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Structure and Function of Enzymes Participating in Metabolism of D-Amino Acid of Bacterial Cell Walls and Development of their Specific Inhibitors

Research Project

Project/Area Number 06454077
Research Category

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

Allocation TypeSingle-year Grants
Research Field 応用微生物学・応用生物化学
Research InstitutionKYOTO UNIVERSITY

Principal Investigator

SODA Kenji  Institute for Chemical Research, Kyoto University Professor, 化学研究所, 教授 (30027023)

Co-Investigator(Kenkyū-buntansha) KURIHARA Tatsuo  Institute for Chemical Research, Kyoto University Instructor, 化学研究所, 助手 (70243087)
ESAKI Nobuyoshi  Institute for Chemical Research, Kyoto University Associate Professor, 化学研究所, 助教授 (50135597)
吉村 徹  京都大学, 化学研究所, 助手 (70182821)
Project Period (FY) 1994 – 1995
Project Status Completed (Fiscal Year 1995)
Budget Amount *help
¥7,900,000 (Direct Cost: ¥7,900,000)
Fiscal Year 1995: ¥2,400,000 (Direct Cost: ¥2,400,000)
Fiscal Year 1994: ¥5,500,000 (Direct Cost: ¥5,500,000)
KeywordsD-Amino acid / Alanine Racemase / D-Amino acid aminotransferase / Glutamate racemase / Suicide substrate / 酵素自殺基質 / UDP-N-アセチルムラミル-L-アラニン / ピリドキサルリン酸 / D-アミノ酸トランスアミナーゼ / セリンO-スルフェート / 酵素阻害剤
Research Abstract

D-Amino acids such as D-alanine and D-glutamate are the indespensable components of the peptidoglycan layr of the bacterial cell walls. Thus, the specific inhibitors for the bacteral enzymes participate in the biosyntheses of D-amino acids can be potent antibiotics. We studied detailed mechanisms of alanine racemase, D-amino acid aminotransferase, and glutamate racemase, to develop the meachanism-based inhibitors for these enzymes. We studied the role of Lys39 of the thermostable alanine racemase of Bacillus stearothermophilus, which is bound to the cofactor, pyridoxal 5'-phosphate (PLP) , and suggested this residue to be a catalytic base by site-directed mutagenesis. The terminal amino group of Lys39 acts as a base to abstract the 2-hydrogen from the substrate. The effects of alkylamines on the K39A mutant enzymes suggest that Lys39 acts as a sole catalytic base to abstract the 2-hydrogen from the substrate and returns it to the 2-carbon of substrate moiety of a deprotonated intermedi … More ate. D-Amino acid aminotransferase catalyzes the transfer of amino group between various D-amino acids and keto acids. We studied the catalytic role of leucine 201 residue of the themostable D-amino acid aminotransferase : the residue was crystallographically shown to be in the vicinity of the active-site to interact with the bound PLP by site-directed mutagenesis. The Leu 201 residue probably regulates the function of cofactor during the conversion of PMP to PLP.Glutamate racemase catalyzes the racemization of glutamate to produce D-glutamate. We compared the enzyme with those of Lactic acid bacteria. The addition of UDP-N-acetylmuramyl-L-alanine, an activator of the E.coli enzyme affected the CD and fluorescence spectra of the E.coli enzyme. In contrast, the enzymes of P.pentosaceus and L.brevis were not activated by UDP-N-acetylmuramyl-L-alanine. The Pediococcus enzyme shows a significant sequence similarity to mammalian myoglobin, and was inhibited by hemin. Glutamate racemase of E.coli was not inhibited by hemin in the absence of UDP-N-acetylmuramyl-L-alanine, but strongly inhibited in the presence of UDP-N-acetylmuramyl-L-alanine. The conformation of the E.coli enzyme is converted to a similar form to that of the Pediococcus enzyme by the addition of UDP-N-acetylmuramyl-L-alanine. Less

Report

(3 results)
  • 1995 Annual Research Report   Final Research Report Summary
  • 1994 Annual Research Report
  • Research Products

    (18 results)

All Other

All Publications (18 results)

  • [Publications] Choi. Soo-Young: "Bacterial Glutamate Racemase has High Sequence Homology with Myoglobins and Forms an Equimolar Inactive Complex with Hemin." Proc. Natl. Acad Sci., USA,. 91. 10144-10147 (1994)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Nishimura Katsushi: "A Simple Method for Detemination of Stereospecificity of Aminotransferase for C-4′Hydrogen Transfer of the Coenzyme." Bioorganic & Medical Chemistry. 2. 605-607 (1994)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Toyama Hirohide: "Reconstitute of Fragmentary Form of Themostable Alanine Racemase" Biosci. Biotech. Biochem.59. 1118-1120 (1995)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Kishimoto Kazuhisa: "Role of Leucine 201 of Thermostable D-Amino Acid Aminotransferase from Thermophile, Bacillus sp. YM-1" J. Biochem.117. 691-696 (1995)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Ashiuchi Makoto: "In Vivo Effect of GroESL on the Folding of Gltamate Racemase of Eschirichia coli." J. Biochem.117. 495-498 (1995)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Yoshimura Tohru: "Alanine Racemase-Structure and Function" KODANSHA, 244 (1994)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Choi, Soo-Young: "Bacterial Glutamate Racemase has High Sequence Homology with Myoglobins and Forms an Equimolar Inactive Complex with Hemin." Proc.Natl.Acad.Sci., USA. 91. 10144-10147 (1994)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Nishimura, Katsushi: "A Simple Method for Determination of Stereospecificity of Aminotransferase for C-4'Hydrogen Transfer of the Coenzyme." Bioorganic & Medical Chemistry. 2. 605-607 (1994)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Yoshimura Tohru: "Alanine Racemase - Structure and Function" (T.Fukui ed.) KODANSHA. 147-163 (1994)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Toyama Hirohide: "Reconstitute of Fragmentary Form of Thermostable Alanine Racemase" Biosci.Biotech.Biochem.59. 1118-1120 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Kishimoto Kazuhisa: "Role of Leucine 201 of Thermostable D-Amino Acid Aminotransferase from Thermophile, Bacillus sp.YM-1" J.Biochem.117. 691-696 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] Ashiuchi Makoto: "In Vivo Effect of GroESL on the Folding of Glutamate Racemase of Eschirichia coli." J.Biochem.117. 495-498 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1995 Final Research Report Summary
  • [Publications] H. Toyama et al.: "Reconstitute of Fragmentary Form of Thermostable Alanine Racemase" Biosci. Biotech. Biochem.59. 1118-1120 (1995)

    • Related Report
      1995 Annual Research Report
  • [Publications] M. Ashiuchi, M et al.: "In Vivo Effect of GroESL on the Folding of Glutamate Racemase of Eschirichia coli" J. Biochem.117. 495-498 (1995)

    • Related Report
      1995 Annual Research Report
  • [Publications] W.M.Jones et al.: "Determination of Free D-Amino Acids with a Bacterial Transaminase:Their Depletion Leads to Inhibition,of Bacterial Growth" Anal.Biochem.218. 204-209 (1994)

    • Related Report
      1994 Annual Research Report
  • [Publications] K.Kishimoto et al.: "Role of Leucine 201 of Thermostable D-Amino Acid Aminotrasferase from a Thermophile." J.Biochem.(印刷中). (1994)

    • Related Report
      1994 Annual Research Report
  • [Publications] H.Toyama et al.: "Reconstitution of Fragmentary Form of Thermostable Alanine Racemase" Biosci.Biotech.Biochem.

    • Related Report
      1994 Annual Research Report
  • [Publications] S.Sawada et al.: "Kinetics of Thermostable Alanine Racemase of bacillus stearothermophilus" Biosci.Biotech.Biochem.

    • Related Report
      1994 Annual Research Report

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Published: 1994-04-01   Modified: 2016-04-21  

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