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Mechanisms of Radical Catalysis in Vitamin B_<12> Enzyme and Reactivation by Molecular Chaperone-like Factor

Research Project

Project/Area Number 13480195
Research Category

Grant-in-Aid for Scientific Research (B)

Allocation TypeSingle-year Grants
Section一般
Research Field Structural biochemistry
Research InstitutionOKAYAMA UNIVERSITY

Principal Investigator

TORAYA Tetsuo  Okayama Univ., Engineering, Professor, 工学部, 教授 (70026318)

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)
Project Period (FY) 2001 – 2003
Project Status Completed (Fiscal Year 2003)
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)
Keywordsvitamin 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>
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- … More 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

Report

(4 results)
  • 2003 Annual Research Report   Final Research Report Summary
  • 2002 Annual Research Report
  • 2001 Annual Research Report
  • Research Products

    (41 results)

All Other

All Publications (41 results)

  • [Publications] Kajiura, H., et al.: "Characterization and Mechanism of Action of a Reactivating Factor for Adenosylcobalamin-dependent Glycerol Dehydratase."J.Biol.Chem.. 276. 36514-36519 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Masuda, J., et al.: "Radical Production Simulated by Photoirradiation of the Diol Dehydratase-Adeni nylpenthylcobalamin Complex"J.Synchrotron Rad. 8. 1182-1185 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Toraya, T., et al.: "Energetic Feasibility of Hydrogen Abstraction and Recombination in Coenzyme B_<12>-dependent Diol Dehydratase Reaction."J.Biochem.-Tokyo. 130. 865-872 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] 虎谷哲夫: "ラジカルによって触媒される酵素反応:ビタミンB_<12>酵素の立体構造と精密触媒機構"生化学. 74(2). 87-102 (2002)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] 鏡山博行ら: "補酵素作用のダイナミズム-酵素の機能を支えるアポタンパクの働き-"ビタミン. 76(5・6). 273-282 (2002)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Toraya, T.: "Enzymatic Radical Catalysis : Coenzyme B12-Dependent Diol Dehydratase."The Chemical Records. 2. 352-366 (2002)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Fukuoka, M., et al.: "Functions of the D-Ribosyl Moiety and the Lower Axial Ligand of the Nucleotide Loop of Coenzyme B12 in Diol Dehydratase and Ethanolamine Ammonia-lyase Reactions."J.Biochem.-Tokyo. 132(6). 935-943 (2002)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Yamanishi, M., et al.: "The crystal structure of coenzyme B12-dependent glycerol dehydratase in complex with cobalamin and 1,2-propanediol."Eur.J.Biochem.. 269. 4484-4494 (2002)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Shibata, N., et al.: "Substrate-Induced Conformational Change of a Coenzyme B12-Dependent Enzyme : Crystal Structure of the Substrate-Free Form of Diol Dehydratase."Biochemistry. 41(42). 12607-12617 (2002)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Eda, M., et al.: "Theoretical study on the mechanism of catalysis of coenzyme B12-dependent diol dehydratase."Bull.Chem.Soc.Jap.. 75(7). 1469-1481 (2002)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Toraya, T.: "Radical Catalysis in Coenzyme B12-Dependent Isomerization (Eliminating) Reactions."Chem.Rev.. 103(6). 2095-2128 (2003)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] 虎谷哲夫: "シンポジウムB群ビタミンはどのように働くか:ビタミンB12酵素の立体構造と精密触媒機構"ビタミン. 77(5・6). 297-312 (2003)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Shibata, N., et al.: "Structural Rationalization for the Lack of Stereospecificity in Coenzyme B12-dependent Diol Dehydratase."J.Biol.Chem.. 278(25). 22717-22725 (2003)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Kajiura, H., et al.: "Characterization and Mechanism of Action of a Reactivating Factor for Adenosylcobalamin-dependent Glycerol Dehydratase."J.Biol.Chem.. 276. 36514-36519 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Masuda, J., et al.: "Radical Production Simulated by Photoirradiation of the Diol Dehydratase-Adeninylpenthylcobalamin Complex"J.Synchrotron Rad.. 8. 1182-1185 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Toraya, T., et al.: "Energetic Feasibility of Hydrogen Abstraction and Recombination in Coenzyme B12-dependent Diol Dehydratase Reaction."J.Biochem.-Tokyo. 130. 865-872 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Toraya, T.: "Radical-catalyzed enzymatic reactions : Structure and mechanism of B12 enzymes"Seikagaku (Japan). 74(2). 87-102 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Kagamiyama, H., et al.: "Dynamic Aspects of Coenzyme Action"Bitamin (Japan). 76(5・6). 273-282 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Toraya, T.: "Enzymatic Radical Catalysis : Coenzyme B12-Dependent Diol Dehydratase."The Chemical Records. 2. 352-366 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Fukuoka, M., et al.: "Functions of the D-Ribosyl Moiety and the Lower Axial Ligand of the Nucleotide Loop of Coenzyme B12 in Diol Dehydratase and Ethanolamine Ammonia-lyase Reactions."J.Biochem.-Tokyo. 132(6). 935-943 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Yamanishi, M., et al.: "The crystal structure of coenzyme B12-dependent glycerol dehydratase in complex with cobalamin and 1,2-propanediol."Eur.J.Biochem.. 269. 4484-4494 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Shibata, N., et al.: "Substrate-Induced Conformational Change of a Coenzyme B12-Dependent Enzyme : Crystal Structure of the Substrate-Free Form of Diol Dehydratase."Biochemistry. 41(42). 12607-12617 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Eda, M., et al.: "Theoretical study on the mechanism of catalysis of coenzyme B12-dependent diol dehydratase."Bull.Chem.Soc.Jap.. 75(7). 1469-1481 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Toraya, T.: "Radical Catalysis, in Coenzyme B12-Dependent Isomerization (Eliminating) Reactions."Chem.Rev.. 103(6). 2095-2128 (2003)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Toraya, T.: "Structure-Based Fine Mechanism of Action of a B12 Enzyme"Bitamin (Japan). 77(5・6). 297-312 (2003)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Shibata, N., et al.: "Structural Rationalization for the Lack of Stereospecificity in Coenzyme B12-dependent Diol Dehydratase."J.Biol.Chem.. 278(25). 22717-22725 (2003)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Toraya, T.: "Radical Catalysis in Coenzyme B12-Dependent Isomerization (Eliminating) Reactions."Chem.Rev.. 103(6). 2095-2128 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] 虎谷哲夫: "シンポジウムB群ビタミンはどのように働くか:ビタミンB12酵素の立体構造と精密触媒機構"ビタミン. 77(5・6). 297-312 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] Shibata, N.: "Structural Rationalization for the Lack of Stereospecificity in Coenzyme B12-dependent Diol Dehydratase."J.Biol.Chem.. 278(25). 22717-22725 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] Toraya, T.: "Novel Pathway for Utilization of Cyclopropanecarboxylate by Rhodococcus rhodochrous."Appl.Environ.Microbiol.. 70(1). 224-228 (2004)

    • Related Report
      2003 Annual Research Report
  • [Publications] 虎谷哲夫: "廣川タンパク質化学 第4巻 酵素"廣川書店. 12, 17 (2003, 2004)

    • Related Report
      2003 Annual Research Report
  • [Publications] Eda, M., Kamachi, T., Yoshizawa, K., Toraya, T.: "Theoretical study on the mechanism of catalysis of coenzyme B12-dependent diol dehydratase"Bull. Chem. Soc. Jap.. 75(7). 1469-1481 (2002)

    • Related Report
      2002 Annual Research Report
  • [Publications] Deshimaru, S., Miyake, Y., Tatsu, Y., Endo, Y., Yamamoto, N., Toraya, T.et al.: "cDNA Cloning, Expression, and Properties of the Catalytic Domain of Starfish Cdc25 Dual-specificity Phosphatase, a Cell Cycle Regulator"J. Biochem.-Tokyo. 131(5). 705-712 (2002)

    • Related Report
      2002 Annual Research Report
  • [Publications] Shibata, N., Masuda, J., Morimoto, Y., Yasuoka, N., Toraya, T.: "Substrate-Induced Conformational Change of a Coenzyme B12-Dependent Enzyme : Crystal Structure of the Substrate-Free Form of Diol Dehydratase"Biochemistry. 41(42). 12607-12617 (2002)

    • Related Report
      2002 Annual Research Report
  • [Publications] Yamanishi, M., Tobimatsu, T., Dokiya, A., Yasuoka, N., Toraya, T.et al.: "The crystal structure of coenzyme B12-dependent glycerol dehydratase in complex with cobalamin and 1,2-propanediol"Eur. J. Biochem.. 269. 4484-4494 (2002)

    • Related Report
      2002 Annual Research Report
  • [Publications] Fukuoka, M., Yamanishi, M., Zou, X., Brown, L.K., Toraya, T.et al.: "Functions of the D-Ribosyl Moiety and the Lower Axial Ligand of the Nucleotide Loop of Coenzyme B12 in Diol Dehydratase and Ethanolamine Ammonia-lyase Reactions"J. Biochem.. 132(6). 935-943 (2002)

    • Related Report
      2002 Annual Research Report
  • [Publications] Toraya, T.: "Enzymatic Radical Catalysis : Coenzyme B12-Dependent Diol Dehydratase"The Chemical Records. 2. 352-366 (2002)

    • Related Report
      2002 Annual Research Report
  • [Publications] Kajiura, H., Mori, K., Tobimatsu, T., Toraya, T: "Characterization and Mechanism of Action of a Reactivating Factor for Adenosylcobalamin-dependent Glycerol Dehydratase"Journal of Biological Chemistry. 276. 36514-36519 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] Masuda, J., Shibata, N., Morimoto, Y., Toraya, T., Yasuoka, N: "Radical Production Simulated by Photoirradiation of the Diol Dehydratase-Adeninylpenthylcobalamin Complex"Journal of Synchrotron Radiation. 8. 1182-1185 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] Toraya, T., Eda, M., Kamachi, T., Yoshizawa, K: "Energetic Feasibility of Hydrogen Abstraction and Recombination in Coenzyme B_<12r>dependent Diol Dehydratase Reaction"Journal of Biochemistry. 130. 865-872 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] 虎谷哲夫: "ラジカルによって触媒される酵素反応:ビタミンB_<12>酵素の立体構造と精密触媒機構"生化学. 74(2). 87-102 (2002)

    • Related Report
      2001 Annual Research Report

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Published: 2001-04-01   Modified: 2016-04-21  

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