Enzyme IIIlac of the staphylococcal phosphoenolpyruvate-dependent phosphotransferase system: site-specific mutagenesis of histidine residues, biochemical characterization and 1H-NMR studies.
The lactose-specific phosphocarrier protein enzyme III of the bacterial phosphoenol-pyruvate-dependent phosphotransferase system of Staphylococcus aureus was modified by site-specific mutagenesis on the corresponding lacF gene in order to replace the histidine residues 78 and 82 of the amino acid sequence with a serine residue. Wild-type and both mutant genes were overexpressed in Escherichia coli and the gene products were purified to homogeneity. The conformation of wild-type and mutant proteins were monitored by 1H-NMR spectroscopy. In vitro phosphorylation studies on mutant lactose-specific enzyme III, as well as evidence from NMR spectroscopy, lead to the conclusion that His78 is the active-site for phosphorylation of lactose-specific enzyme III by phospho-HPr (histidine-containing protein). The role of His82 probably is the enhancement of velocity and efficiency of the phosphotransfer from lactose-specific enzyme III to lactose-specific enzyme II. This result refutes the conclusion of former work based on data by protelytic cleavage and sequencing of the 32P-labeled peptide of lactose-specific enzyme III that His82 is the active-site for phosphorylation.
Finkeldei
U
U
Abteilung Biologie der Ruhr, Universität Bochum, Germany.
Kalbitzer
H R
HR
Eisermann
R
R
Stewart
G C
GC
Hengstenberg
W
W
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites; D005798:Genes, Bacterial; D006639:Histidine; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000737:chemistry / Q000235:genetics*; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics*; D010766:Phosphorylation; D015202:Protein Engineering; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D013211:Staphylococcus aureus / Q000201:enzymology* / Q000235:genetics
D001426:Bacterial Proteins; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; D006639:Histidine; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C055489:phosphoenolpyruvate-lactose phosphocarrier protein, Lactobacillus