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

β-内酰胺抗性微生物产生的定点突变金属β-内酰胺酶的制备以及野生型和突变型酶水解β-内酰胺的机制

• 批准号: 13672257
• 负责人: MORI Hiromasa
• 金额: $ 1.98万
• 依托单位: Kumamoto University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13672257/
• 关键词: 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; メタロ-β-ラクタマーゼ; 部位特異的変異酵素

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.

本研究的目的是研究金属β-内酰胺酶IMP-1水解抗生素β-内酰胺类抗生素的机制，通过测定β-内酰胺在不同pH和Zn（II）浓度下的水解速率，发现β-内酰胺水解活性因Zn（II）离子的释放而丧失，对β-内酰胺水解起重要作用的氨基酸残基也在不同pH和Zn（II）浓度下的水解速率有所不同IMP-1的脱辅基酶经CoCl_2溶液滴定法制备钴（II）取代酶，并测定其紫外-可见光谱。这些钴（II）取代的酶获得两个钴离子。突变体IMP-1的金属结合位点与野生型IMP-1不同，为5配位结构。制备突变体IMP-1的晶体，并对其结构进行X射线衍射分析。突变体D81 E的整个结构与野生型IMP-1几乎相同。D81 E的金属结合位点中Zn（II）-Zn（II）的距离为3.7 μ m，而野生型IMP-1中为3.3 μ m。

项目成果

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)

Masafumi Goto：“与抗 HIV-EP1 DNA 结合的金属螯合抑制剂相关的三种新型五齿铜 (II) 配合物的合成、表征和光谱特性”J.

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)

Hiromasa Kurosaki：“带有吡啶基甲基和 N,N-二甲基氨基甲酰基甲基基团的 1, 8-双官能化不对称仙客来铜 (II) 络合物的合成和表征”Inorg。

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

M.Goto、H.Yasuzawa、T.Higashi、Y.Yamaguchi、A.Kawanami、S.Mifune、H.Mori、H.Nakayama、K.Harada、Y.Arakawa：“β-内酰胺水解的依赖性粘质沙雷菌 (IMP-1) 产生的锌 (II) β-内酰胺酶对 pH 值和锌 (II) 离子浓度的影响

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)

Masafumi Goto：“1,11-双(2-吡啶甲基)-1,4,8,11-四氮杂环十四烷-5,7-二酮钯(II)配合物的合成和X射线晶体结构”J。

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)

Masafumi Goto：“N-(4-硝基-2-羟基)苯基甲基-N-(2-吡啶基乙基)-N-(2-吡啶基甲基)胺双(μ-羟基)二铁(III)配合物的合成和表征烷烃的羟基化反应”Bioorg.Med.Chem.Lett..11.785-788(2001)