2003 Fiscal Year Final Research Report Summary
Structure and function complex flavo-proteins which produce free radicals
| Project/Area Number |
13480212
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Functional biochemistry
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| Research Institution | NIPPON MEDICAL SCHOOL |
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Principal Investigator |
NISHINO Takeshi NIPPON MEDICAL SCHOOL, Dept. Biochemistry and Molecular Biology, professor, 大学院・医学研究科, 教授 (40094312)
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| Co-Investigator(Kenkyū-buntansha) |
OKAMOTO Ken NIPPON MEDICAL SCHOOL, Dept, Biochemistry and Molecular Biology, instructor, 医学部, 助手 (60267143)
NISHINO Tomoko NIPPON MEDICAL SCHOOL, Dept. Biochemistry and Molecular Biology, instructor, 医学部, 助手 (80075613)
IWASAKI Toshio NIPPON MEDICAL SCHOOL, Dept. Biochemistry and Molecular Biology, lecturer, 医学部, 講師 (40277497)
MATSUMURA Tomohiro NIPPON MEDICAL SCHOOL, Dept, Biochemistry and Molecular Biology, instructor, 医学部, 助手 (20297930)
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| Project Period (FY) |
2001 – 2003
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| Keywords | Active Oxygen / Xanthine Dehydrogenase / Xanthine Oxidase / Metallo protein / X-ray crystallography / Non-heme iron / Peroxiredoxin / Iron-binding protein |
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| Research Abstract |
Mammalian xanthine oxidoreductase is synthesized as a dehydrogenase (XDH) but can be readily converted to its oxidase form (XO), either by proteolysis or modification of cysteine residues. The crystal structures of bovine milk XDH and XO demonstrated previously. We identify a unique cluster of amino acids, which plays a dual role by forming the core of a relay system for the XDH/XO transition and by gating a solvent channel leading toward the FAD ring. A more detailed structural comparison and site-directed mutagenesis analysis experiments showed that Phe549, Arg335, Trp336 and Arg427 sit at the center of a relay system that transmits modifications of the linker peptide to the active site loop (Gln423-Lys433). Further, we solved the crystal structure of the key intermediate in the hydroxylation reaction of xanthine oxidoreductase with a slow substrate, in which the carbon-oxygen bond of the product is formed, yet the product remains complexed to the molybdenum. This intermediate displays a stable, broad charge-transfer band at around 640 nm. The crystal structure of the complex indicates that the catalytically labile Mo-OH oxygen has formed a bond with a carbon atom of the substrate. A water molecule usually seen in the active site of the enzyme is absent in the present structure, which probably accounts for the stability of this intermediate toward ligand displacement by hydroxide.
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