| Project/Area Number |
14580616
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Bioorganic chemistry
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| Research Institution | RIKEN (2003-2004)
Osaka City University (2002) |
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Principal Investigator |
SHIRO Yoshitsugu (2003-2004) RIKEN, Biometal Science Lab., Chief Scientist, 城生体金属科学研究室, 主任研究員 (70183051)
松永 勇 (2002) 大阪市立大学, 大学院・医学研究科, 講師 (00254425)
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| Co-Investigator(Kenkyū-buntansha) |
SUGIMOTO Hiroshi RIKEN, Biometal Science Lab., Research Scientist, 城生体金属科学研究室, 研究員 (90344043)
KIM Misa RIKEN, Biometal Science Lab., 城生体金属科学研究室, 基礎科学特別研究員 (40373295)
城 宣嗣 理化学研究所, 生体物理化学研究室, 主任研究員 (70183051)
小倉 壽 大阪市立大学, 大学院・医学研究科, 教授 (10115222)
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| Project Period (FY) |
2002 – 2004
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| Project Status |
Completed (Fiscal Year 2004)
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| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2004: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 2003: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 2002: ¥1,500,000 (Direct Cost: ¥1,500,000)
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| Keywords | Cytochrome P450 / Hydroxylation of Fatty Acids / Hydrogen Peroxide / Heme-enzymes / Oxygenation / X-ray Crystallography / シトクロムP450 / ペルオキシゲナーゼ / ペルオキシダーゼ / モノオキシゲナーゼ / 反応機構 / 脂肪酸 |
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| Research Abstract |
Our research aim in this project was to understand the relationship of function and structure of cytochrome P450Bsβ from Bacillus subtilis, which can catalyze the hydroxylation of α- and β-positions of fatty acids using hydrogen peroxide H_2O_2 as an oxygen donor. We succeeded in making the single crystal and afterward in determining the structure (2.1Å resolution) of the ferric (Fe^<3+>) enzyme in the presence of a substrate (palimitic acid). On the basis of the structure, we prepared some mutants, and measured their enzymatic activities and spectral properties, to reveal roles of the mutated residues in the enzymatic functions (specificities in the substrate recognition, the catalytic reactions, the reaction sites, etc.). Then we proposed the molecular mechanism of the reaction catalyzed by P450Bsβ. In the mechanism, Arg242 binds with the carboxylate of the substrate fatty acid, which acts as a general acid-base catalyst in the peroxide O-O bond cleavage. The CO complex of the substrate-bound enzyme was determined. It was found that Phe79 was moved, but others were not upon the CO binding to the ferrous iron. To obtain a crystal of this enzyme in the substrate-free form, we examined the purification procedures for removal of the bound substrate by gel-filteration or by the product formation with H_2O_2. But we have not yet obtained the crystal of the substrate-free enzyme. In pursuing the structural determination of the short-lived reaction intermediate (possibly the Fe^<5+>=O state), we tried to stabilize the oxy complex of the enzyme in solution state, and then to inject two electrons with the X-ray reduction technique. We found the partial formation of the oxy complex. Finally, we discussed details of the reaction mechanism of heme-containing enzymes which utilize hydrogen peroxide.
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