydroxylated xanthate product could be formed, which could inhibit in a competitive manner the hydroxylation of LA. The observed differences of xanthates interactions with the active sites of the two similar cytochrome P450 isoforms (CYP4A11 and CYP2E1) involved in the metabolism of FA, which lead to different changes in the enzyme activity, suggest that xanthates can be used as probing tools for analyzing enzyme active sites when exploring useful and selective compounds influencing FA homeostasis.

Dataset Information

Coumarin Derivatives as Substrate Probes of Mammalian Cytochromes P450 2B4 and 2B6: Assessing the Importance of 7-Alkoxy Chain Length, Halogen Substitution, and Non-Active Site Mutations.

Publications

Coumarin Derivatives as Substrate Probes of Mammalian Cytochromes P450 2B4 and 2B6: Assessing the Importance of 7-Alkoxy Chain Length, Halogen Substitution, and Non-Active Site Mutations.

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