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J. Biol. Chem., Vol. 279, Issue 34, 35630-35637, August 20, 2004

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The Structure of Human Cytochrome P450 2C9 Complexed with Flurbiprofen at 2.0-Å Resolution^*

Michael R. Wester, Jason K. Yano, Guillaume A. Schoch, Christine Yang, Keith J. Griffin, C. David Stout¶||, and Eric F. Johnson**

From the Departments of Molecular and Experimental Medicine and ^¶Molecular Biology, The Scripps Research Institute, La Jolla, California 92037

The structure of human P450 2C9 complexed with flurbiprofen was determined to 2.0 Å by x-ray crystallography. In contrast to other structurally characterized P450 2C enzymes, 2C5, 2C8, and a 2C9 chimera, the native catalytic domain of P450 2C9 differs significantly in the conformation of the helix F to helix G region and exhibits an extra turn at the N terminus of helix A. In addition, a distinct conformation of the helix B to helix C region allows Arg-108 to hydrogen bond with Asp-293 and Asn-289 on helix I and to interact directly with the carboxylate of flurbiprofen. These interactions position the substrate for regioselective oxidation in a relatively large active site cavity and are likely to account for the high catalytic efficiency exhibited by P450 2C9 for the regioselective oxidation of several anionic non-steroidal anti-inflammatory drugs. The structure provides a basis for interpretation of a number of observations regarding the substrate selectivity of P450 2C9 and the observed effects of mutations on catalysis.

Received for publication, May 17, 2004 , and in revised form, June 3, 2004.

^* This work was supported by National Institutes of Health Grant GM031001 (to E. F. J.). Facilities for computer-assisted sequence analysis, DNA sequencing, and the synthesis of oligonucleotides were supported in part by General Clinical Research Center Grant M01 RR00833 and by the Sam and Rose Stein Charitable Trust. Portions of this research were carried out at the Stanford Synchrotron Radiation Laboratory (SSRL), a national user facility operated by Stanford University on behalf of the United States Department of Energy, Office of Basic Energy Sciences. The SSRL Structural Molecular Biology Program is supported by the United States Department of Energy, Office of Biological and Environmental Research and by the National Center for Research Resources, Biomedical Technology Program, and NIGMS of the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The atomic coordinates and structure factors (code 1R9O) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

Both authors contributed equally to this work.

^|| To whom correspondence may be addressed: Dept. of Molecular Biology, The Scripps Research Institute, 10550 N. Torrey Pines Rd., MB8, La Jolla, CA 92037. Tel.: 858-784-8738; Fax: 858-784-2857; E-mail: dave{at}scripps.edu. ** To whom correspondence may be addressed: Dept. of Molecular and Experimental Medicine, The Scripps Research Institute, 10550 N. Torrey Pines Rd., MEM-255, La Jolla, CA 92037. Tel.: 858-784-7918; Fax: 858-784-7978; E-mail: johnson{at}scripps.edu.

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