| Project/Area Number |
13125206
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| Research Category |
Grant-in-Aid for Scientific Research on Priority Areas
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| Allocation Type | Single-year Grants |
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| Review Section |
Science and Engineering
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| Research Institution | KUMAMOTO UNIVERSITY |
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Principal Investigator |
MIURA Retsu Kumamoto Univ., Graduate School of Medical Sciences, Professor, 大学院・医学薬学研究部, 教授 (70093466)
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| Co-Investigator(Kenkyū-buntansha) |
TAMAOKI Haruhiko Kumamoto Univ., Graduate School of Medical Sciences, Instructor, 大学院・医学薬学研究部, 助手 (80264290)
SETOYAMA Chiaki Kumamoto Univ., Graduate School of Medical Sciences, Assoc. Prof., 大学院・医学薬学研究部, 助手 (60040250)
NISHINA Yasuzo Kumamoto Univ., Graduate School of Medical Sciences, Assoc. Prof., 大学院・医学薬学研究部, 助教授 (50112553)
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| Project Period (FY) |
2001 – 2003
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| Project Status |
Completed (Fiscal Year 2003)
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| Budget Amount *help |
¥30,000,000 (Direct Cost: ¥30,000,000)
Fiscal Year 2003: ¥9,400,000 (Direct Cost: ¥9,400,000)
Fiscal Year 2002: ¥9,400,000 (Direct Cost: ¥9,400,000)
Fiscal Year 2001: ¥11,200,000 (Direct Cost: ¥11,200,000)
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| Keywords | flavoenzymes / D-amino acid oxidase / acyl-CoA oxidase / acyl-CoA dehydrogenase / artificial flavins / X-ray crystallography / density functional theory method (8) / 中鎖アシルCoAデヒドロゲナーゼ / 酵素-基質複合体 / 分子軌道計算 / 電荷移動相互作用 / 遷移状態アナログ / 3-チアオクタノイルCoA / 酸素との反応 / 水素結合 / 部位特異的変異 |
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| Research Abstract |
The three-dimensional structure of acyl-CoA oxidase was solved by X-ray crystallography for the first time. This structure has been long-sought with the expectation of clarifying the mechanism of flavin-oxygen reaction. The detailed structure of the active site provided structural basis of control mechanism of its reaction with molecular oxygen.
On the basis of the three-dimensional structure of D-amino acid oxidase (DAO), the substrate recognition site was mutated by genetic engineering and novel enzymes with capability of oxidizing acidic D-amino acids, which the wild type DAO cannot oxidize, were obtained.
The three-dimensional structure of 3-thiaoctanoyl-CoA-acyl-CoA dehydrogenase (MCAD) complex was solved by X-ray crystallography. This complex has been assigned as a transition-state analog by means of various experimental methods. The crystal structure provided unique information on the interaction between 3-thiaoctanoyl-CoA and the flavin ring. This interaction was verified theoretically by the density functional theory calculations. The results from the calculation was extended to the normal reaction between substrate and enzyme and revealed the important role of the substrate-flavin interaction in enhancing the catalytic process of MCAD.
Artificial flavins with different redox potential were incorporated to FAD bound to MCAD and its reactivity was changed. When 8-cyano-flavin was used as an artificial flavin, MCAD so obtained gained novel reactivity to dehydrogenate propionyl-CoA and 3-ketooctanoyl-CoA, both of which native MCAD is incapable of dehydrogenating. This novel reactivity was obtained by the high redox potential of 8-cyano-flavin compared to native flavin.
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