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Structure and Function of Quinoprotein Dehydrogenase

Research Project

Project/Area Number 09044228
Research Category

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

Allocation TypeSingle-year Grants
Section一般
Research Field 応用微生物学・応用生物化学
Research InstitutionYamaguchi University

Principal Investigator

MATSUSHITA Kazunobu  Faculty of Agriculture, Yamaguchi University, Professor, 農学部, 教授 (50107736)

Co-Investigator(Kenkyū-buntansha) MIGITA Taiko  School of Allied Health Sciences, Yamaguchi University, Associate Professor, 医療技術短期大学部, 助教授 (90159161)
MIYOSHI Hideo  Graduate school of Agriculture, Kyoto University, Associate Professor, 大学院・農学研究科, 助教授 (20190829)
YAMADA Mamoru  Faculty of Agriculture, Yamaguchi University, Associate Professor, 農学部, 助教授 (30174741)
TAYAMA Hirohide  Faculty of Agriculture, Yamaguchi University, Assistant Professor, 農学部, 助手 (60240884)
Project Period (FY) 1997 – 1999
Project Status Completed (Fiscal Year 1999)
Budget Amount *help
¥7,300,000 (Direct Cost: ¥7,300,000)
Fiscal Year 1999: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 1998: ¥2,800,000 (Direct Cost: ¥2,800,000)
Fiscal Year 1997: ¥3,200,000 (Direct Cost: ¥3,200,000)
KeywordsPyrroloquinoline quinone / Quinoprotein dehydrogenase / Glucose dehydrogenase / Alcohol Dehydrogenase / X ray crystallography / Pseudomonas putida / Escherichia coli / Acetic acid bacteria
Research Abstract

Pyrroloquinoline quinone-dependent quinoprotein dehydrogenases, glucose dehydrogenase (GDH) of Escherichia coli and two alcohol dehydrogenases (ADH) of Pseudomonas putida and Gluconobacter suboxydans have been investigated to elucidate their structure and function, which was performed by several different means. 1)Structural and functional study of E. coli GDH : Several mutant enzymes were prepared by using random and site-directed mutageneses, and their reaction kinetics and oxido-reduction spectra were examined after purification. Compared with the structural model, the function of following amino acid residues, His-262, His-775, Trp-404, Asp-466, Asp-730 and Lys-493, have been identifisd. 2)Electron transfer study of P. putida ADH : (ADH IIB) was purified together with a blue copper protein, azurin, from the soluble fraction of P. putida and then the reaction between both proteins was examined by kinetic, fluorometric and redox titration analyses. As a result, the electron transfer between both proteins was shown to occur by a hydrophobic interaction and also by a freely reversible on and off binding process. Furthermore, the azurin-dependent alcohol oxidation activity could be reconstituted on the membrane vesicles and also with purified cytochrome oxidase. 3)Ubiquinone reaction site of E. coli GDH and G. suboxydans ADH : The ubiquinone-binding site of GDH was shown to be located near the surface of cytoplasmic membrane by reconstituting GDH with phospholipids having ubiquinone analogs at different depth. In G. suboxydans ADH, besides the ubiquinone reduction site, ubiquinol oxidation site was found to present separated from the site of ubiquinone reduction. 4)X ray crystallography of P. putida ADH and G. suboxydans ADH : The structure of ADH IIB of P. putida has already been successfully determined at 1.9 A. Whereas, G. suboxydans ADH has been crystallized and now under determination of the structure.

Report

(4 results)
  • 1999 Annual Research Report   Final Research Report Summary
  • 1998 Annual Research Report
  • 1997 Annual Research Report
  • Research Products

    (32 results)

All Other

All Publications (32 results)

  • [Publications] J.Frebortova et al.: "Intramolecular electron transport in quinoprotein alcohol dehydrogenas of Acetobacter methanolicus:A redox-titration study"BicChim.Biophys.Acta. 1363. 24-34 (1998)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] M.Yamada et al.: "Mutat isolation of the Escherichia coli quinoprotein glucose dehydrogenase and analysis of crucial reaidues Asp-730 and His-775 for-its function"J.Biol.Chem.. 273. 22021-22027 (1998)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] K.Matsushita et al.: "The quihohemoprotein alcoholdehydrogenase of Gluconobacter suboxydans has ubiquinol oxidation activity at a site different from the ubiquinone reduction site"BicChim.Biophys.Acta. 1409. 154-164 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] H.Miyoshi et al.: "Topographical characterization of the udiquimone reduction site of glucose dehydrogenase of in Escherhia coil using depth-dependcnt fluorescent inhibitors"BicChim.Biophys.Acta. 1412. 29-36 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] K.Matsushita et al.: "Electron transfer from quinohemoprotein alcohol dehydrogenase to blue copper protein azurin,in the alcohol oxidase respiratory chain of Pseudomonas putida HK5"Biochemistry. 38. 6111-6118 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] 山田守、松下一信: "キノプロテインおよびキノヘモグロビン脱水素酵素の分子内および分子間電子移動"生化学. 71. 413-428 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] 松下一信(共著): "新版微生物学実験法:5.7細胞活性の測定"講談社サイエンティフィク(杉山純多ら編). 324 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] 松下一信(共著): "広川タンパク質化学第4巻酵素4.1オキシドレダクターゼ アルコールデヒドロゲナーゼ(キノプロテイン)"(広川書店)野崎光洋ら(編). 353 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] J, Frebortova, MATSUSHITA, K, YAKUSHI, T, TOYAMA, H & ADACHI, O: "Quinoprotein alcohol dehydrogenase of acetic acid bacteria : Kinetic study on the enzyme purified from Acetobacter methanolicus"Biosci. Biotech. Biochem.. 61. 459-465 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Frebortova, MATSUSHITA, K, ARATA, H & ADACHI, O: "Intramolecular electron transport in quinoprotein alcohol dehydrogenase of Acetobacter methanolicus : A redox-titration study"Biochim. Biophys. Acta. 1363. 24-34 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] YAMADA, INBE, H, TANAKA, M, SUMI, K, MATSUSITA, K & ADACHI, O: "Mutant isolation of the Escherichia coli quinoprotein glucose dehydrogenase and analysis of crucial residues Asp-730 and His-775 for its function"J. Biol. Chem.. 273. 22021-22027 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] MATSUSHITA, K, YAKUSHI, T, TOYAMA, H & ADACHI, O, MIYOSHI, H, TAGAMI, E & SAKAMOTO, K :: "The quinohemoprotein alcohol dehydrogenase of Gluconobater suboxydans has ubiquinol oxidation activity at a site different from the ubiquinone reduction site"Biochim. Biophys. Acta. 1409. 154-164 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] MATSUSHITA, K, YAMASHITA, T, AOKI, N, TOYAMA, H & ADACHI, O: "Electron transfer from quinohemoprotein alcohol dehydrogenaseto blue copper protein azurin in the alcohol oxidase respiratory chain of Pseudomonas putida HK5"Biochemistry. 38. 6111-6118 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] MIYOSHI, H, NIITOME, Y, MATSUSITA, K, YAMADA, M & IWAMURA, H: "Topographical characterization of the ubiquinone reduction site of glucose dehydrogenase in Escherichia coli using depth-dependent fluorescent inhibitors"Biochem. Biophys. Acta. 1412. 29-36 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] YAMADA, M & MATSUSHITA, K: "Intra-and intermoleuclar electron transfer in quinoprotein and quinohomoprotein dehydrogenases (in Japanese)"Seikagaku. 71. 413-428 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] CHEN, Zhi-wei, BARUCH, Petra, F, Scott Mathews, MATSUSITA, K, YAMASHITA, T, TOYAMA & ADACHI, O: "Crystallization and preliminary differation studies of two quinoprotein alcohol dehydrogenases(ADHs) : a soluble monomeric ADH from Pseudomonas putida HK5(ADH-IIB)and a heterotrimeric membrane-bound ADH from Gluconobacter suboxydans (ADH-GS) ;"Acta Cryst. D. 55. 1933-1936 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] L. D. Elias, TANAKA, M, IZU, H, MATSUSITA, K, ADACHI, O & YAMADA, M: "Functions of amino acid residues in the active site of Escherichia coli PQQ-containing quinoprotein glucose dehydrogenase"J. Biol. Chem. 275,(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] H.Miyoshi et al.: "Topographical characterization of the ubiquinone reduction site of glucose dehydrogenase of in Escherichia coli using depty-dependent fluorescent inhibitors"Biochim.Biophys.Acta. 1412. 29-36 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] K.Matsushita et al.: "Electron transfer from quinohemoprotein alcohol dehydrogenase to blue copper protein azurin,in the alcohol oxidase respiratory chain of Pseudomonas putida"Biochemistry. 38. 6111-6118 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Zhi-wei Chen et al.: "Crystallization and preliminary diffraction studies of two quinoprotein alcohol dehydro-genases(ADHs):a soluble monomeric ADH from Pseudomonas putida HK5(ADH-IIB)and a heterotrimeric membraned ADH from Gluconobacter suboxydans(ADH-GS)"Acta Cryst.D. 55. 1933-1936 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] M.D.Elias et al.: "Functions of amino acid residues in the active site of Esherichia coli PQQ-containing quinoprotein glucose dehydrogenase"J.Niol.Chem.. 275. in press (2000)

    • Related Report
      1999 Annual Research Report
  • [Publications] 山田守、松下一信: "キノプロテインおよびキノヘモプロテイン脱水素酵素の分子内および分子間電子移動"生化学. 71. 413-428 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] 松下一信(共著): "新版微生物学実験法:5.7細胞活性の測定"講談社サイエンティフィク(杉山純多ら編). 324 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] 松下一信(共著): "広川タンパク質化学第4巻酵素4.1オキシドレダクターゼアルコールデヒドロゲナーゼ(キノプロテイン)"(広川書店)野崎光洋ら(編). 353 (2000)

    • Related Report
      1999 Annual Research Report
  • [Publications] Y.Nomura et al.: "Preparation and preservation of freeze-drined cells of acetic acid bacteria with aldehyde oxidase activity" Biosci Biotech Biochem. 62. 1134-1137 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] O.Adachi et al.: "Characterization of quinohemoprotein amine dehydrogenase from Pseudomonas putida" Biosci Biotech Biochem. 62. 469-478 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] M.Yamada et al.: "Mutat isolation of the Escherichia coli quinoprotein glucose dehydrogenase and analysis of crucial residues Asp-730 and His-775 for its function" J.Biol.Chem.273. 22021-22027 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] K.Matsushita et al.: "The quinohemoprotein alcohol dehydrogenase of Gluconobacter suboxydans has ubiquinol oxidation activity at a site different from the ubiquinone reduction site" Biochim.Biophys.Acta. 1409. 154-164 (1999)

    • Related Report
      1998 Annual Research Report
  • [Publications] H.Miyoshi et al.: "Topographical characterization of the ubiquinone reduction site of glucose dehydrogenase of in Escherichia coli using depht-dependent fluorescent inhibitors" Biochim.Biophys.Acta. (in press). (1999)

    • Related Report
      1998 Annual Research Report
  • [Publications] T.Ikeda et al.: "Electrocatalytic properties of Acetobacter aceti cells immobilized on electrodes for the quinone-mediated oxidation of ethanol" J.Electroanal.Chem.430. 197-204 (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] J.Frebortova et al.: "Intramolecular electron transport in quinoprotein alcohol dehydrogenase of Acetobacter methanolicus : A redox-tiration study" Biochim.Biophys.Acta. 1363. 24-34 (1998)

    • Related Report
      1997 Annual Research Report
  • [Publications] H.Ohshima et al.: "Characterization of the ubiquinone reduction site of mitochondrial complex I using bulky synthetic ubiquinones" Biochemistry. (in press). (1998)

    • Related Report
      1997 Annual Research Report

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Published: 1997-04-01   Modified: 2016-04-21  

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