Cloning and Characterization of Leucyl‐tRNA Synthetase from Pseudomonas aeruginosa

Abstract

IntroductionPseudomonas aeruginosa is an opportunistic pathogen and a common cause of nosocomial infections. Aminoacyl‐tRNA synthetases (aaRSs) are a class of enzymes that catalyze the covalent attachment of amino acids to their cognate tRNAs during protein biosynthesis. We describe here the cloning and enzymatic characterization of the class I leucyl tRNA synthetase (LeuRS) from P. aeruginosa.ResultsLeuRS from P. aeruginosa was cloned and expressed in E. coli and purified to greater than 98% homogeneity. Sequence analysis shows that this protein contains the characteristic motifs of class I aminoacyl‐tRNA synthetases. The ability of LeuRS to function in aminoacylation of its cognate tRNA was determined in tRNA aminoacylation assays. The kinetic parameters for the interaction of P. aeruginosa LeuRS with its three substrates (tRNA, ATP, leucine) were determined. Initial velocities were determined for charging of tRNA using tRNA concentrations between 6.5 and 10 μM tRNALeu. The KM and Vmax for the interaction of LeuRS with the substrate tRNALeu was determined to be 7.14 μM and 0.36 μM min−1, respectively. The ATP:PPi exchange reaction was used to monitor interaction with ATP and leucine.ConclusionLeuRS identified in P. aeruginosa was cloned, expressed and purified and shown to be functional in assays suggesting that it is functional in protein synthesis. Research was partially supported by NIH grant 1SC3GM098173–01A1.