Divergence in Noncognate Amino Acid Recognition between Class I and Class II Lysyl-tRNA Synthetases

Abstract

Lysine insertion during coded protein synthesis requires lysyl-tRNA Lys , which is synthesized by lysyl-tRNA synthetase (LysRS). Two unrelated forms of LysRS are known: LysRS2, which is found in eukaryotes, most bacteria, and a few archaea, and LysRS1, which is found in most archaea and a few bacteria. To compare amino acid recognition between the two forms of LysRS, the effects of L-lysine analogues on aminoacylation were investigated. Both enzymes showed stereospecificity toward the L-enantiomer of lysine and discriminated against noncognate amino acids with different R-groups (arginine, ornithine). Lysine analogues containing substitutions at other positions were generally most effective as inhibitors of LysRS2. For example, the K i values for aminoacylation of S-(2-aminoethyl)-L-cysteine and L-lysinamide were over 180-fold lower with LysRS2 than with LysRS1. Of the other analogues tested, only ␥-aminobutyric acid showed a significantly higher K i for LysRS2 than LysRS1. These data indicate that the lysinebinding site is more open in LysRS2 than in LysRS1, in agreement with previous structural studies. The physiological significance of divergent amino acid recognition was reflected by the in vivo resistance to growth inhibition imparted by LysRS1 against S-(2-aminoethyl)-L-cysteine and LysRS2 against ␥-aminobutyric acid. These differences in resistance to naturally occurring noncognate amino acids suggest the distribution of LysRS1 and LysRS2 contributes to quality control during protein synthesis. In addition, the specific inhibition of LysRS1 indicates it is a potential drug target.

(R)-β-Lysine-modified Elongation Factor P Functions in Translation Elongation

“…Intein-tagged proteins were purified on a chitin affinity column (New England Biolabs) and stored in 25 mM Tris-HCl, pH 8.0, 150 mM NaCl, 4 mM 2-mercaptoethanol, 20% glycerol. E. coli BL21(DE3)/pQE31-Ec-lysS and E. coli BL21(DE3)/pQE31-Ec-pheS-pheT were used to prepare lysyltRNA synthetase and phenylalanyl-tRNA synthetase, respectively (16,17). Native tRNAs were purchased from Chemical Block (Moscow, Russia).…”

Discrimination of Cognate and Noncognate Substrates at the Active Site of Class I Lysyl-tRNA Synthetase

“…Previous studies indicated that LysRS1 and LysRS2 are markedly different in their abilities to discriminate lysine analogues (18). For example, the competitive inhibitor AEC has a K i for the aminoacylation reaction that is several hundred-fold lower for LysRS2 than LysRS1.…”

“…Aminoacylation was performed at 37 °C in 100 mM Hepes (pH 7.2), 25 mM KCl, 10 mM Sample disks were washed and radioactivity counted as described previously (18). The steady-state kinetic parameters given represent the average of at least two independent experiments where values deviated by no more than 10% between individual determinations.…”

“…The B. burgdorferi lysK encoded LysRS1 and mutants cloned into the pET15b vector were expressed in E. coli BL21(DE3) cells as described previously (18). Cells were harvested by centrifugation and washed in column buffer (20 mM Tris-HCl, pH 8, 300 mM NaCl, 30 mM Imidazole, 1 mM MgCl 2 , 10% glycerol).…”

“…Point mutations were confirmed by sequencing each gene completely. In vitro transcribed B. burgdorferi tRNA Lys (UUU anticodon) was prepared and purified as described before (18).…”