A paralog of lysyl-tRNA synthetase aminoacylates a conserved lysine residue in translation elongation factor P

Abstract

Aminoacyl-tRNA synthetase (aaRS) paralogs with unknown functions exist in various species. We now report novel 'protein lysylation' by an Escherichia coli lysyl-tRNA synthetase paralog, GenX/PoxA/YjeA. X-ray crystallographic analysis shows that the structure of the GenX protein resembles that of a class II aaRS. Further in vitro studies reveal that it specifically aminoacylates EF-P with lysine. The shape of the protein substrate mimics that of the L-shaped tRNA, and its lysylation site corresponds to the tRNA 3' end. Thus, we show how the aaRS architecture can be adapted to achieve aminoacylation of a specific protein. Moreover, in vivo analyses reveal that the translation elongation factor P (EF-P) lysylation by GenX is enhanced by YjeK (lysine 2,3-aminomutase paralog), which is encoded next to the EF-P gene, and might convert alpha-lysyl-EF-P to beta-lysyl-EF-P. In vivo analyses indicate that the EF-P modification by GenX and YjeK is essential for cell survival.

Loss of Elongation Factor P Disrupts Bacterial Outer Membrane Integrity

“…Notably, the modification of EF-P occurs at a lysyl residue (lysine 34) that corresponds to the same position as the lysyl residue that is converted to hypusine in aIF5A and eIF5A. Two other laboratories have independently corroborated aspects of these findings (6,60). YjeK belongs to the family of 2,3-lysine aminomutases, ironsulfur cluster-containing enzymes that catalyze the interconversion of L-lysine and 3,6-diaminohexanoic acid (also known as ␤-lysine) (8).…”

“…While PoxA bears close homology to the catalytic domain of the class II lysyl-tRNA synthetase (LysRS) family of enzymes that catalyze the addition of lysine to its cognate tRNA Lys , a number of studies have failed to show that PoxA can aminoacylate a tRNA (1,36,37). PoxA instead catalyzes the ligation of (R)-␤-lysine to the side chain of the conserved lysyl residue in EF-P to yield an unusual lysyl-␤-lysine moiety (42,48,60). Salmonella poxA and yjeK mutants display nearly identical phenotypes, including increased resistance to S-nitrosoglutathione (GSNO) and hypersusceptibility to a large number of unrelated antimicrobial compounds (7,42,55).…”

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

“…However, two recent studies also indicated that EF-P could act during elongation by facilitating the synthesis of proteins containing stretches of consecutive proline residues (5,6). EF-P is similar in size and shape to a tRNA and can be post-translationally modified by a lysyl-tRNA synthetase paralog, PoxA (7,8). In a reaction analogous to tRNA aminoacylation, PoxA activates (R)-␤-lysine and subsequently transfers it to a conserved residue (Lys-34) in EF-P (9).…”

Distinct XPPX sequence motifs induce ribosome stalling, which is rescued by the translation elongation factor EF-P

“…In bacteria, the translational arrest is relieved by the translation elongation factor P (EF-P), which binds to the stalled ribosomes and stimulates peptide bond formation (5,6). In Escherichia coli, EF-P is posttranslationally modified by YjeA, YjeK, and YfcM (EpmA, EpmB, and EpmC) (8)(9)(10) and the resulting lysinylation modification has been shown to be critical for the rescue activity of EF-P in vivo and in vitro (5,6). EF-P homologs exist in all archaea and eukaryotes, termed aIF-5A and eIF-5A, respectively (11).…”