A Leucyl-tRNA Synthetase Urzyme: Authenticity of tRNA Synthetase Catalytic Activities and Promiscuous Phosphorylation of Leucyl-5′ AMP

Aminoacyl-tRNA synthetase (aaRS)/tRNA cognate pairs translate the genetic code by synthesizing specific aminoacyl-tRNAs that are assembled on messenger RNA by the ribosome. Deconstruction of the two distinct aaRS superfamilies (Classes) has provided conceptual and experimental models for their early evolution. Urzymes, containing similar to 120-130 amino acids excerpted from regions where genetic coding sequence complementarities have been identified, are key experimental models motivated by the proposal of a single bidirectional ancestral gene. Previous reports that Class I and Class II urzymes accelerate both amino acid activation and tRNA aminoacylation have not been extended to other synthetases. We describe a third urzyme (LeuAC) prepared from the Class IA Pyrococcus horikoshii leucyl-tRNA synthetase. We adduce multiple lines of evidence for the authenticity of its catalysis of both canonical reactions, amino acid activation and tRNA(Leu )aminoacylation. Mutation of the three active-site lysine residues to alanine causes significant, but modest reduction in both amino acid activation and aminoacylation. LeuAC also catalyzes production of ADP, a non-canonical enzymatic function that has been overlooked since it first was described for several full-length aaRS in the 1970s. Structural data suggest that the LeuAC active site accommodates two ATP conformations that are prominent in water but rarely seen bound to proteins, accounting for successive, in situ phosphorylation of the bound leucyl-5'AMP phosphate, accounting for ADP production. This unusual ATP consumption regenerates the transition state for amino acid activation and suggests, in turn, that in the absence of the editing and anticodon-binding domains, LeuAC releases leu-5'AMP unusually slowly, relative to the two phosphorylation reactions.

Automated summary

Aminoacyl-tRNA synthetases (aaRS)/tRNA pairs play a crucial role in translating the genetic code. The study of urzymes, which are experimental models for the early evolution of aaRS, has provided insights into their catalytic activities. In this study, a new urzyme (LeuAC) was described and shown to catalyze both amino acid activation and tRNA aminoacylation. Additionally, LeuAC was found to have a non-canonical enzymatic function of producing ADP.