Multiple catalytic activities of Escherichia coli lysyl-tRNA synthetase (LysU) are dissected by site-directed mutagenesis
Multiple catalytic activities of Escherichia coli lysyl-tRNA synthetase (LysU) are dissected by site-directed mutagenesis
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DOI：
10.1111/febs.12053
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发表时间：
2013-01-01
期刊：
影响因子：
5.4
通讯作者：
Miller, Andrew D.
中科院分区：
文献类型：
--
作者：
Chen, Xiaolong;Boonyalai, Nonlawat;Miller, Andrew D.
来自大肠杆菌的热诱导赖氨酰-tRNA合成酶（LysU; EC6/1/1/6.html）在l-赖氨酸/Mg 2 +/Zn 2+存在下将ATP转化为二腺苷三磷酸和二腺苷四磷酸（Ap 3A/Ap 4A）。为了更详细地理解LysU，制备了26个突变体：6个E264，4个R269和16个通过丙氨酸扫描内壳/基序2环的突变体。在甘油存在且不存在外源添加的Zn 2 +/l-赖氨酸的情况下，我们意外地发现E264 K催化甘油-3-磷酸的产生，其由ATP向ADP的周转提供动力。E264 Q和E264 N也能够具有这种活性，但在正常条件下（额外的Zn 2 +/l-赖氨酸/Mg 2+），所有三种都显示几乎不形成Ap 4A/Ap 3A。相比之下，野生型LysU具有较弱的甘油激酶样的能力，在不存在的Zn 2+和主导的Ap 4A/Ap 3A合成在其存在下。动力学和等温滴定量热结果表明E264是Zn 2+促进Ap 4A/Ap 3A合成的关键残基。这与E264为与活性位点络合的Zn 2+离子提供锚点的假设一致，同时与酶结合的赖氨酰-腺苷酸中间体和二级底物ATP/ADP配位。甘油激酶样活性在破坏这种特定的协调时被发现。
The heat-inducible lysyl-tRNA synthetase from Escherichia coli (LysU; EC6/1/1/6.html) converts ATP to diadenosine tri- and tetraphosphates (Ap3A/Ap4A) in the presence of l-lysine/Mg2+/Zn2+. To understand LysU in more detail, 26 mutants were prepared: six of E264, four of R269 and sixteen mutants by alanine-scanning of the inner shell/motif 2 loop. In the presence of glycerol and absence of exogenously added Zn2+/l-lysine, we unexpectedly found that E264K catalysed the production of glycerol-3-phosphate, powered by ATP turnover to ADP. E264Q and E264N are also capable of this activity, but all three show little formation of Ap4A/Ap3A under normal conditions (additional Zn2+/l-lysine/Mg2+). By contrast, wild-type LysU has a weaker glycerol kinase-like capability in the absence of Zn2+ and is dominated by Ap4A/Ap3A synthesis in its presence. Kinetic and isothermal titration calorimetry results suggest that E264 is a crucial residue for Zn2+ promotion of Ap4A/Ap3A synthesis. This is consistent with the hypothesis that E264 provides an anchor point for a Zn2+ ion complexed to the active site, with simultaneous coordination to the enzyme bound lysyl-adenylate intermediate and secondary substrate ATP/ADP. The glycerol kinase-like activity is uncovered on disruption of this specific coordination.