耐辐射奇球菌Mur蛋白保守功能位点的研究  

Identification of Catalytic Residues of Mur Protein From Deinococcus radiodurans

作　　者：Amir Miraj Ul Hussain Shah 杨粟[1] 张慧[1] 华跃进[1] 

机构地区：[1]浙江大学原子核农业科学研究所/农业部和浙江省核农学重点实验室,浙江杭州310029

出　　处：《核农学报》2016年第3期451-459,共9页Journal of Nuclear Agricultural Sciences

基　　金：公益性行业(农业)科研专项(201103007);国家基金面上项目(31370102);浙江省重点创新团队(2010R50033)

摘　　要：为进一步明确耐辐射奇球菌中锰调蛋白Mur的具体结构与功能,采用序列比对分析和定点突变技术构建了耐辐射奇球菌的锰调蛋白Mur(DR0865)的3个保守氨基酸位点突变株,并分析3个点突变菌株的金属离子敏感性、Mn2+结合对功能的影响、外界胁迫耐受性及点突变蛋白的DNA体外结合活性。结果表明,3个定点突变H77A、H78A和H79A都是Mur蛋白重要的活性位点,对Mur调控细胞吸收外源性锰离子的功能起到决定性的作用,同时影响了Mur的Mn2+结合活性。凝胶迁移率实验表明H78与H79位点对Mur的DNA结合活性至关重要。本研究为探究耐辐射奇球菌的极端抗性和锰调蛋白的具体作用机制提供了重要的理论依据。For further understanding the precise structural features and functions of manganese uptake regulator（ Mur） in Deinococcus radiodurans,sequence alignment and site-directed mutagenesis technology were applied and mutations in three conserved amino acid residues of Mur were constructed. For exploration of the contribution of Mur in stress resistance of D. radiodurans,metal ions sensitivity,affection of manganese ion binding and stress resistance activity of each site mutation strains were analysed and DNA binding activity of each mutant proteins were also examined. The results indicated that the three residues,including H77A、H78A and H79 A,were all critical for Mur protein activity and played important parts in the manganese uptake regulation of Mur,meanwhile,these three residues acted significant roles in Mn2 ＋binding activity of Mur. Electrophoretic mobility shift assay indicated that amino acid residue H78 and H79 were quite important for the DNA binding activity of Mur. The above experiments provide important theoretical references for understanding the extreme stress resistance of D. radiodurans and specific mechanism of Mur.

关 键 词：耐辐射奇球菌 锰调蛋白 金属离子敏感性 DNA结合活性 

分 类 号：Q936[生物学—微生物学]