磷酸乙醇胺转移酶MCR1结构与功能的研究

本实验克隆了来源于E.coli的MCR1基因酶催化结构域,以E.coli表达系统表达蛋白.采用亲和层析、阴离子交换层析、分子筛层析等纯化方法获得纯度高、均一性好的蛋白;采用座滴和悬滴法,获得酶催化区域的蛋白质晶体;收集X射线数据后,分子置换法解析出酶催化区域的结构,其分辨率达到1.63埃.反常散射信号检测到4个锌离子信号,结构分析锌离子与周围Thr285、His465、His466、His395位氨基酸联系紧密,且Thr285被磷酸化.Thr285、His465、His466和His395点突变为丙氨酸后,宿主菌粘菌素抗性显著降低,表明该区域与酶活密切相关.本实验解析出MCR1酶活性区域的结构,鉴定出酶活性中心,为寻找抗MCR1靶向药物提供可用信息.

Study on structure and function of phosphoethanolamine transferase MCR1

In this experiment, the MCR1 gene enzyme catalytic domain derived from E.coli was cloned and expressed in E.coli expression system. Purified proteins with high purity and good homogeneity were obtained by affinity chromatography, anion exchange chromatography and molecular sieve chromatography. The protein crystals of the enzyme catalyzed region were screened by the method of seating drop and the hanging drop. After collecting x-ray data, the structure of enzyme catalytic region was analyzed by molecular displacement method, and the resolution reached 1.63 angstrom. Four zinc ion signals were detected by the anomalous scattering signal. The structural analysis found that zinc ions was closely related to the surrounding amino acids of Thr285, His465, His466 and His395 , and Thr285 was phosphorylated. After the mutation of Thr285, His465, His466 and His395 to alanine, the resistance of host bacteria to colistin decreased significantly, indicating that the region was closely related to enzyme activity. In this experiment, the structure of MCR1 enzywas analyzed, me active region and the active center of enzyme was identified, which provided useful information for searching for anti MCR1 targeted drugs.