Enzymatic characterization of a class II lysyl-tRNA synthetase, LysS, from Myxococcus xanthus

• LysS is monomeric and requires Mn2+ for the synthesis of Ap4A.
• LysS has Ap4A, Ap3A and ADP synthase activity without the requirement for Zn2+.
• LysS has symmetrical Ap4A hydrolase activity.
• LysS has different properties from class II synthetases previously reported.

Lysyl-tRNA synthetases efficiently produce diadenosine tetraphosphate (Ap4A) from lysyl-AMP with ATP in the absence of tRNA. We characterized recombinant class II lysyl-tRNA synthetase (LysS) from Myxococcus xanthus and found that it is monomeric and requires Mn2+ for the synthesis of Ap4A. Surprisingly, Zn2+ inhibited enzyme activity in the presence of Mn2+. When incubated with ATP, Mn2+, lysine, and inorganic pyrophosphatase, LysS first produced Ap4A and ADP, then converted Ap4A to diadenosine triphosphate (Ap3A), and finally converted Ap3A to ADP, the end product of the reaction. Recombinant LysS retained Ap4A synthase activity without lysine addition. Additionally, when incubated with Ap4A (minus pyrophosphatase), LysS converted Ap4A mainly ATP and AMP, or ADP in the presence or absence of lysine, respectively. These results demonstrate that M. xanthus LysS has different enzymatic properties from class II lysyl-tRNA synthetases previously reported.