Multiple catalytic activities of Escherichia coli lysyl-tRNA synthetase (LysU) are dissected by site-directed mutagenesis

作者：Chen, Xiaolong; Boonyalai, Nonlawat; Lau, Catherine; Thipayang, Salinthip; Xu, Yuhong; Wright, Michael; Miller, Andrew D.^*

来源：FEBS Journal, 2013, 280(1): 102-114.

DOI：10.1111/febs.12053

• Ap(4)A
• Ap(3)A
• glycerol-3-phosphate
• kinase
• LysU
• multiple catalytic activities

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.

• 出版日期2013-1
• 单位浙江大学