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, 7 (10), 667-9

The tRNA Synthetase Paralog PoxA Modifies Elongation factor-P With (R)-β-lysine

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The tRNA Synthetase Paralog PoxA Modifies Elongation factor-P With (R)-β-lysine

Hervé Roy et al. Nat Chem Biol.

Abstract

The lysyl-tRNA synthetase paralog PoxA modifies elongation factor P (EF-P) with α-lysine at low efficiency. Cell-free extracts containing non-α-lysine substrates of PoxA modified EF-P with a change in mass consistent with addition of β-lysine, a substrate also predicted by genomic analyses. EF-P was efficiently functionally modified with (R)-β-lysine but not (S)-β-lysine or genetically encoded α-amino acids, indicating that PoxA has evolved an activity orthogonal to that of the canonical aminoacyl-tRNA synthetases.

Figures

Figure 1
Figure 1. Amino acid recognition by PoxA
(a) The 20 amino acids forming the lysyl-adenylate binding pocket of PoxA and LysRS are identical, apart from the two positions indicated in red. For clarity, only 14 residues are represented. Crystal structures are LysRS from Bacillus stearothermophilus and PoxA from Salmonella enterica Typhimurium. The Van der Waals surface of the lysyl-adenylate is represented. S. enterica Ala298 corresponds to E. coli Ala294. (b) PoxA-catalyzed ATP/PPi exchange in the presence of non-cognate amino acids (single letter code) and lysine analogs. The concentration of each amino acid was 20 mM. Errors correspond to the standard deviation from three independent experiments. (c) Metabolite fractionation by LC on silica gel. Fractions were separately tested for stimulation of ATP/PPi exchange catalyzed by PoxA (○) or LysRS (□).
Figure 2
Figure 2. Modification of EF-P with β-lysine
(a) Lysine structures (b) PoxA catalyzed aminoacylation of EF-P with (R)-β-lysine. Aminoacylation of EF-P or the inactive K34A variant was assayed in the presence or absence of 20µM (R)-β-lysine, (S)-β-lysine, or α-lysine. Modified EF-P containing an additional –NH3+ group was separated from the unmodified EF-P by 1D isoelectric focusing and detected by western blotting. (c) Complementation of Salmonella ΔyjeK strain with (R)-β-lysine. Growth in liquid LB with (+) or without (−) 8 µg/ml gentamicin for 24 hours at 37 °C. Growth was monitored in the presence of either 0.8 mM (R)-β-lysine, 0.8 mM α-lysine, or no lysine (−). Optical densities of replicate cultures are plotted. Growth rates were consistently slower on β-lysine than on α-lysine, suggesting a modest inhibitory effect at the concentrations used, the reason for which is presently unclear.

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