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Studies on the Reaction Mechanisms of Flavoenzymes by Multi-Nuclear NMR

Research Project

Project/Area Number 01480525
Research Category

Grant-in-Aid for General Scientific Research (B)

Allocation TypeSingle-year Grants
Research Field 物質生物化学
Research InstitutionKansai Medical University

Principal Investigator

MIURA Retsu  Kansai Med. Univ., Faculty of Med. Professor, 医学部, 教授 (70093466)

Co-Investigator(Kenkyū-buntansha) KURODA Kiyo  Kansai Med. Univ., Faculty of Med. Assistant Professor, 医学部, 助手 (30131436)
FUJII Shigeru  Kansai Med. Univ., Faculty of Med. Associate Professor, 医学部, 助教授 (60144482)
Project Period (FY) 1989 – 1991
Project Status Completed (Fiscal Year 1991)
Budget Amount *help
¥6,400,000 (Direct Cost: ¥6,400,000)
Fiscal Year 1991: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 1990: ¥2,300,000 (Direct Cost: ¥2,300,000)
Fiscal Year 1989: ¥3,400,000 (Direct Cost: ¥3,400,000)
KeywordsFlavoenzyme / Reaction Mechanism / Electron Transfer / Nuclear Magnetic Resonance / NMR / Multi-nuclear NMR / Stable Isotope Label
Research Abstract

1. D-AMINO ACID OXIDASE(DAO)
Substrate analogs, o-, m-, and p-flucrobenzoate were used for elucidating the electron transfer from a substrate to oxidized flavin by means of F(19)NMR. Of the NMR signals of these analogs, the signal of o-derivative exhibited the largest chemical shift change when bound to DAO, while those ofm- and p-derivativesexhibitedmoderateandminorchanges, respectively. This is consistent with the electron transfer from the amino nitrogen of a substrate to the oxidized flavin.
NADPH-ADRENODOXIN REDUCTASE(ADR)
ADR was reconstituted with C(13)-enriched FAD and the C- 13 NMR spectra of the reconstituted ADR in the free state and in the complexed state with NADP were measured. The positions enriched were 2, 4, 4a, and loa-positions of the flavin nucleus. In the complexed form with NADP, the 4a-signal-was down-field shifted, suggesting that when electrons are transferred from NADPH to oxidized flavin the NADPH-binding lowers the electron density at the 4a-position thereby facilitating the electron transfer.
3. OLD YELLOW ENZYME(OYE)
When OYE reconstituted with 8-fluoro-FMN was subjected to limited proteolysis by chymotrypsin, the protein was specificaly cleaved to yield 14KD and 34KD fragments. Subsequently, the amino group of the leucine residue of the N-terTninus of the 14KD fragment generated by the cleavage attacked the 8-position of 8-fluoro-FMN nucleophilically and a covalent bond was fontied between the flavin moiety and the 14KD fragment. From these results we could deduce the position of FMN binding and the mode of binding of FMN in OYF-

Report

(4 results)
  • 1991 Annual Research Report   Final Research Report Summary
  • 1990 Annual Research Report
  • 1989 Annual Research Report
  • Research Products

    (57 results)

All Other

All Publications (57 results)

  • [Publications] Retsu Miura: "19F-NMR study on the interaction of fluorobenzoate with pocine kidney D-amino acid oxidase." Journal of Biochemistry. 105. 318-322 (1989)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] Toshio Yamano: "Rate enhancement of the electron transfer of the adrenodoxin-adrenodoxin reductase system by dicarboxylic acids." Biochemical and Biophysical Research Communications. 162. 168-174 (1989)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] 三浦 洌: "フラビン酵素活性域への多核NMRの応用" 蛋白質核酸酵素. 35. 825-835 (1990)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] Toshio Yamano: "Rate enhancement of the electron transfer of the adrenodoxin-adrenodoxin reductase system by inorganic and nucleotide phosphates." FEBS Letters. 264. 138-140 (1990)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] 藤井 茂: "旧黄色酵素の活性域について" ビタミン. 64. 555-566 (1990)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] Shigeru Fujii: "Studies on the flavin-binding region of old yellow enzyme with and active site probe,8-fluoro-8-demethyl FMN." Journal of Biochemistry. 109. 55-60 (1991)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] Shigeru Fujiii: "On the ligand-protein and ligand-flavin interactions in NADPH-adrenodoxin reductase as studied by 31P-and13C-NMR.Use of 13C-enriched FAD as a probe." Journal of Biochemistry. 109. 144-149 (1991)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] Sabu Kasai: "Structure of FP390 including its prosthetic group(Q-flavin):physiological significance of light emitting reaction in luminous bacteria" Flavins and Flavoproteins 1990(B.Curti,S.Ronchi,G.Zanetti,eds.,Walter de Gruyter). 285-288 (1991)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] Kunio Matsui: "Photproduct of 8-hydroxyflavin." Flavins and Flavoproteins 1990(B.Curti,S.Ronchi,G.Zanetti,eds.,Walter de Gruyter). 33-36 (1991)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] Shigeru Fujii: "13C-NMR study of NADPH-adrenodoxin reductase reconstituted with 13C-enriched FAD." Flavins and Flavoproteins 1990(B.Curti,S.Ronchi,G.Zanetti,eds.,Walter de Gruyter). 381-384 (1991)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] Retsu Miura: "Stdies on old yellow enzyme reconstitutedwith the active site probe 8-fluoro-8-demethyl FMN.Covalent modification and 19F-NMR." Flavins and Flavoproteins 1990(B.Curti,S.Ronchi,G.Zanetti,eds.,Walter de Gruyter). 357-360 (1991)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] Miho Ohta: "Bovine adrenal cytochrome P-450(11β)-mediated conversion of 11-deoxycortisol to 18-and 19-hydroxy derivatives;structural analysis by 1H-NMR." Journal of Steroid Biochemistry. 39. 911-920 (1991)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] Yasuki Nonaka: "NADPH-Adrenodoxin reductase" Chemistry and Biochemistry of Flavoenzymes(Franz Muller,ed.,CRC Press). Vol.3. 329-341 (1991)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] Yasuzo Nishina: "Resonance Raman study on the complexes of medium-chain acyl-CoA dehydrogenase." Journal of Biochemistry. (1992)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] Retsu Miura: "19F-NMR study on the interaction of fluorobenzoate with porcine kidney D-amino acid oxidase." J. Biochem.105. 318-322 (1989)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] Toshio Yamano: "Rate enhancement of the electron transfer of the adrenodoxin-adrenodoxin reductase system by dicarboxylic acids." Biochem. Biophys. Res. Commun.162. 168-174 (1989)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] Retsu Miura: "The application of multinuclear NMR to the active sites of flavoenzymes." Protein, Nucleic Acid and Enzyme. 35. 825-830 (1990)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] Toshio Yamano: "Rate enhancement of the electron transfer of the adrenodoxin-adrenodoxin reductase system by inorganic and nucleotide phosphates." FEBS Lett.264. 138-140 (1990)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] Shigeru Fujii: "On the active site region of old yellow enzyme." Vitamins. 64. 555-566 (1990)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] Shigeru Fujii: "Studies on the flavin-binding region of old yellow enzyme with an active site probe, 8-fluoro-8-demethyl FMN." J. Biochem.109. 55-60 (1990)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] Shigeru Fujii: "On the ligand-protein and ligand-flavin interactions in NADPH-adrenodoxin reductase as studied by 31P-ad 13C-NMR. Use of 13C-enriched FAD as a probe." J. Biochem.109. 144-149

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] Sabu Kasai: "Structure of FP390 including its prosthetic group (Q-flavin) : physiological significance of light emitting reaction in luminous bacteria." Flavins and Flavoproteins 1990. (B. Curti, S. Ronchi, G. Zanetti, eds., Walter de Gruyter). 285-288 (1991)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] Kunio Matsui: "Photoproduct of 8-hydroxyflavin." Flavins and Flavoproteins 1990. (B. Curti, S. Ronchi, G. Zanetti, eds., Walter de Gruyter). 33-36 (1991)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] Retsu Miura: "Studies on old yellow enzyme reconstituted with 8-fluoro-8-demethyl FMN. Covalent modification and 19F-NMR." Flavins and Flavoproteins 1990. (B. Curti, S. Ronchi, G. Zanetti, eds., Walter de Gruyter). 357-360 (1991)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] Shigeru Fujii: "13C-NMR study of NADPH-adrenodoxin reductase reconstituted with 13C-enriched FAD." Flavins and Flavoproteins 1990. (B. Curti, S. Ronchi, G. Zanetti, eds., Walter de Gruyter). 381-384 (1991)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] Miho Ohta: "Bovine adrenal cytochrome P-450 (11beta)-mediated conversion of 11-deoxycortisol to 18- and 19- hydroxy derivatives ; structural analysis by 1H-NMR." J. Steroid Biochem.39. 911-920 (1991)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] Yasuki Nonaka: "NADPH-Adrenodoxin reductase." Chemistry and Biochemistry of Flavoenzymes. (F. Muller, ed., CRC Press). 329-341 (1991)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] Yasuzo Nishina: "Resonance Raman study on the complexes of medium-chain acyl-CoA dehydrogenase." J. Biochem.

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1991 Final Research Report Summary
  • [Publications] Retsu Miura: "19F-NMR study on the interaction of fluorobenzoate with pocine kidney D-amino acid oxidase." Journal of Biochemistry. 105. 318-322 (1989)

    • Related Report
      1991 Annual Research Report
  • [Publications] Toshio Yamano: "Rate enhancement of the electron transfer of the adrenodoxin-adrenodoxin reductase system by dicarboxylic acids." Biochemical and Biophysical Research Communications. 162. 168-174 (1989)

    • Related Report
      1991 Annual Research Report
  • [Publications] 三浦 洌: "フラビン酵素活性域への多核NMRの応用" 蛋白質核酸酵素. 35. 825-835 (1990)

    • Related Report
      1991 Annual Research Report
  • [Publications] Toshio Yamano: "Rate enhancement of the electron transfer of the adrenodoxin-adrenodoxin reductase system by inorganic and nucleotide phosohates." FEBS Letters. 264. 138-140 (1990)

    • Related Report
      1991 Annual Research Report
  • [Publications] 藤井 茂: "旧黄色酵素の活性域について" ビタミン. 64. 555-566 (1990)

    • Related Report
      1991 Annual Research Report
  • [Publications] Shigeru Fujii: "Studies on the flavin-binding region of old yellow enzyme with an active site probe,8-fluoro-8-demethyl FMN." Journal of Biochemistry. 109. 55-60 (1991)

    • Related Report
      1991 Annual Research Report
  • [Publications] Shigeru Fujiii: "On the ligand-protein and ligand-flavin interactions in NADPH-adrenodoxin reductase as studied by 31P-and 13C-NMR.Use of 13C-enriched FAD as a probe." Journal of Biochemistry. 109. 144-149 (1991)

    • Related Report
      1991 Annual Research Report
  • [Publications] Sabu Kasai: "Structure of FP390 including its prosthetic group(Q-flavin):physiological significance of light emitting reaction in luminous bacteria" Flavins and Flavoproteins 1990 (B.Curti,S.Ronchi,G.Zanetti,eds.,Walter de Gruyter). 285-288 (1991)

    • Related Report
      1991 Annual Research Report
  • [Publications] Kunio Matsui: "Photproduct of 8-hydroxyflavin." Flavins and Flavoproteins 1990 (B.Curti,S.Ronchi,G.Zanetti,eds.,Walter de Gruyter). 33-36 (1991)

    • Related Report
      1991 Annual Research Report
  • [Publications] Shigeru Fujii: "13C-NMR study of NADPH-adrenodoxin reductase reconstituted with 13C-enriched FAD." Flavins and Flavoproteins 1990 (B.Curti,S.Ronchi,G.Zanetti,eds.,Walter de Gruyter). 381-384 (1991)

    • Related Report
      1991 Annual Research Report
  • [Publications] Retsu Miura: "Stdies on old yellow enzyme reconstitutedwith the active site probe 8-fluoro-8-demethyl FMN.Covalent modification and 19F-NMR." Flavins and Flavoproteins 1990 (B.Curti,S.Ronchi,G.Zanetti,eds.,Walter de Gruyter). 357-360 (1991)

    • Related Report
      1991 Annual Research Report
  • [Publications] Miho Ohta: "Bovine adrenal cytochrome P-450(11β)-mediated comversion of 11-Deoxycortisol to 18-and 19-hydroxy derivaties;structural analysis by 1H-NMR." Journal of Steroid Biochemistry. 39. 911-920 (1991)

    • Related Report
      1991 Annual Research Report
  • [Publications] 三浦 洌: " ^<19>FーNMR study of the interaction of fluorobenzoate with porcine kidney Dーamino acid oxidase" J.Biochem.105. 318-322 (1989)

    • Related Report
      1990 Annual Research Report
  • [Publications] 山野 俊雄: "Rate enhancement of the electron transfer of the adrenodoxinーadrenodoxin reductase system by dicarboxylic acids" Biochem.Biophys.Res.Commun.162. 168-174 (1989)

    • Related Report
      1990 Annual Research Report
  • [Publications] 三浦 洌: " ^<13>Cー標識フラビンを用いたアドレノドキシン還元酵素の ^<13>CーNMR" 生化学. 61. 965 (1989)

    • Related Report
      1990 Annual Research Report
  • [Publications] 藤井 茂: "8ーFー置換FMNを用いた旧黄色酵素の活性域の研究" 生化学. 61. 965 (1989)

    • Related Report
      1990 Annual Research Report
  • [Publications] 山野 俊雄: "Rate ennancement of the electron transfor of the adrenodoxinーadrenodoxin reductase system by inorganic and nucleotide phosphates" FEBS Lett.264. 138-140 (1990)

    • Related Report
      1990 Annual Research Report
  • [Publications] 三浦 洌: "フラビン酵素活性域への多核NMRの応用" 蛋白質核酸酵素. 35. 825-835 (1990)

    • Related Report
      1990 Annual Research Report
  • [Publications] 藤井 茂: "旧黄色酵素の活性域について" ビタミン. 64. 555-566 (1990)

    • Related Report
      1990 Annual Research Report
  • [Publications] 藤井 茂: "多核NMRによるフラビン酵素反応機構の研究" 生化学. 62. 698 (1990)

    • Related Report
      1990 Annual Research Report
  • [Publications] 藤井 茂: "8ーFー置換FMNをプロ-ブとした旧黄色酵素の活性域の研究" 生化学. 62. 760 (1990)

    • Related Report
      1990 Annual Research Report
  • [Publications] 藤井 茂: "Studies on the flavinーbinding region of old yellow enzyme with an acitve site probe,8ーfluoroー8ーdemethyl FMN" J.Biochem.109. 55-60 (1991)

    • Related Report
      1990 Annual Research Report
  • [Publications] 藤井 茂: "On the ligandーprotein and ligandーflavin interactions in NADPHーadrenodoxin reductase as studied by 31Pーand 13CーNMR." J.Biochem.109. 144-149 (1991)

    • Related Report
      1990 Annual Research Report
  • [Publications] 三浦洌: "^<19>F-NMR study of the interaction of fluorobenzoate with porcine kidney D-amino acid oxidase" J.Biochem.105. 318-322 (1989)

    • Related Report
      1989 Annual Research Report
  • [Publications] 山野俊雄: "Rate enhancement of the electron transfer of the adrenodoxin-adrenodoxin reductase system by dicarboxylic acids" Biochem.Biophys.Res.Commun.162. 168-174 (1989)

    • Related Report
      1989 Annual Research Report
  • [Publications] 三浦洌: "^<13>Cー標識フラビンを用いたアドレノドキシン還元酵素の^<13>CーNMR" 生化学. 61. 965 (1989)

    • Related Report
      1989 Annual Research Report
  • [Publications] 藤井茂: "8ーFー置換FMNを用いた旧黄色酵素の活性域の研究" 生化学. 61. 965 (1989)

    • Related Report
      1989 Annual Research Report
  • [Publications] 山野俊雄: "Rate enhancement of the electron transfer of the adrenodoxin-adrenodoxin reductase system by inorganic and nucleotide phosphates" FEBS Lett. (1990)

    • Related Report
      1989 Annual Research Report
  • [Publications] 三浦洌: "フラビン酵素活性域への多核NMRの応用" 蛋白質核酸酵素. 35. (1990)

    • Related Report
      1989 Annual Research Report

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Published: 1989-04-01   Modified: 2016-04-21  

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