Post-translational Modification by β-Lysylation Is Required for Activity of Escherichia coli Elongation Factor P (EF-P)

Bacterial elongation factor P (EF-P) is the ortholog of archaeal and eukaryotic initiation factor 5A (eIF5A). EF-P shares sequence homology and crystal structure with eIF5A, but unlike eIF5A, EF-P does not undergo hypusine modification. Recently, two bacterial genes, yjeA and yjeK, encoding truncated homologs of class II lysyl-tRNA synthetase and of lysine-2,3-aminomutase, respectively, have been implicated in the modification of EF-P to convert a specific lysine to a hypothetical beta-lysyl-lysine. Here we present biochemical evidence for beta-lysyl-lysine modification in Escherichia coli EF-P and for its role in EF-P activity by characterizing native and recombinant EF-P proteins for their modification status and activity in vitro. Mass spectrometric analyses confirmed the lysyl modification at lysine 34 in native and recombinant EF-P proteins. The beta-lysyl-lysine isopeptide was identified in the exhaustive Pronase digests of native EF-P and recombinant EF-P isolated from E. coli coexpressing EF-P, YjeA, and YjeK but not in the digests of proteins derived from the vectors encoding EF-P alone or EF-P together with YjeA, indicating that both enzymes, YjeA and YjeK, are required for beta-lysylation of EF-P. Endogenous EF-P as well as the recombinant EF-P preparation containing beta-lysyl-EF-P stimulated N-formyl-methionyl-puromycin synthesis similar to 4-fold over the preparations containing unmodified EF-P and/or alpha-lysyl-EF-P. The mutant lacking the modification site lysine (K34A) was inactive. This is the first report of biochemical evidence for the beta-lysylation of EF-P in vivo and the requirement for this modification for the activity of EF-P.