title: Post-translational modification by β-lysylation is required for activity of Escherichia coli elongation factor P (EF-P).
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.
authors: Oral and Pharyngeal Cancer Branch, NIDCR, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors: Jong-Hwan
authors: JH
authors: Park
authors: Hans E
authors: HE
authors: Johansson
authors: Hiroyuki
authors: H
authors: Aoki
authors: Bill X
authors: BX
authors: Huang
authors: Hee-Yong
authors: HY
authors: Kim
authors: M Clelia
authors: MC
authors: Ganoza
authors: Myung Hee
authors: MH
authors: Park
mesh_terms: D003851:Deoxyribonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008239:Lysine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013058:Mass Spectrometry; D010445:Peptide Elongation Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D010445:Peptide Elongation Factors; D011994:Recombinant Proteins; C020746:factor EF-P; D003851:Deoxyribonucleases; C527632:yjeA protein, E coli; D008239:Lysine