Domain acquisition by class I aminoacyl-tRNA synthetase urzymes coordinated the catalytic functions of HVGH and KMSKS motifs

Leucyl-tRNA synthetase (LeuRS) is a Class I aminoacyl-tRNA synthetase (aaRS) that synthesizes leucyl-tRNA(leu) for codon-directed protein synthesis. Two signature sequences, HxGH and KMSKS help stabilize transition-states for amino acid activation and tRNA aminoacylation by all Class I aaRS. Separate alanine mutants of each signature, together with the double mutant, behave in opposite ways in Pyrococcus horikoshii LeuRS and the 129-residue urzyme ancestral model generated from it (LeuAC). Free energy coupling terms, & UDelta;(& UDelta;G(& DDAG;)), for both reactions are large and favourable for LeuRS, but unfavourable for LeuAC. Single turnover assays with P-32 & alpha;-ATP show correspondingly different internal products. These results implicate domain motion in catalysis by full-length LeuRS. The distributed thermodynamic cycle of mutational changes authenticates LeuAC urzyme catalysis far more convincingly than do single point mutations. Most importantly, the evolutionary gain of function induced by acquiring the anticodon-binding (ABD) and multiple insertion modules in the catalytic domain appears to be to coordinate the catalytic function of the HxGH and KMSKS signature sequences. The implication that backbone elements of secondary structures achieve a major portion of the overall transition-state stabilization by LeuAC is also consistent with coevolution of the genetic code and metabolic pathways necessary to produce histidine and lysine sidechains.

Automated summary

LeuRS is a Class I aaRS that synthesizes leucyl-tRNA for protein synthesis. Signature sequences HxGH and KMSKS stabilize transition-states in amino acid activation and tRNA aminoacylation. Mutations in these signatures have opposite effects in LeuRS and its ancestral model LeuAC, suggesting domain motion in catalysis by LeuRS. The gain of function in LeuAC is attributed to the coordination of HxGH and KMSKS by the anticodon-binding and multiple insertion modules.