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

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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.