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Preparation of site-directed mutated metallo β-lactamase produced from a microorganism resistant for β-lactams and mechanism of hydrolysis for β-lactams by wild-type and mutated enzymes

Research Project

Project/Area Number 13672257
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field Physical pharmacy
Research InstitutionKumamoto University

Principal Investigator

MORI Hiromasa  Pharmaceutical Sciences Associated Professor, 薬学部, 助教授 (40040315)

Co-Investigator(Kenkyū-buntansha) KUROSAKI Hiromasa  Pharmaceutical Sciences Assistant, 薬学部, 助手 (70234599)
GOTO Masafumi  Pharmaceutical Sciences Professor, 薬学部, 教授 (50080180)
Project Period (FY) 2001 – 2002
Project Status Completed (Fiscal Year 2002)
Budget Amount *help
¥3,100,000 (Direct Cost: ¥3,100,000)
Fiscal Year 2002: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 2001: ¥2,100,000 (Direct Cost: ¥2,100,000)
Keywordsdrug-resistant microorganism / metallo β-lactamase / hydrolysis of β-lactams / PCR / site-directed mutated enzyme / Co(II)-substituted enzymes / crystal structure analysis by X-ray diffraction / UV-vis spectrum / メタロ-β-ラクタマーゼ / 部位特異的変異酵素
Research Abstract

The object of this study is the research of the mechanisms for hydrolysis of antibiotics, β-lactams, by a metallo β-lactamase, IMP-1, produced from Serratia marcescens.
It was obtained the result that the hydrolyzing activity was lost by release of Zn(II) ions by measuring the rate of the hydrolysis of β-lactams in varying pH and Zn(II) concentration.
The amino acid residues that play the important role for hydrolysis of β-lactams were identified by preparation of site-directed mutants of IMP-1.
The cobalt(II)-substituted enzymes from apo-enzyme of IMP-1 were prepared by titration of CoCl_2 solution and its site-directed mutants, and were measured the UV-vis spectrum. These cobalt(II)-substituted enzymes obtained two cobalt ions. The structure of the metal binding site in mutants was 5-coordinated structure different from wild-type IMP-1.
The crystal of mutant of IMP-1 was prepared and the structure was analyzed by X-ray diffraction analysis. The whole structure of mutant, D81E, was almost the same compared wild-type IMP-1. The distance of Zn(II)-Zn(II) in the metal binding site of D81E was 3.7Å compared 3.3Å in wild-type IMP-1.

Report

(3 results)
  • 2002 Annual Research Report   Final Research Report Summary
  • 2001 Annual Research Report
  • Research Products

    (9 results)

All Other

All Publications (9 results)

  • [Publications] M.Goto, H.Yasuzawa, T.Higashi, Y.Yamaguchi, A.Kawanami, S.Mifune, H.Mori, H.Nakayama, K.Harada, Y.Arakawa: "Dependence of Hydrolysis of β-Lactams with a Zinc(II)β-Lactamase Produced from Serratia marcescens (IMP-1) on pH and Concentration of Zinc(II) Ion : Dissociation of Zn(II) from IMP-1 in Acidic Medium"Biol.Pharm.Bull.. 26(5). (2003)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] M. Goto, H. Yasuzawa, T. Higashi, Y. Yamaguchi, A. Kawanami, S. Mifune, H. Mori, H. Nakayama, K. Harada, Y. Arakawa: "Dependence of Hydrolysis of β-Lactams with a Zinc(II) β-Lactamase Produced from Serratia marcescens (IMP-1) on pH and Concentration of Zinc(II) Ion: Dissociation of Zn(II) from IMP-1 in Acidic Medium"Biol. Pharm. Bull.. 26(5) (in press). (2003)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] M.Goto, H.Yasuzawa, T.Higashi, Y.Yamaguchi, A.Kawanami, S.Mifune, H.Mori, H.Nakayama, K.Harada, Y.Arakawa: "Dependence of Hydrolysis of β-Lactams with a Zinc(II) β-Lactamase Produced from Serratia marcescens (IMP-1) on pH and Concentration of Zinc(II) Ion : Dissociation of Zn(II) from IMP-1 in Acidic Medium"Biol. Pharm. Bull.. 26(5). (2003)

    • Related Report
      2002 Annual Research Report
  • [Publications] Masafumi Goto: "DNA Cleavage by Pentadentate Iron(II) Complexes Containing Fluoro-Substituted Phenyl Groups"Bioorg. Med. Chem. Lett.. 12. 201-203 (2002)

    • Related Report
      2001 Annual Research Report
  • [Publications] Masafumi Goto: "Successive Intermolecular Transiminations in an Iron(II) Complex with Chiral Tridentate Ligands"Chem. Commun.. 2388-2389 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] Masafumi Goto: "Synthesis, Characterization of Bis(μ-hydroxo)diiron(III) Complex of N-(4-Nitro-2-hydroxy)phenylmethyl-N-(2-pyridylethyl)-N-(2-pyridylmethyl)amine and Hydroxylation Reaction of Alkane"Bioorg. Med. Chem. Lett.. 11. 785-788 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] Masafumi Goto: "Synthesis and X-ray Crystal Structures of Palladium(II) Complexes of 1,11-bis(2-pyridylmethyl)-1,4,8,11-tetraazacyclotetradecane-5,7-dione"J. Chem. Soc., Dalton Trans.. 898-901 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] Masafumi Goto: "Synthesis, characterization, and spectroscopic properties of three novel pentadentate copper(II) complexes related to the metal-chelating inhibitors against the DNA-Binding with HIV-EP1"J. Chem. Soc., Dalton Trans.. 441-447 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] Hiromasa Kurosaki: "Synthesis and characterization of a 1,8-difunctionalized dissymmetrical cyclam copper(II) complex bearing pyridylmethyl and N, N-dimethylcarbamoylmethyl groups"Inorg. Chim. Acta.. 322. 145-149 (2001)

    • Related Report
      2001 Annual Research Report

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Published: 2001-04-01   Modified: 2016-04-21  

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