Structural and functional insights into polymorphic enzymes of cytochrome P450 2C8
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The cytochrome P450 (CYP) superfamily plays a key role in the oxidative metabolism of a wide range of drugs and exogenous chemicals. CYP2C8 is the principal enzyme responsible for the metabolism of the anti-cancer drug paclitaxel in the human liver. Nearly all previous works about polymorphic variants of CYP2C8 were focused on unpurified proteins, either cells or human liver microsomes; therefore their structure–function relationships were unclear. In this study, two polymorphic enzymes of CYP2C8 (CYP2C8.4 (I264M) and CYP2C8 P404A) were expressed in E. coli and purified. Metabolic activities of paclitaxel by the two purified polymorphic enzymes were observed. The activity of CYP2C8.4 was 25% and CYP2C8 P404A was 30% of that of WT CYP2C8, respectively. Their structure–function relationships were systematically investigated for the first time. Paclitaxel binding ability of CYP2C8.4 increased about two times while CYP2C8 P404A decreased about two times than that of WT CYP2C8. The two polymorphic mutant sites of I264 and P404, located far from active site and substrate binding sites, significantly affect heme and/or substrate binding. This study indicated that two important nonsubstrate recognition site (SRS) residues of CYP2C8 are closely related to heme binding and/or substrate binding. This discovery could be valuable for explaining clinically individual differences in the metabolism of drugs and provides instructed information for individualized medication.
KeywordsStructure–function relationship Polymorphism CYP2C8 Drug metabolism Individualized medication
We thank Prof. E.F. Johnson (The Scripps Research Institute, California, USA) for the generous gift of the pCWOri + plasmid, encoding CYP 2C8dH and Prof. J.M. Chen (Fudan University, Shanghai, China) for the generous support of paclitaxel. This work is supported in part by Shanghai Pujiang Talent Project (08PJ14017), the National Science Foundation of China (20771029), the Shanghai Leading Academic Discipline Project (B108), the Program for the Platform of new medicine creation (NO. 2009ZX09301-011), the Specialized Research Fund for the Doctoral Program of Higher Education, and Beijing Synchrotron Radiation Facility (BSRF) in the Institute of High Energy Physics, Chinese Academy of Sciences.
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