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

Abstract

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, Δ(Δ G ^‡), for both reactions are large and favourable for LeuRS, but unfavourable for LeuAC. Single turnover assays with ³²Pα-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.

Graphical Abstract

Graphical Abstract

Thermodynamic cycle analysis of the free energy contributions of the HxGH and KMSKS catalytic signatures in Leucyl-tRNA synthetase and its 129-residue urzyme identify dramatic changes in the relative contributions of the KMSKS sequence and the HVGH*KMSKS two-way interaction. Structural analysis shows that a conserved network of non-polar packing in the anticodon binding domain functions as an “Enforcer” to impose coupled behavior in the full-length enzyme.