title: 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.
abstract: 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).
authors.affiliation: Department of Bacteriology, University of Wisconsin, Madison, Wisconsin 53706, USA.
authors.forename: Shaun R
authors.initials: SR
authors.lastname: Brinsmade
authors.forename: Jorge C
authors.initials: JC
authors.lastname: Escalante-Semerena
mesh_terms: D019943:Amino Acid Substitution; D007700:Kinetics; D009243:NAD / Q000737:chemistry* / Q000378:metabolism; D010709:Phosphate Acetyltransferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020836:Protein Structure, Quaternary / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D019779:Salmonella enterica / Q000201:enzymology* / Q000235:genetics
chemical_list: D009243:NAD; D010709:Phosphate Acetyltransferase