X-ray Crystallographic Study of Antibiotics resistant Proteins

本研究包括4个项目：1)β-内酰胺酶：1-a)SHV-1：美罗培南复合体：解决了SHV-1与美罗培南共价络合的高分辨(1.05A)X射线结构。高分辨率数据提供了一些重要氢原子的电子密度。其中一个有趣的结果是质子化的Glu166和相关的氢键网络围绕脱酰基水。1-b)gc1β-内酰胺酶：氨苄西林复合体：阴沟肠杆菌gc1与良好底物氨苄西林的C类β-内酰胺酶复合体被蛋白质修饰捕获，并通过X射线结晶学进行解析。2)β/APH双功能酶：AAc/APH双功能酶来源于金黄色葡萄球菌，在氨基糖苷类抗生素中加入乙酰基或/和磷酸基团进行灭活。将AAc/APH双功能酶基因克隆到pCOLD载体上进行高效表达。从1株L培养液中可获得3~5 mg蛋白质。采用卡那霉素亲和层析和凝胶过滤进行纯化。进行了初步的结晶筛选。3)大环内酯磷酸转移酶：利用聚合酶链式反应将大环内酯磷酸转移酶基因(MphA)克隆到pCoD载体上进行高效表达。目前，这种酶的数量不足以形成晶体。另一方面，调节蛋白MPHR可以高效表达为GST融合蛋白。4)Jeprae分枝杆菌二氢翼酸合成酶：二氢翼酸合成酶(DHPS)是重要的抗菌药物DAPSON的主要靶标。麻风药。为了解决DHPS-DAPSON的复合体，我们在大肠杆菌中进行了高效表达系统的测试。在所有病例中，DHPs都是以包涵体形式表达的，与伴侣蛋白共表达可以有效减少DHPs的表达。

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.