2002 Fiscal Year Final Research Report Summary
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
| Project/Area Number |
13672257
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Physical pharmacy
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| Research Institution | Kumamoto University |
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Principal Investigator |
MORI Hiromasa Pharmaceutical Sciences Associated Professor, 薬学部, 助教授 (40040315)
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| 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)
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| Project Period (FY) |
2001 – 2002
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| Keywords | drug-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 |
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| 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.
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Research Products
(2 results)
