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Studies on the Structure and the Mechanism of Radical Enzymes

Research Project

Project/Area Number 10680611
Research Category

Grant-in-Aid for Scientific Research (C)

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

Principal Investigator

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

Co-Investigator(Kenkyū-buntansha) YAMANISHI Mamoru  Okayama Univ., Faculty of Engineering, Assistant, 工学部, 助手 (30240063)
TOBIMATSU Takamasa  Okayama Univ., Faculty of Engineering, Associate Professor, 工学部, 助教授 (30188768)
Project Period (FY) 1998 – 1999
Project Status Completed (Fiscal Year 1999)
Budget Amount *help
¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 1999: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 1998: ¥3,000,000 (Direct Cost: ¥3,000,000)
Keywordscobalamin / diol dehydratase / glycerol dehydratase / crystal structure / potassium ion / density functional theory / reactivating factor / molecular chaperone / 電子常磁性共鳴 / シアノコバラミン / 結晶構造分析
Research Abstract

Electron paramagnetic resonance (EPR) measurements were conducted to elucidate the mode of binding of cobalamin to diol dehydratase. It was demonstrated that this enzyme binds cobalamin in the ゛base-on″mode, and that the bulkiness of the lower axial base is important for protection of reactive radical intermediates. Two forms of crystals of the diol dehydratase-cyanocobalamin complex were obtained, which diffracted up to 2.2 and 3.0 A resolution. The crystal structure of the complex revealed that cobalamin is bound to the enzyme in the base-on mode. The active site exists inside the TIM barrel which may protect reactive radical intermediates from side reactions. The two hydroxyl groups of the substrate coordinate directly to KィイD1+ィエD1 in the active site. Density functional theory computations indicated that the hydroxyl group migration from C-2 to C-1 proceeds by a concerted pathway through a cyclic transition state. KィイD1+ィエD1 seems to be important not only in stabilizing the transition state but also in labilizing the Co-C bond indirectly by increasing the substrate binding energy.
The products of putative reactivating factor genes were purified and confirmed in vitro to function as a reactivating factor for glycerol-inactivated and oィイD22ィエD2-inactivated holoenzymes. The mechanism of action of the diol dehydratase-reactivating factor was investigated. It was demonstrated that ATP- and ADP -forms of this factor are low and high affinity forms for diol dehydratase, and that the modified coenzyme in the inactivation is released from the enzyme and, as a result, substituted by intact coenzyme, reconstituting catalytically active holoenzyme. This factor is thus considered as a sort of molecular chaperone. The two genes in proximity to the glycerol dehydratase genes were identified as the genes for a glycerol dehydratase-reactivating factor. Their amino acid sequences showed high homology with the diol dehydratase-reactivating factor.

Report

(3 results)
  • 1999 Annual Research Report   Final Research Report Summary
  • 1998 Annual Research Report
  • Research Products

    (34 results)

All Other

All Publications (34 results)

  • [Publications] Yamanishi, M., et al.: "EPR Spectroscopic Evidence for the Mechanism-based Inactivation of Adenosylcobalamin-dependent Diol Dehydratase by Coenzyme Analogs"Journal of Biochemistry. 124. 598-601 (1998)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Toraya, T., and Mori, K.: "A Reactivating Factor for Coenzyme B12-Dependent Diol Dehydratase"Journal of Biological Chemistry. 247. 3372-3377 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Masuda, J., et al.: "Crystallization and Preliminary X-ray Study of Two Crystal Forms of Klebsiella oxytoca Diol Dehydratase-cyanocobalamin Complex"Acta Crystallographica. D55. 907-909 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Tobimatsu, T., et al.: "Identification and Expression of the Genes Encoding a Reactivating Factor for Adenosylcobalamin-dependent Glycerol dehydratase"Journal of Bacteriology. 181. 4110-4113 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Shibata, N., et al.: "A New Mode of B12 Binding and the Direct participation of a Potassium Ion in enzymatic Catalysis :X-ray Structure of Diol Dehydratase"Structure. 7. 997-1008 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Toraya, T., et al.: "Direct Participation of Potassium Ion in the Catalysis of Coenzyme B12-dependent Diol Dehydratase"Journal of Biochemistry. 126. 650-654 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Mori, K., and Toraya, T.: "Mechanism of Reactivation of Coenzyme B12-dependent Diol Dehydratase by a Molecular Chaperon-like Reactivating Factor"Biochemistry. 38. 13170-13178 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Yamada, K., et al.: "Heterologous High Level Expression, Purification, and Enzymological Properties of Recombinant Rat Cobalamin-dependent Methionine synthase"Journal of Biological Chemistry. 274. 35571-35576 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] 虎谷哲夫: "ビタミンB12酵素の遺伝子クローン化と立体構造の解明(総説)"ビタミン. 73. 561-566 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Toraya, T.: "Radical Catalysis of B12 Enzymes : Structure, Mechanism, Inactivation and Reactivation of Diol and Glycerol Dehydratases (総合論文)"Cellular and Molecular Life Sciences. 106-127 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Toraya, T.: "The Structure and the Mechanism of Action of Coenzyme B12-dependent Diol Dehydratases"Journal of Molecular Catalysis. (in press). (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] M. Yamanishi, et al.: "EPR Spectroscopic Evidence for the Mechanism-Based Inactivation of Adenosylcobalamin-Dependent Diol Dehydratase by Coenzyme Analogs"Journal of Biochemistry. 124(3). 598-601 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] T. Toraya and K. Mori: "A Reactivating Factor for Coenzyme B12-Dependent Diol Dehydratase"Journal of Biological Chemistry. 274. 3372-3377 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] J. Masuda, et al.: "Crystallization and Preliminary X-ray Study of Two Crystal Forms of Klebsiella oxytoca Diol Dehydratase-cyanocobalamin Complex"Acta. Crystallogr. D55. 907-909 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] T. Tobimatsu, et al.: "Identification and Expression of the Genes Encoding a Reactivating Factor for Adenosylcobalamin-dependent Glycerol Dehydratase"J. Bacteriol.. 181. 4110-4113 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] N. Shibata, et al.: "A New Mode of B12 Binding and the Direct Participation of a Potassium Ion in Enzyme Catalysis. X-ray Structure of Diol Dehydratase"Structure. 7. 997-1008 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] T. Toraya, et al.: "Direct Participation of Potassium Ion in the Catalysis of Coenzyme B12-Dependent Diol Dehydratase"Journal of Biological Chemistry. 126. 650-654 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] K. Mori, and T. Toraya: "Mechanism of Reactivation of Coenzyme B12-Dependent Diol Dehydratase by a Molecular Chaperone-Like Reactivating Factor"Biochemistry. 38. 13170-13178 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] K. Yamada, et al.: "Heterologous High level Expression, Purification, and Enzymological Properties of Recombinant Rat Cobalamin-dependent Methionine Synthase"Journal of Biological Chemistry. 274. 35571-35576 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] T. Toraya: "Gene cloning and structural determination of vitamin B12 enzymes"Vitamins. 73. 561-666 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] T. Toraya: "Radical Catalysis of B12 Enzymes : Structure, Mechanism, Inactivation and Reactivation of Diol and Glycerol Dehydratases"Cell. Mol. Life Sci.. 57. 106-127 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] T. Toraya: "Radical Catalysis of B12 Enzymes : Structure, Mechanism, Inactivation and Reactivation of Diol and Glycerol Dehydratases"Journal of Molecular Catalysis. (in press). (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] T.Toraya: "Radical Catalysis of B12 Enzymes: Structure, Mechanism, Inactivation and Reactivation of Diol and Glycerol Dehydratases"Cell. Mol. Life Sci. (in press). (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] K.Yamada, et al.: "Heterologous High level Expression, Purification, and Enzymological Properties of Recombinant Rat Cobalamin-dependent Methionine Synthase"Journal of Biological Chemistry. 274. 35571-35576 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] K.Mori, and T.Toraya,: "The Mechanism of Reactivation of Coenzyme B12-Dependent Diol Dehydratase by a Molecular Chaperone-Like Reactivating Factor"Biochemistry. 38. 13170-13178 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] T.Toraya, et al.: "Direct Participation of Potassium Ion in the Catalysis of Coenzyme B12-Dependent Diol Dehydratase"Journal of Biological Chemistry. 126. 650-654 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] J.Masuda, et al.: "Crystallization and Preliminary X-ray Study of Two Crystal Forms of Klebsiella oxytoca Diol Dehydratase-cyanocobalamin Complex"Acta. Crystallogr.. D55. 907-909 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] N.Shibata, et al.: "A New Mode of B12 Binding and the Direct Participation of a Potassium Ion in Enzyme Catalysis. X-ray Structure of Diol Dehydratase"Structure. 7. 997-1008 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] T.Tobimatsu, et al.: "Identification and Expression of the Genes Encoding a Reactivating Factor for Adenosylcobalamin-dependent Glycerol Dehydratase"J. Bacter. 181. 4110-4113 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] M.Yamanishi,et al.: "EPR Spectroscopic Evidence for the Mechanism-Based Inactivation of Adenosylcobalamin-Dependent Diol Dehydratase by Coenzyme Analogs" Journal of Biochemistry. 124(3). 598-601 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] T.Toraya and K.Mori: "A Reactivating Factor for Coenzyme B_<12>-Dependent Diol Dehydratase" Journal of Biological Chemistry. 274(6). 3372-3377 (1999)

    • Related Report
      1998 Annual Research Report
  • [Publications] J.Masuda,et al.: "Crystallization and Preliminary X-ray Study of Two Crystal Forms of Klebsiella oxytoca Diol Dehydratase-cyanocobalamin Complex" Acta Crystallographica D. in press. (1999)

    • Related Report
      1998 Annual Research Report
  • [Publications] M.Yamanishi,et al.: "Evidence for Axial Coordination of 5,6-Dinethylbenzimidazole tothe Cabalt Atom of Adencsyloobalamin Bourd to Diol Dehydratase" Biochemistry. 37(14). 4799-4803 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] T.Tobimatsu,et al.: "Molecular Cloning Sequencing and Charactenzation of the Genes for Adenosylcobalamin-dependent Diol Dehydratase of kebsiella pneumonae" Biosci.Biotechnol.Biochem.62(9). 1774-1777 (1998)

    • Related Report
      1998 Annual Research Report

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Published: 1998-04-01   Modified: 2016-04-21  

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