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Structural and functional analysis of bacterial D-amino acid metabolic enzymes to develop their inhibitors

Research Project

Project/Area Number 08456052
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 InstitutionKansai University

Principal Investigator

SODA Kenji  Kansai University・Faculty of Engineering, Professor, 工学部, 教授 (30027023)

Co-Investigator(Kenkyū-buntansha) ESAKI Nobuyoshi  Kyoto University・Institute for Chemical Research, Professor, 化学研究所, 教授 (50135597)
OIKAWA Tadao  Kansai University・Faculty of Engineering, Associate Professor, 工学部, 講師 (80233005)
Project Period (FY) 1996 – 1997
Project Status Completed (Fiscal Year 1997)
Budget Amount *help
¥8,000,000 (Direct Cost: ¥8,000,000)
Fiscal Year 1997: ¥2,800,000 (Direct Cost: ¥2,800,000)
Fiscal Year 1996: ¥5,200,000 (Direct Cost: ¥5,200,000)
KeywordsD-amino acid aminotransafearse / alanine racemase / peptidoglycan / glutamate racemase / reaction mechanism / D-アミノ酸
Research Abstract

D-Amino acid aminotransferase (D-AAT), alanine racemase (AlaR), amino acid racemase of broad substrate specificities (AAR), and glutamate racemase (GluR) participate in the metabolism of D-amino acids, some of which are indispensable for bacteria as components of the peptidoglycan layr of cell walls. Physiological roles of these D-amino acids in mammals are not known, thus these enzymes have been regarded as a target for the development of novel antibacterial agents serving, for example, as suicide substrates. To develop the mechanism-based inhibitors, we studied the structure-function relationship of these enzymes. We studied the role of Arg98 of D-AAT,the binding site for the alpha-carboxyl group of substrates. This is presumably crucial for the unique stereospecificity of the enzyme. Replacement of Arg98 by replacement by methionine and lysine, resulted in decreases in the kmax values and increases in the Kd values for both amino donors and amino acceptors. The introduction of another mutation, replacement of Tyr88 being located near Arg98 by arginine, in addition to the above Arg98 mutation, resulted in increases in the kmax values but little change in the Kd values. We cloned the glutamate racemase gene (murl) of Bacillus pumilus cells into E.coli WM335, a D-glutamate auxotroph, by means of a genetic complement method. MurI of B.pumilus encodes a 272-amino acid protein with an unusual initiation codon, TTG,which is probably the reason for the low expression efficiency of the gene. We also studied the half and overall transaminations catalyzed by AAR,and found that the enzyme catalizes the non-stereospecific transaminations.

Report

(3 results)
  • 1997 Annual Research Report   Final Research Report Summary
  • 1996 Annual Research Report
  • Research Products

    (23 results)

All Other

All Publications (23 results)

  • [Publications] 吉村,徹: "D-アミノ酸の役割 生理作用と代謝をめぐって" 現代化学. 12号. 14-19 (1996)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Yoshimura, T.: "Stereospecificity for the Hydrogen Transfer and Molecular Evolution of Pyridoxal Enzymes" Biosci.Biotech.Biochem.60巻2号. 181-187 (1996)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] 吉村,徹: "細菌のD-アミノ酸代謝関連酵素の構造と機能の特性" 日本農芸化学会誌. 70巻1号. 15-20 (1996)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Liu, L.: "Cloning and Expression of the Glutamate Racemase Gene of Bacillus pumilus" Journal of Biochemistry. 121巻6号. 1155-1161 (1997)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Liu, L.: "Compensation for D-Glutamate Auxotrophy of Escherichia coli WM335 by D-Amino Acid Aminotransferase Gene and Regulation of murI expression" Bioscience Biotechnology Biochemistry. 62巻1号. 193-195 (1998)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Lim, Y.-H.: "Nonstereospecific Transamination Catalyzed by Pyridoxal Phosphatedependent Amino Acid Racemases of Broad Substrate Specificity" The Journal of Biological Chemistry. 273巻7号. 4001-4005 (1998)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Yoshimura, T.et al.: "Role of D-Amino Acids" Gendai Kagaku. 12 (309). 14-19 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Yoshimura, T.et al.: "Stereospecificity for the Hydrogen Transfer and Molecular Evolution of Pyridoxal Enzymes" Biosci.Biotech.Biochem.60 (2). 181-187 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Yoshimura, T.: "Structure and Function of Bacterial D-Amino Acid-Related Enzymes Enzymes" Nippon Nougeikagaku kaishi. 70 (1). 15-20 (1996)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Liu, L.et al.: "Cloning and Expression of the Glutamate Racemase Gene of Bacillus pumilus" J.Biochem.121 (6). 1155-1161 (1997)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Liu, L.et al.: "Compensation for D-Glutamate Auxotrophy of Escherichia coli WM335 by D-Amino Acid Aminotransferase Gene and Regulation of murI Expression" Biocsci.Biotech.Biochem.62 (1). 193-195 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] Lim, Y.-H.: "Nonstereospecific Transamination Catalyzed by Pyridoxal Phosphate-dependent Amino Acid Racemases of Broad Substrate Specificity" J.Biol.Chem.273 (7). 4001-4015 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1997 Final Research Report Summary
  • [Publications] 吉村,徹: "D-アミノ酸の役割 生理作用と代謝をめぐって" 現代化学. 12号. 14-19 (1996)

    • Related Report
      1997 Annual Research Report
  • [Publications] Yoshimura,T.: "Stereospecificity for the Hydrogen Transfer and Molecular Evolution of Pyridoxal Enzymes" Biosci.Biotech.Biochem.60巻2号. 181-187 (1996)

    • Related Report
      1997 Annual Research Report
  • [Publications] 吉村,徹: "細菌のD-アミノ酸代謝関連酵素の構造と機能の特性" 日本農芸化学会誌. 70巻1号. 15-20 (1996)

    • Related Report
      1997 Annual Research Report
  • [Publications] Liu,L.: "Cloning and Expression of the Glutamate Racemase Gene of Bacillus pumilus" Journal of Biochemistry. 121巻6号. 1155-1161 (1997)

    • Related Report
      1997 Annual Research Report
  • [Publications] Liu,L.: "Compensation for D-Glutamate Auxotrophy of Escherichia coli WM335 by D-Amino Acid Aminotransferase Gene and Regulation of murI expression" Bioscience Biotechnology Biochemistry. 62巻1号. 193-195 (1998)

    • Related Report
      1997 Annual Research Report
  • [Publications] Lim,Y.-H.: "Nonstereospecific Transamination Catalyzed by Pyridoxal Phosphate-dependent Amino Acid Racemases of Broad Substrate Specificity" The Journal of Biological Chemistry. 273巻7号. 4011-4005 (1998)

    • Related Report
      1997 Annual Research Report
  • [Publications] 左右田健次: "D-アミノ酸代謝とビタミンB6酵素" ビタミン. 70・3. 103-113 (1996)

    • Related Report
      1996 Annual Research Report
  • [Publications] T.Yoshimura: "Stereospecificity for the Hydrogen Transfer and Molecular Evolution of Pyridoxal Enzymes" Biosci. Biotech. Biochem.60・2. 181-187 (1996)

    • Related Report
      1996 Annual Research Report
  • [Publications] Kwang-Hwan Jhee: "Stereospecificity of Thermostable Ornithine 5-Aminotransferase for the Hydrogen Transfer in the L-and D-Ornithine Transamination" Biochemistry. 35・30. 9792-9796 (1996)

    • Related Report
      1996 Annual Research Report
  • [Publications] 吉村徹: "D-アミノ酸の役割・生理作用と代謝をめぐって" 現代化学. 12. 14-19 (1996)

    • Related Report
      1996 Annual Research Report
  • [Publications] Hyeon-Su Ro: "Site-directed Mutagenesis of the Amino Acid Residues in β-strand III [Val30Val36]of D-amino Acid Aminotransferase of Bacillus sp. YM-1" FEBS Letters. 398. 141-145 (1996)

    • Related Report
      1996 Annual Research Report

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Published: 1996-04-01   Modified: 2016-04-21  

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