In vivo and in vitro analyses of single-amino acid variants of the Salmonella enterica phosphotransacetylase enzyme provide insights into the function of its N-terminal domain

J Biol Chem. 2007 Apr 27;282(17):12629-40. doi: 10.1074/jbc.M611439200. Epub 2007 Mar 5.


The function of the N-terminal domain ( approximately 350 residues) of the Pta (phosphotransacetylase) enzyme of Salmonella enterica is unclear. Results from in vivo genetic and in vitro studies suggest that the N-terminal domain of Pta is a sensor for NADH and pyruvate. We isolated 10 single-amino acid variants of Pta that, unlike the wild-type protein, supported growth of a strain of S. enterica devoid of Acs (acetyl-CoA synthetase; AMP-forming) activity on 10 mm acetate. All mutations were mapped within the N-terminal domain of the protein. Kinetic analyses of the wild type and three variant Pta proteins showed that two of the variant proteins were faster enzymes (k(cat) 2.5-3-fold > k(cat) Pta(WT). Results from sedimentation equilibrium experiments are consistent with Pta(WT) being a trimer. Pta variants formed more hexamer than the Pta(WT) protein. NADH inhibited Pta(WT) activity by inducing a conformational change detectable by limited trypsin proteolysis; NADH did not inhibit variant protein Pta(R252H). Pyruvate stimulated Pta(WT) activity, and its effect was potentiated in the variants, being most pronounced on Pta(R252H).

Publication types

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

MeSH terms

  • Amino Acid Substitution*
  • Kinetics
  • NAD / chemistry*
  • NAD / metabolism
  • Phosphate Acetyltransferase / chemistry*
  • Phosphate Acetyltransferase / genetics
  • Phosphate Acetyltransferase / metabolism
  • Protein Structure, Quaternary / genetics
  • Protein Structure, Tertiary / genetics
  • Salmonella enterica / enzymology*
  • Salmonella enterica / genetics


  • NAD
  • Phosphate Acetyltransferase