Cys-113 and Cys-422 form a high affinity metalloid binding site in the ArsA ATPase
Cys-113 and Cys-422 form a high affinity metalloid binding site in the ArsA ATPase
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DOI：
10.1074/jbc.m600125200
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发表时间：
2006-04-14
影响因子：
4.8
通讯作者：
Rosen, BP
中科院分区：
文献类型：
--
作者：
Ruan, X;Bhattacharjee, H;Rosen, BP
大肠杆菌质粒R773的arsRDABC操纵子编码三价类金属As（III）和Sb（III）的ArsAB挤出泵。ArsA，催化亚基有两个同源的半部分，A1和A2。每一半具有将核苷酸结合结构域物理连接到类金属结合结构域的共有信号转导结构域。ArsA与类金属结合和ArsB转运之间的关系尚不清楚。在这项研究中，直接类金属结合ArsA进行了检查。结果表明，ArsA仅在Mg ~（2+）-核苷酸存在下以高亲和力结合单个Sb（III）。Cys-113和Cys-422的密码子突变消除了Sb（III）与纯化的ArsA的结合。C113 A/C422 A ArsA具有与野生型相似的基础ATP酶活性，但缺乏类金属刺激活性。在完整细胞中测定类金属的积累，其中减少的摄取是由ArsAB泵的主动挤出引起的。表达arsA（C113 A/C422 A）B基因的细胞在仅表达单独的arsB的细胞和表达野生型arsAB基因的细胞之间具有中等水平的类金属抗性和蓄积。结果表明，而类金属刺激ArsA活性增强的泵的能力，以减少细胞内的类金属浓度，高亲和力结合的类金属ArsA是不是强制性的运输或电阻。然而，在亚毒性浓度的亚砷酸盐中生长的携带arsAB或arsA（C113 A/C422 A）B的细胞混合群体中，携带野生型arsAB的细胞在不到1周的时间内用突变型arsA（C113 A/C422 A）B取代细胞，表明类金属结合位点赋予进化优势。
The arsRDABC operon of Escherichia coli plasmid R773 encodes the ArsAB extrusion pump for the trivalent metalloids As(III) and Sb(III). ArsA, the catalytic subunit has two homologous halves, A1 and A2. Each half has a consensus signal transduction domain that physically connects the nucleotide-binding domain to the metalloid-binding domain. The relation between metalloid binding by ArsA and transport through ArsB is unclear. In this study, direct metalloid binding to ArsA was examined. The results show that ArsA binds a single Sb( III) with high affinity only in the presence of Mg2+-nucleotide. Mutation of the codons for Cys-113 and Cys-422 eliminated Sb( III) binding to purified ArsA. C113A/ C422A ArsA has basal ATPase activity similar to that of the wild type but lacks metalloid-stimulated activity. Accumulation of metalloid was assayed in intact cells, where reduced uptake results from active extrusion by the ArsAB pump. Cells expressing the arsA(C113A/C422A)B genes had an intermediate level of metalloid resistance and accumulation between those expressing only arsB alone and those expressing wild type arsAB genes. The results indicate that, whereas metalloid stimulation of ArsA activity enhances the ability of the pump to reduce the intracellular concentration of metalloid, high affinity binding of metalloid by ArsA is not obligatory for transport or resistance. Yet, in mixed populations of cells bearing either arsAB or arsA(C113A/C422A)B growing in subtoxic concentrations of arsenite, cells bearing wild type arsAB replaced cells with mutant arsA(C113A/C422A)B in less than 1 week, showing that the metalloid binding site confers an evolutionary advantage.