title: Multiple catalytic activities of Escherichia coli lysyl-tRNA synthetase (LysU) are dissected by site-directed mutagenesis.
abstract: The heat-inducible lysyl-tRNA synthetase from Escherichia coli (LysU; EC6/1/1/6.html) converts ATP to diadenosine tri- and tetraphosphates (Ap(3)A/Ap(4)A) in the presence of L-lysine/Mg(2+)/Zn(2+). 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 Zn(2+)/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 Ap(4)A/Ap(3)A under normal conditions (additional Zn(2+)/L-lysine/Mg(2+)). By contrast, wild-type LysU has a weaker glycerol kinase-like capability in the absence of Zn(2+) and is dominated by Ap(4)A/Ap(3)A synthesis in its presence. Kinetic and isothermal titration calorimetry results suggest that E264 is a crucial residue for Zn(2+) promotion of Ap(4)A/Ap(3)A synthesis. This is consistent with the hypothesis that E264 provides an anchor point for a Zn(2+) 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.
authors.affiliation: Zhejiang California International NanoSystems Institute, Zhejiang University, Hangzhou, China.
authors.forename: Xiaolong
authors.initials: X
authors.lastname: Chen
authors.forename: Nonlawat
authors.initials: N
authors.lastname: Boonyalai
authors.forename: Catherine
authors.initials: C
authors.lastname: Lau
authors.forename: Salinthip
authors.initials: S
authors.lastname: Thipayang
authors.forename: Yuhong
authors.initials: Y
authors.lastname: Xu
authors.forename: Michael
authors.initials: M
authors.lastname: Wright
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Miller
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry; D019943:Amino Acid Substitution; D020134:Catalytic Domain; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics; D005991:Glycerol Kinase / Q000737:chemistry; D006860:Hydrogen Bonding; D006868:Hydrolysis; D007700:Kinetics; D008250:Lysine-tRNA Ligase / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D015032:Zinc / Q000737:chemistry
chemical_list: D029968:Escherichia coli Proteins; D000255:Adenosine Triphosphate; D005991:Glycerol Kinase; D008250:Lysine-tRNA Ligase; D015032:Zinc