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J. Biol. Chem., Vol. 280, Issue 26, 24888-24894, July 1, 2005

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Mechanism of the Conversion of Xanthine Dehydrogenase to Xanthine Oxidase

IDENTIFICATION OF THE TWO CYSTEINE DISULFIDE BONDS AND CRYSTAL STRUCTURE OF A NON-CONVERTIBLE RAT LIVER XANTHINE DEHYDROGENASE MUTANT^*

Tomoko Nishino, Ken Okamoto, Yuko Kawaguchi, Hiroyuki Hori, Tomohiro Matsumura, Bryan T. Eger¶, Emil F. Pai¶, and Takeshi Nishino||

From the Department of Biochemistry and Molecular Biology, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo 113-8602, Japan and ^¶Departments of Biochemistry, Medical Biophysics, and Molecular and Medical Genetics, Division of Molecular and Structural Biology, University of Toronto, Ontario Cancer Institute/University Health Network, Toronto, Ontario M5G 2M9, Canada

Mammalian xanthine dehydrogenase can be converted to xanthine oxidase by modification of cysteine residues or by proteolysis of the enzyme polypeptide chain. Here we present evidence that the Cys⁵³⁵ and Cys⁹⁹² residues of rat liver enzyme are indeed involved in the rapid conversion from the dehydrogenase to the oxidase. The purified mutants C535A and/or C992R were significantly resistant to conversion by incubation with 4,4'-dithiodipyridine, whereas the recombinant wild-type enzyme converted readily to the oxidase type, indicating that these residues are responsible for the rapid conversion. The C535A/C992R mutant, however, converted very slowly during prolonged incubation with 4,4'-dithiodipyridine, and this slow conversion was blocked by the addition of NADH, suggesting that another cysteine couple located near the NAD⁺ binding site is responsible for the slower conversion. On the other hand, the C535A/C992R/C1316S and C535A/C992R/C1324S mutants were completely resistant to conversion, even on prolonged incubation with 4,4'-dithiodipyridine, indicating that Cys¹³¹⁶ and Cys¹³²⁴ are responsible for the slow conversion. The crystal structure of the C535A/C992R/C1324S mutant was determined in its demolybdo form, confirming its dehydrogenase conformation.

Received for publication, February 17, 2005 , and in revised form, March 28, 2005.

The atomic coordinates and structure factors (code 1WYG) 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 Grants-in-aid 08249104 and 11169231 (to Takeshi Nishino) for Science Research on Priority Areas and a Grant-in-aid 09480167 (to Takeshi Nishino) for Science Research from the Ministry of Education, Science, Sports, and Culture of Japan and a grant from the Canadian Institutes for Health Research (to E. F. P.). 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 on-line version of this article (available at http://www.jbc.org) contains supplemental text and references, Tables S1 and S2, and Figs. S1-S4.

Present address: Dept. of Applied Chemistry, Faculty of Engineering, Ehime University, Matsuyama 790-8577, Japan.

^|| To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo 113-8602, Japan. Tel.: 81-3-3822-2131; Fax: 81-3-5685-3054; E-mail: nishino{at}nms.ac.jp.

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