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J. Biol. Chem., Vol. 283, Issue 8, 4818-4824, February 22, 2008

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Substrate Orientation in Xanthine Oxidase

CRYSTAL STRUCTURE OF ENZYME IN REACTION WITH 2-HYDROXY-6-METHYLPURINE^*

James M. Pauff, Jinjin Zhang, Charles E. Bell, and Russ Hille¹

From the Department of Biochemistry, University of California, Riverside, California 92521 and the Department of Molecular & Cellular Biochemistry, The Ohio State University, Columbus, Ohio 43210

Xanthine oxidoreductase catalyzes the final two steps of purine catabolism and is involved in a variety of pathological states ranging from hyperuricemia to ischemia-reperfusion injury. The human enzyme is expressed primarily in its dehydrogenase form utilizing NAD⁺ as the final electron acceptor from the enzyme's flavin site but can exist as an oxidase that utilizes O₂ for this purpose. Central to an understanding of the enzyme's function is knowledge of purine substrate orientation in the enzyme's molybdenum-containing active site. We report here the crystal structure of xanthine oxidase, trapped at the stage of a critical intermediate in the course of reaction with the slow substrate 2-hydroxy-6-methylpurine at 2.3Å_. This is the first crystal structure of a reaction intermediate with a purine substrate that is hydroxylated at its C8 position as is xanthine and confirms the structure predicted to occur in the course of the presently favored reaction mechanism. The structure also corroborates recent work suggesting that 2-hydroxy-6-methylpurine orients in the active site with its C2 carbonyl group interacting with Arg-880 and extends our hypothesis that xanthine binds opposite this orientation, with its C6 carbonyl positioned to interact with Arg-880 in stabilizing the Mo^V transition state.

Received for publication, September 21, 2007 , and in revised form, October 24, 2007.

The atomic coordinates and structure factors (code 3B9J) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported by National Institutes of Health Grant GM 075036 (to R. H.). 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.

¹ To whom correspondence should be addressed: Dept. of Biochemistry, University of California, 2466 Boyce Hall, Riverside, California 92521. Tel.: 951-827-6354; Fax: 951-827-3719; E-mail: russ.hille{at}ucr.edu.

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