A bacterial ortholog of class II lysyl-tRNA synthetase activates lysine

Abstract

Aminoacyl-tRNA synthetases produce aminoacyl-tRNAs, essential substrates for accurate protein synthesis. Beyond their central role in translation some of these enzymes or their orthologs are recruited for alternative functions, not always related to their primary cellular role. We investigate here the enzymatic properties of GenX (also called PoxA and YjeA), an ortholog of bacterial class II lysyl-tRNA synthetase. GenX is present in most Gram-negative bacteria and is homologous to the catalytic core of lysyl-tRNA synthetase, but it lacks the amino terminal anticodon binding domain of the latter enzyme. We show that, in agreement with its well-conserved lysine binding site, GenX can activate in vitro l-lysine and lysine analogs, but does not acylate tRNA(Lys) or other cellular RNAs.

Fig. 1

Sequence alignment of GenX from various bacterial species (Styphi, Ecoli, Ypest, Hinf, Smeli, Lint, refers to the following organisms: Salmonella typhimurium, E. coli, Yersinia pestis, Haemophilus influenzae, Sinorhizobium meliloti, Leptospira interrogans). EcoliS refers to the E. coli LysRS (encoded by lysS). Class II AARS defining motifs I, II and III are indicated. Residues involved in ATP and lysine binding are indicated by an open circle and a star respectively.

Fig. 2

GenX activates L-lysine in vitro (A) in a magnesium concentration manner (B). (A): amino acid activation reaction catalyzed by GenX was performed in the presence of 10 mM lysine and either 2 mM ATP (■) or a mix of CTP/GTP/UTP (2 mM each, □) and in the presence of a 19 amino acids mix (1 mM each, deprived of lysine) and either 2 mM ATP (▲) or a mix of CTP/GTP/UTP (2 mM each, △). Lysine activation of E. coli LysRS in the presence of ATP was used as control (○). (B): Lysine (10 mM) activation by GenX in the presence of 2 mM ATP and 10 mM (■), 5 mM (▲), 1 mM (◆), 0.5 mM (●) and 0 mM (○) magnesium chloride.

Fig. 3

Activation of L-lysine and L-lysine analogs. A: Bar graph representing the activation of S-2 aminoethyl L-cysteine (1), L-Lysine hydroxamide (2), Lysinamide (3), 1–5 diaminopentane (4), ornithine (5), L-lysine methyl ester (6), D-lysine (7), 5-hydroxy L-lysine (8), alpha-aminobutyric acid (9), gamma-aminobutyric acid (10), diaminopimelic acid (11) and L-Lysine (K), all present at 25 mM concentration by E. coli LysRS (black bars) and GenX (grey bars). B: molecular structure of L-lysine and 5-hydroxy L-lysine (drawn with chemdraw-3D).

Fig. 4

Comparison of GenX (PDB code 3g1z, shown in tan) with the solved LysRS structure (A) (PDB code: 1bbu shown in blue) shows three regions that are structurally distinct. These include the complete absence of the N-terminal OB-fold tRNA binding domain, and two loops in GenX (residues 49–53 and 272–279, highlighted in yellow) that are adjacent to the active site. The lysyl-adenylate binding pockets (B) are nearly identical in GenX and LysRS with the only exception being residues located on the structurally distinct loop containing GenX residues 49–53 (yellow).