Study on the active site function of the cephalosporin hydrolysing beta-lactamase.

• Project/Area Number: 05671808
• Research Category: Grant-in-Aid for General Scientific Research (C)
• Allocation Type: Single-year Grants
• Research Field: Biological pharmacy
• Research Institution: Chiba University
• Principal Investigator: TSUKAMOTO Kikuo Facul.Pharmaceutical Sciences, Chiba University Research Associates, 薬学部, 助手 (20183478)
• Project Period (FY): 1993 – 1994
• Project Status: Completed(Fiscal Year 1994)
• Budget Amount: ¥2,100,000 (Direct Cost : ¥2,100,000); Fiscal Year 1994 : ¥700,000 (Direct Cost : ¥700,000); Fiscal Year 1993 : ¥1,400,000 (Direct Cost : ¥1,400,000)
• Keywords: beta-lactamase / active site / catalytic mechanism / beta-lactam antibiotics / drug resistance / protein-ligand interaction / molecular dynamics / molecular modelling / 基質特異性

Research Abstract

In the clinical field, cephalosporin hydrolysing class C beta-lactamase are of importance because clinical isolates producing class C beta-lactamase show high levels of resistance to third generation oxyimino cephalosporins. To survey functional amino acids of the class C beta-lactamase, a series of mutant gense of the class C beta-lactamase of Citrobacter freundii GN346 were constructed by site-directed mutagenesis. Three conserved residues, i.e., Lys67, Tyr150 and Lys315 were identified to be the catalytic residues because the substitution of these residues to non-conserved amino acids resulted in a dramatic decrease in the enzyme activity. Change of kinetic parameters to favorable substrates, the kinetic analysis of the acyl-enzyme intermediate formation by using ^<14>C-benzylpenicillin as a substrate indicated that these three residues play important role in catalytic mechanism. To estimate the interaction between residues lying in the active site pocket and a favorable beta-lactam substrate, molecular dynamic symulation was performed. This result sugested that Lys67 assists the active site Ser64 in the acylation step, and the other two residues, Tyr150 and Lys315, may involed in the deacylation step.

Research Products

• [Publications] Nukaga,M.et al.: "A survey of a functional amino acid of class C β-lactamase corresponding to Glu166 of class A β-lactamases." FEBS Lett.332. 93-98 (1993)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Nukaga,M. et al.: "Interaction of oxyimino β-lactams with a class C β-lactamase and a mutant with a spectrum extended to β-lactams." Antimicrob.Agents Chemother.38. 1374-1377 (1994)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Nukaga, M., Tanimoto, K., Tsukamoto, K., Imajo, S., Ishiguro, M.and Sawai, T.: "A survey of a functional amino acid of class C beta-lactamase corresponding to Glu166 of class A beta-lactamases." FEBS Lett.322. 93-98 (1993)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Nukaga, M., Tsukamoto, K., Yamaguchi, H.and Sawai, T.: "Interaction of oxyimino beta-lactams with a class C beta-lactamase and a mutant with a spectrum extended to beta-lactams." Antimicrob. Agents Chemother.38. 1374-1377 (1994)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Nukaga,M.et al.: "Interaction of oxyimino β-lactams with a class C β-lactamase and a multant with a spectrum extended to β-lactams." Antimicrob.Agents Chemother.38. 1374-1377 (1994)
• [Publications] Nukaga,M.et al.: "A survey of a functional amino acid of class C β-lactamase corresponding to Glu166 of class A β-lactamases." FEBS Lett.332. 93-98 (1993)