Journal of Biochemistry Advance Access first published online on February 14, 2007
This version published online on February 15, 2007
Journal of Biochemistry, doi:10.1093/jb/mvm054
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© 2007 The Japanese Biochemical Society
Two Mutations Convert Mammalian Xanthine Oxidoreductase to Highly Superoxide-Productive Xanthine Oxidase
From the 1Department of Biochemistry and Molecular Biology, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo 113-8602, 2Department of Biomaterials Sciences, School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, and 3Departments of Biochemistry, Medical Biophysics, and Molecular and Medical Genetics, University of Toronto and Division of Cancer Genomics & Proteomics, Ontario Cancer Institute/University Health Network, MaRS/TMDT, 101 College Street, Toronto, ON, Canada M5G 1L7.
Address correspondence to: Takeshi Nishino, Department 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
Received January 9, 2007; Accepted January 30, 2007
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Abstract |
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Reactive oxygen species are generated by various systems, including NADPH oxidases, xanthine oxidoreductase (XOR), and mitochondrial respiratory enzymes, and contribute to many physiological and pathological phenomena. Mammalian xanthine dehydrogenase (XDH) can be converted to xanthine oxidase (XO), which produces both superoxide anion and hydrogen peroxide in a molar ratio of about 1:3, depending upon the conditions. Here, we present a mutant of rat XOR that displays mainly XO activity with a superoxide:hydrogen peroxide production ratio of about 6:1. In the mutant, tryptophan 335, which is a component of the amino acid cluster crucial for switching from the XDH to the XO conformation, was replaced with alanine, and phenylalanine 336, which modulates FAD’s redox potential through stacking interactions with the flavin cofactor, was changed to leucine. When the mutant was expressed in Sf9 cells, it was obtained in the XO form, and dithiothreitol treatment only partially restored the pyridine nucleotide-binding capacity. The crystal structure of the dithiothreitol-treated mutant at 2.3 Å resolution showed the enzyme’s two subunits to be quite similar, but not identical: the cluster involved in conformation-switching was completely disrupted in one subunit, but remained partly associated in the other one. The chain trace of the active site loop in this mutant is very similar to that of the bovine XO form. These results are consistent with the idea that the XDH and XO forms of the mutant are in an equilibrium that greatly favors the XO form, but the equilibrium is partly shifted towards the XDH form upon incubation with dithiothreitol.