Analysis of Human Cytochrome P450 2C8 Substrate Specificity Using a Substrate Pharmacophore and Site-Directed Mutants
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- 作者：Armelle Melet ; Cristina Marques-Soares ; Guillaume A. Schoch ; Anne-Christine Macherey ; Maryse Jaouen ; Patrick M. Dansette ; Marie-Agnè ; s Sari ; Eric F. Johnson ; and Daniel Mansuy
- 刊名：Biochemistry
- 出版年：2004
- 出版时间：December 14, 2004
- 年：2004
- 卷：43
- 期：49
- 页码：15379 - 15392
- 全文大小：501K
- 年卷期：v.43,no.49(December 14, 2004)
- ISSN：1520-4995
文摘
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 substratepharmacophore and a three-dimensional (3D) model. Analysis of the structural features common to CYP2C8substrates exhibiting a micromolar Km led to a substrate pharmacophore in which the site of oxidation byCYP2C8 is 12.9, 8.6, 4.4, and 3.9 Å from features that could establish ionic or hydrogen bonds, andhydrophobic interactions with protein amino acid residues. Comparison of this pharmacophore with a 3Dmodel of CYP2C8 constructed using the X-ray structure of CYP2C5 suggested potential CYP2C8 aminoacid residues that could be involved in substrate recognition. Twenty CYP2C8 site-directed mutants wereconstructed and expressed in yeast to compare their catalytic activities using five CYP2C8 substrates thatexhibit 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 anionicsubstrates of CYP2C8 such as retinoic acid and fluvastatin. Serine 100 appears to be involved in hydrogenbonding 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 favorablehydrophobic interactions or to steric clashes with substrates. For instance, the S114F mutant was unableto catalyze the 6
-hydroxylation of paclitaxel. The S114F and F205A mutants were the best catalysts forretinoic acid and paclitaxel (or fluvastatin) hydroxylation, respectively, with kcat/Km values 5 and 2.1 (or2.4) times higher, respectively, than those found for CYP2C8. Preliminary experiments of docking of thesubstrate into the experimentally determined X-ray structure of substrate-free CYP2C8, which becameavailable quite recently [Schoch, G. A., et al. (2004) J. Biol. Chem. 279, 9497], were consistent with keyroles for S100, S114, and F205 residues in substrate binding. The results suggest that the effects of mutationof arginine 241 on anionic substrate hydroxylation could be indirect and result from alterations of thepacking of helix G with helix B'.
-hydroxylation of paclitaxel. The S114F and F205A mutants were the best catalysts forretinoic acid and paclitaxel (or fluvastatin) hydroxylation, respectively, with kcat/Km values 5 and 2.1 (or2.4) times higher, respectively, than those found for CYP2C8. Preliminary experiments of docking of thesubstrate into the experimentally determined X-ray structure of substrate-free CYP2C8, which becameavailable quite recently [Schoch, G. A., et al. (2004) J. Biol. Chem. 279, 9497], were consistent with keyroles for S100, S114, and F205 residues in substrate binding. The results suggest that the effects of mutationof arginine 241 on anionic substrate hydroxylation could be indirect and result from alterations of thepacking of helix G with helix B'.