ABSTRACT
Elongation factor P is modified with (R)-ß-lysine by the lysyl-tRNA synthetase (LysRS) paralog PoxA. PoxA specificity is orthogonal to LysRS, despite their high similarity. To investigate α- and ß-lysine recognition by LysRS and PoxA, amino acid replacements were made in the LysRS active site guided by the PoxA structure. A233S LysRS behaved as wild type with α-lysine, while the G469A and A233S/G469A variants decreased stable α-lysyl-adenylate formation. A233S LysRS recognized ß-lysine better than wildtype, suggesting a role for this residue in discriminating α- and ß-amino acids. Both enantiomers of ß-lysine were substrates for tRNA aminoacylation by LysRS, which, together with the relaxed specificity of the A233S variant, suggest a possible means to develop systems for in vivo co-translational insertion of ß-amino acids.
Subject(s)
Bacillus cereus/enzymology , Bacterial Proteins/chemistry , Lysine-tRNA Ligase/chemistry , Lysine/chemistry , Peptide Elongation Factors/chemistry , Amino Acid Substitution , Bacillus cereus/genetics , Bacterial Proteins/genetics , Lysine/genetics , Lysine-tRNA Ligase/genetics , Mutation, Missense , Peptide Elongation Factors/geneticsABSTRACT
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 ß-lysine, a substrate also predicted by genomic analyses. EF-P was efficiently functionally modified with (R)-ß-lysine but not (S)-ß-lysine or genetically encoded α-amino acids, indicating that PoxA has evolved an activity orthogonal to that of the canonical aminoacyl-tRNA synthetases.
