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1990 Fiscal Year Final Research Report Summary

Study on function of the active-site of cephalosporinase type beta-lactamase

Research Project

Project/Area Number 01571194
Research Category

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

Allocation TypeSingle-year Grants
Research Field Biological pharmacy
Research InstitutionChiba University

Principal Investigator

SAWAI Tetsuo  Chiba Univ. Fac. of Pharm. Sci., Professor, 薬学部, 教授 (70009174)

Co-Investigator(Kenkyū-buntansha) TSUKAMOTO Kikuo  Chiba Univ. Fac. of Pharm. Sci., Assistant, 薬学部, 助手 (20183478)
Project Period (FY) 1989 – 1990
Keywordsbeta-Lactamase / Cephalosporinase / Active-site / beta-Lactam Antibiotics / Drug Resistance / Site-directed Mutagenesis / Gram-negative Bacteria
Research Abstract

This study was carried out for elucidating the molecular structure of the active-site of a cephalosporinase type bata-lactamase and the function of amino acid residues located in the active-site. The results of this study will contribute to the improvelopments in bata-lactam antibiotics and specific inhibitors for beta-lactamases. The beta-lactamase gene was cloned from the chromosomal DNA of Citrobacter Freundii to a vector plasmid and the nucleotide sequence was completely determined. From the nuclotide sequence, the amino acid sequence of the mature enzyme was deduced (361 amino acids). Ser-64 was also confirmed to be the catalytic site. On the basis of the comparison between the amino acid sequence of the C.freeundii beta-lactamase and those of known beta-lactamases, the amino acid residues constructing the active-site space were speculated. Those residues were expected to participate in the catalytic action or to determine the substrate spectrum. Lys-67, Tyr-150 and Lys-315 which were assumed to be situated in positions close to Ser-64 in the three-dimensional structure of the enzyme protein, were converted into other amino acids by site-directed mutagenesis, respectively. The kinetic investigations of these mutant enzymes revealed the contribution of the individual residues to the enzyme activity. Asp-217 and Glu-219 were assumed to be located at the rim of the active-site hollow, and be unable to interact directly with the substrate in the active-site hollow. However, we found an interesting fact that the substrate spectrum of the mutant enzymes were extended to oxyimino cephalosporins. On the other hand, the mutant enzymes showed a lower activity to cephamycins.

  • Research Products

    (8 results)

All Other

All Publications (8 results)

  • [Publications] T.Tsukamoto: "Role of lysineー67 in the active site of class C βーlactamase from Citrobacter freundii GN346" Eur.J.Biochem.188. 15-22 (1990)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] T.Tsukamoto: "Substitution of aspartic acidー217 of Citrobacter freundii cephalosporinase and properties of the mutant enzymes" FEBS Lett.246. 211-214 (1990)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] T.Tsukamoto: "Extension of the substrate spectrum by an amino acid substitution at residue 219 in the Citrobacter freundii cephalosporinase" J.Bacteriol.172. 4348-4351 (1990)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] T.Tsukamoto: "Function of the conserved triad residues in the class C βーlactamase from Citrobacter freundii GN346" FEBS Lett.271. 243-246 (1990)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] T. Tsukamoto: "Role of lysine-67 in the active site of class C beta-lactamase from Citrobacter freundii GN346" Eur. J. Biochem.188. 15-22 (1990)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] T. Tsukamoto: "Substitution of aspartic acid-217 of Citrobacter freundii cephalosporinase and properties of the mutant enzymes" FEBS Lett.246. 211-214 (1990)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] T. Tsukamoto: "Extension of the substrate spectrum by amino acid substitution at residue 219 in the Citrobacter freundii cephalosporinase" J. Bacteriol.172. 4348-4351 (1990)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] T. Tsukamoto: "Function of the conserved triad residues in the class C beta-lactamase from Citrobacter freundii GN346" FEBS Lett.271. 243-246 (1990)

    • Description
      「研究成果報告書概要(欧文)」より

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Published: 1993-08-12  

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