| Project/Area Number |
13480195
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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 |
Structural biochemistry
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| Research Institution | OKAYAMA UNIVERSITY |
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Principal Investigator |
TORAYA Tetsuo Okayama Univ., Engineering, Professor, 工学部, 教授 (70026318)
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| Co-Investigator(Kenkyū-buntansha) |
YAMANISHI Mamoru Okayama Univ., Engineering, Assistant, 工学部, 助手 (30240063)
TOBIMATSU Takamasa Okayama Univ., Engineering, Associate Professor, 工学部, 助教授 (30188768)
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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 |
¥9,300,000 (Direct Cost: ¥9,300,000)
Fiscal Year 2003: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 2002: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 2001: ¥6,500,000 (Direct Cost: ¥6,500,000)
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| Keywords | vitamin B12 coenzyme / radical enzyme / reactivation / molecular chaperone / X-ray analysis / theoretical calculation / mechanism-based inactivation / diol dehydratase / 再活性化因子 / エタノールアミンアンモニアリアーゼ / 自殺不活性化 / ビタミンB_<12> |
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| Research Abstract |
1.Mechanism of radical catalysis by vitamin B12 enzymes
(1) Recombinant glycerol dehydratase was purified, and its enzymological properties were investigated. Its X-ray structure was solved for the first time. (2) The formation of the adenine-anchored radical was crystallographically demonstrated upon illumination of the diol dehydratase-adeninylpentylcobalamin complex with visible light. (3) The structure of substrate-free form of diol dehydratase was determined. It was strongly suggested that substrate triggers the homolysis of the coenzyme Co-C bond by inducing further steric strain to the Co-C bond that had been already strained to some extent. (4) Coenzymic activity of coenzyme analogs in which the base moiety of the coezyme B12 was replaced by other bases was correlated with the bulkiness of the base. It was also suggested that the nucleotide moiety is required for stabilizing radical intermediates. (5) The X-ray structures of the complexes of diol dehydratase with B12 and R-or S-
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enantiomer were analyzed, and The stereochemical _courses of the steps of the conversion of each enantiomeric substrate to product was completely elucidated based on the X-ray structures. (6) Theoretical calculations with a simplified model indicated that the activation energies of each steps of diol dehydratase reaction are small enough to be supplied by substrate binding energy.
2. Mechanism of reactivation of a B12 enzyme by molecular chaperone-like Factor
(1) The two ORFs near the glycerol dehydratase genes were identified as putative reactivating factor genes. The purified gene products actually reactivated the inactivated holoenzyime by a molecular chaperone-like manner. (2) The gene encoding a reactivating factor for ethanolamine ammonia-lyric was identified. The purified gene product was shown to serve as a reactivating factor in the presence of B12 coenzyme and ATP.
The results obtained by this study was summarized and published as a review in the special issue of "Radical Enzymology" in Chemical Reviews. The fact that I was invited to write this review for this most authoritative journal in chemistry indicates that the scientific merit of this study rated excellent among the international community in this field. Less
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