Post-translational modification by β-lysylation is required for activity of Escherichia coli elongation factor P (EF-P)

J Biol Chem. 2012 Jan 20;287(4):2579-90. doi: 10.1074/jbc.M111.309633. Epub 2011 Nov 29.

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.

Publication types

  • Research Support, N.I.H., Intramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • 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

Substances

  • Escherichia coli Proteins
  • Peptide Elongation Factors
  • Recombinant Proteins
  • factor EF-P
  • Deoxyribonucleases
  • yjeA protein, E coli
  • Lysine