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 catalyzes synthesis of leucyl-tRNAleu for codon-directed protein synthesis on the ribosome. Class I aaRS, which were key to the evolution of genetic coding, contain two discrete signature sequences, HIGH and KMSKS, that participate in transition-state stabilization by the entire eleven-enzyme Class I aaRS superfamily. Combinatorial mutagenesis and thermodynamic cycle analyses of these catalytic signatures in full-length Pyrococcus horikoshii LeuRS and the 129-residue urzyme ancestral model generated from it (LeuAC) provide quantitative insight into the evolutionary gain of function induced by acquisition of the anticodon-binding (ABD) and multiple insertion modules in the catalytic domain. The free energy coupling terms, {Delta}({Delta}G{ddagger}), are small and unfavorable for LeuAC, but large and favorable for LeuRS. Thus, the ABD and other insertion modules induce strong cooperativity between the two signature sequences, which are uncoupled in LeuAC. These results further substantiate the authenticity of LeuAC urzyme catalysis and implicate domain motion in catalysis by the full-length LeuRS. Most importantly, 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. Bullet PointsO_LIThe LeuRS HVGH and KMSKS signature motifs are energetically coupled by -1.6 kcal/mole. C_LIO_LIThe same motifs are anti-coupled by +0.8 kcal/mole in the 129 residue urzyme, LeuAC. C_LIO_LIAncestral Class I aaRS did not require either histidine or lysine for catalysis. C_LI