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Analysis of Human Cytochrome P450 2C8 Substrate Specificity Using a Substrate Pharmacophore and Site-Directed Mutants

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Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, UMR 8601 CNRS, Université Paris 5, 45 Rue des Saints-Pères, 75270 Paris Cedex 06, France, and Department of Molecular and Experimental Medicine, The Scripps Research Institute, 10550 North Torrey Pines Road, MEM-255, La Jolla, California 92037
Cite this: Biochemistry 2004, 43, 49, 15379–15392
Publication Date (Web):November 18, 2004
Copyright © 2004 American Chemical Society

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    The structural determinants of substrate specificity of human liver cytochrome P450 2C8 (CYP2C8) were investigated using site-directed mutants chosen on the basis of a preliminary substrate pharmacophore and a three-dimensional (3D) model. Analysis of the structural features common to CYP2C8 substrates exhibiting a micromolar Km led to a substrate pharmacophore in which the site of oxidation by CYP2C8 is 12.9, 8.6, 4.4, and 3.9 Å from features that could establish ionic or hydrogen bonds, and hydrophobic interactions with protein amino acid residues. Comparison of this pharmacophore with a 3D model of CYP2C8 constructed using the X-ray structure of CYP2C5 suggested potential CYP2C8 amino acid residues that could be involved in substrate recognition. Twenty CYP2C8 site-directed mutants were constructed and expressed in yeast to compare their catalytic activities using five CYP2C8 substrates that exhibit different structures and sizes [paclitaxel, fluvastatin, retinoic acid, a sulfaphenazole derivative (DMZ), and diclofenac]. Mutation of arginine 241 had marked effects on the hydroxylation of anionic substrates of CYP2C8 such as retinoic acid and fluvastatin. Serine 100 appears to be involved in hydrogen bonding interactions with a polar site of the CYP2C8 substrate pharmacophore, as shown by the 3−4-fold increase in the Km of paclitaxel and DMZ hydroxylation after the S100A mutation. Residues 114, 201, and 205 are predicted to be in close contact with substrates, and their mutations lead either to favorable hydrophobic interactions or to steric clashes with substrates. For instance, the S114F mutant was unable to catalyze the 6α-hydroxylation of paclitaxel. The S114F and F205A mutants were the best catalysts for retinoic acid and paclitaxel (or fluvastatin) hydroxylation, respectively, with kcat/Km values 5 and 2.1 (or 2.4) times higher, respectively, than those found for CYP2C8. Preliminary experiments of docking of the substrate into the experimentally determined X-ray structure of substrate-free CYP2C8, which became available quite recently [Schoch, G. A., et al. (2004) J. Biol. Chem. 279, 9497], were consistent with key roles for S100, S114, and F205 residues in substrate binding. The results suggest that the effects of mutation of arginine 241 on anionic substrate hydroxylation could be indirect and result from alterations of the packing of helix G with helix B‘.

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     The research was supported by funds from CNRS and Ministry of Research (France) (group of D.M.) and by the U.S. National Institutes of Health (Grant GM031001, group of E.F.J.).

     UMR 8601 CNRS.


     The Scripps Research Institute.


     To whom correspondence should be addressed. Telephone: 33 1 42 86 21 69. Fax:  33 1 42 86 83 87. E-mail:  daniel.mansuy@

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