title: Purification and characterization of WaaP from Escherichia coli, a lipopolysaccharide kinase essential for outer membrane stability.
abstract: In Escherichia coli, Salmonella enterica, and Pseudomonas aeruginosa, the waaP (rfaP) gene product is required for the addition of phosphate to O-4 of the first heptose residue of the lipopolysaccharide (LPS) inner core region. This phosphate substitution is particularly important to the biology of these bacteria; it has previously been shown that WaaP is necessary for resistance to hydrophobic and polycationic antimicrobials in E. coli and that it is required for virulence in invasive strains of S. enterica. WaaP function is also known to be essential for the viability of P. aeruginosa. The predicted WaaP protein shows low levels of similarity (10-15% identity) to eukaryotic protein kinases, but its kinase activity has never been tested. Here we report the purification of WaaP and the reconstitution of its enzymatic activity in vitro. The purified enzyme catalyzes the incorporation of 33P from [gamma-33P]ATP into acceptor LPS purified from a defined E. coli waaP mutant. Enzymatic activity is dependent upon the presence of Mg2+ and is maximal from pH 8.0 to 9.0. The apparent Km (determined at saturating concentrations of the second substrate) is 0.13 mm for ATP and 76 microm for LPS. These data are the first proof that WaaP is indeed an LPS kinase. Further, site-directed mutagenesis of a predicted catalytic residue suggests that WaaP shares a common mechanism of action with eukaryotic protein kinases.
authors.affiliation: Department of Microbiology, University of Guelph, Guelph, Ontario N1G 2W1, Canada.
authors.forename: J A
authors.initials: JA
authors.lastname: Yethon
authors.forename: C
authors.initials: C
authors.lastname: Whitfield
mesh_terms: D000595:Amino Acid Sequence; D000942:Antigens, Bacterial / Q000096:biosynthesis; D001426:Bacterial Proteins / Q000302:isolation & purification / Q000378:metabolism; D002240:Carbohydrate Sequence; D002462:Cell Membrane / Q000502:physiology*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins; D006863:Hydrogen-Ion Concentration; D008070:Lipopolysaccharides / Q000096:biosynthesis*; D008274:Magnesium / Q000494:pharmacology; D008969:Molecular Sequence Data; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
chemical_list: D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008070:Lipopolysaccharides; D017853:Phosphotransferases (Alcohol Group Acceptor); C499634:WaaP protein, E coli; D017868:Cyclic AMP-Dependent Protein Kinases; D008274:Magnesium