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Analysis and alteration of the substrate recognition machinery of stereospecific 2-hydroxyacid dehydrogenases from lactic acid bacteria.

Research Project

Project/Area Number 15580067
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field Applied microbiology
Research InstitutionFaculty of Science and Technology, Tokyo University of Science

Principal Investigator

TAGUCHI Hayao  Tokyo University of Science, Department of Applied Biological Science, Faculty of Science and Technology, 理工学部, 教授 (90188136)

Project Period (FY) 2003 – 2004
Project Status Completed (Fiscal Year 2004)
Budget Amount *help
¥3,300,000 (Direct Cost: ¥3,300,000)
Fiscal Year 2004: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 2003: ¥2,400,000 (Direct Cost: ¥2,400,000)
KeywordsLactate dehydrogenase / Formate dehydrogenase / 2-Hydroxyacid dehydrogenase / Substrate specificity / Substrate recognition / Lactic acid bacteria / Allosteric enzyme / Catalysis / Lactobacillus / アロステリック酵素 / タンパク質工学
Research Abstract

For L.pentosus D-LDH, replacement of Tyr52 with Leu, Val, and Ala induced size-dependent changes in the specificity of the enzyme to aliphatic or aromatic 2-ketoacid substrates, indicating that the size or shape of hydrophobic side chain at position 52 determines the space of the binding site for hydrophobic side chains of 2-ketoacid substrates. The replacements of Tyr52 with Arg, Thr and Asp, and those of Phe299 with Gly and Ser greatly reduced the enzyme activities toward all 2-ketoacids tested, and induced slow but significant catalysis of NADH oxidation without substrate. However, the double mutations for positions 52 and 299 did not additively injured the enzyme function, but compensated each other for substrate-independent NADH oxidation and catalytic function for some substrates. Replacement of Asn97 with Asp did not markedly change the overall protein structure, but markedly perturbed the conformation of the active site loop in Lactobacillus pentosus D-LDH. The Asn97Asp mutant … More D-LDH exhibited virtually the same k_<cat>, but about 70-fold higher K_M value for pyruvate than the wild-type enzyme. For Paracoccus sp.12-A FDH, in contrast, replacement of Glu141 with Gln and Asn induced only 5.5- and 4.3-fold increases in the K_M value, but 110 and 590-fold decreases in the k_<cat> values for formate, respectively. Furthermore, these mutant FDHs, particularly the Glu141Asn enzyme, exhibited markedly enhanced catalytic activity for glyoxylate reduction, indicating that FDH is converted to a 2-hydroxyacid dehydrogenase on the replacement of Glu 141. These results indicate that the active site loops play different roles in the catalytic reactions of D-LDH and FDH-stabilization of substrate binding and promotion of hydrogen transfer, respectively-and that Asn97 and Glu141,which stabilize suitable loop conformations, are essential elements for proper loop functioning.
For L.casei allosteric L-LDH, which exhibits relatively wide substrate specificity for 2-ketoacids, replacements of Arg173 with Gln and of His188 with Ala or Asp revealed that some 2-ketoacids can be bound to both the catalytic and allosteric sites of the enzyme, and the binding to the allosteric site can exhibit significant activation effects. These replacements also indicated that Arg173 is essential for the 2-ketoacid binding to the allosteric site, and His188 determines the specificity toward 2-ketoacids. Less

Report

(3 results)
  • 2004 Annual Research Report   Final Research Report Summary
  • 2003 Annual Research Report
  • Research Products

    (7 results)

All 2005 2004 2003 Other

All Journal Article (5 results) Book (1 results) Publications (1 results)

  • [Journal Article] Distinct conformation-mediated functions of an active site loop in the catalytic reactions and formate dehydrogenase of NAD-dependent D-lactate dehydrogenase2005

    • Author(s)
      篠田 剛
    • Journal Title

      J.Biol.Chem. 280

      Pages: 17068-17075

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Distinct conformation-mediated functions of an active site loop in the catalytic reactions of NAD-dependent D-lactate dehydrogenase and formate dehydrogenase2005

    • Author(s)
      Takeshi Shinoda
    • Journal Title

      J.Biol.Chem. 280

      Pages: 17068-17075

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Conversion of Lactobacillus pentosus D-lactate dehydrogenase to a D-hydroxyiso-caproate dehydrogenase through a single amino acid replacement.2003

    • Author(s)
      徳田千束
    • Journal Title

      Journal of Bacteriology 185

      Pages: 5023-5026

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Conversion of Lactobacillus pentosus D-lactate dehydrogenase to a D-hydroxyisocaproate dehydrogenase through a single amino acid replacement.2003

    • Author(s)
      Chizuka Tokuda
    • Journal Title

      Journal of Bacteriology 185

      Pages: 5023-5026

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Distinct conformation-mediated functions of an active site loop in the catalytic reactions of NAD-dependent D-lactate dehydrogenase and formate dehydrogenase

    • Author(s)
      田口速男 他9名
    • Journal Title

      Journal of Biological Chemistry (印刷中)

    • Related Report
      2004 Annual Research Report
  • [Book] タンパク質工学の基礎2004

    • Author(s)
      松澤 洋
    • Total Pages
      262
    • Publisher
      東京化学同人
    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Publications] 田口速男 他10名: "Conversion of Lactobacillus pentosus D-lactate dehydrogenase to a D-hydroxyisocaproate dehydrogenase through a single amino acid replacement."Journal of Bacteriology. 185・16. 5023-5026 (2003)

    • Related Report
      2003 Annual Research Report

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Published: 2003-04-01   Modified: 2016-04-21  

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