PMID:22128152

Citation	Park, JH, Johansson, HE, Aoki, H, Huang, BX, Kim, HY, Ganoza, MC and Park, MH (2012) Post-translational modification by β-lysylation is required for activity of Escherichia coli elongation factor P (EF-P). J. Biol. Chem. 287:2579-90
Abstract	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 β-lysyl-lysine. Here we present biochemical evidence for β-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 β-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 β-lysylation of EF-P. Endogenous EF-P as well as the recombinant EF-P preparation containing β-lysyl-EF-P stimulated N-formyl-methionyl-puromycin synthesis ∼4-fold over the preparations containing unmodified EF-P and/or α-lysyl-EF-P. The mutant lacking the modification site lysine (K34A) was inactive. This is the first report of biochemical evidence for the β-lysylation of EF-P in vivo and the requirement for this modification for the activity of EF-P.
Links	PubMed PMC3268417 Online version:10.1074/jbc.M111.309633
Keywords	Deoxyribonucleases/chemistry; Deoxyribonucleases/genetics; Deoxyribonucleases/metabolism; Escherichia coli/genetics; Escherichia coli/metabolism; Escherichia coli Proteins/chemistry; Escherichia coli Proteins/genetics; Escherichia coli Proteins/metabolism; Lysine/chemistry; Lysine/genetics; Lysine/metabolism; Mass Spectrometry; Peptide Elongation Factors/chemistry; Peptide Elongation Factors/genetics; Peptide Elongation Factors/metabolism; Protein Processing, Post-Translational/physiology; Recombinant Proteins/chemistry; Recombinant Proteins/genetics; Recombinant Proteins/metabolism
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
LACF3:EFP		GO:0003746: translation elongation factor activity	ECO:0000314:		F		Mass spectrometric analyses confirmed the lysyl modification at lysine 34 in native and recombinant EF-P proteins.	complete

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