X-ray Crystallographic Study of Antibiotics resistant Proteins

抗生素抗性蛋白的 X 射线晶体学研究

• 批准号: 17590090
• 负责人: NUKAGA Michiyoshi
• 金额: $ 2.3万
• 依托单位: Josai International University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2005
• 资助国家: 日本
• 起止时间: 2005 至 2006
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-17590090/
• 关键词: Infection Disease; Antibiotics; Enzyme; Protein; X-ray Crystallography

4 Projects are included in this study.1) β-Lactamase:1-a) SHV-1 : meropenem complex:High resolution (1.05 A) x-ray structure of SHV-1 covalently complexed with meropenem was solved. High resolution data provides some electron densities for important hydrogen atoms. One of the interesting results is protonated Glu166 and associated hydrogen bonding network around deacylation water. They suggest the deacylation water with the nucleophilicity decreased.1-b) GC1 β-Lactamase : ampicillin complex:Complex of class C β-lactamase from Enterobacter cloacae GC1 with good substrate ampicillin was trapped using protein modification and solved by x-ray crystallography. This is the first report in which acyl-enzyme of substrate could be trapped in classC β-lactamase.2) AAC/APH bifunctional enzyme:AAC/APH bifunctional enzyme was from MRSA and it add acetyl or/and phosphate group to amino glycoside antibiotics to inactivate. AAC/APH bifunctional enzyme gene was cloned to pCold vector for high expression. 3-5 mg protein could be obtained from 1 L culture. Kanamycin affinity column and gel filtration were used for purification. Preliminary crystallization screening was done. Although some crystals could be obtained, further optimizations are required.3) Macrolide phosphotransferase:Macrolide phosphotransferase gene (mphA) was cloned using PCR to pCold vector for high expression. Currently, the amount of the enzyme is not enough to make crystals. On the other hand, regulatory protein, mphR, could be highly expressed as GST fusion protein.4) Dihydropteroate synthase from Mycobacterium Jeprae:Dihydropteroate synthase (DHPS) is main target of dapson which is the important anti-M. leprae drug. To solve the complex of DHPS-dapson, high expression systems in E.coli were tested. In all of case, DHPS was expressed as inclusion body at first, co-expression with chaperone protein was effective to reduce them.

1）β-内酰胺酶：1-a）SHV-1：美罗培南复合物：解析了SHV-1与美罗培南共价复合物的高分辨（1.05 A）X射线结构。高分辨率数据提供了一些重要的氢原子的电子密度。有趣的结果之一是质子化的Glu 166和脱酰水周围的相关氢键网络。1-B）GC 1 β-内酰胺酶：氨苄青霉素复合物：使用蛋白质修饰捕获来自阴沟肠杆菌GC 1的C类β-内酰胺酶与良好底物氨苄青霉素的复合物，并通过X射线晶体学解析。2）AAC/APH双功能酶：AAC/APH双功能酶来源于MRSA，它是在氨基糖苷类抗生素上加乙酰基或/和磷酸基，使之水解。将AAC/APH双功能酶基因克隆到pCold载体中进行高效表达。3-5每升培养液可获得50 mg蛋白。采用卡那霉素亲和柱和凝胶过滤进行纯化。进行初步结晶筛选。3）大环内酯磷酸转移酶：利用PCR技术将大环内酯磷酸转移酶基因（mphA）克隆到pCold载体上进行高效表达。目前，这种酶的量还不足以制造晶体。另一方面，调控蛋白mphR可作为GST融合蛋白得到高效表达。4）二氢蝶酸合酶：二氢蝶酸合酶（DHPS）是重要的抗结核分枝杆菌药物dapson的主要作用靶点。治疗麻风病的药为了解决DHPS-dapson的复合物，在大肠杆菌中测试了高表达系统。在所有情况下，DHPS首先以包涵体形式表达，与伴侣蛋白共表达可有效降低其表达量。