Purification and characterization of WaaP from Escherichia coli, a lipopolysaccharide kinase essential for outer membrane stability

In Escherichia coli Salmonella enterica, and Pseudomonos aeruginosa, the wa aP (rfaP) gene product is required for the addition of phosphate to 0-4 of the first heptose residue of the lipopolysaccharide (LPS) inner core region . This phosphate substitution is particularly important to the biology of t hese bacteria; it has previously been shown that WaaP is necessary for resi stance to hydrophobic and polycationic antimicrobials in E. coli and that i t is required for virulence in invasive strains of S. enterica. WaaP functi on is also known to be essential for the viability of P. aeruginosa. The pr edicted WaaP protein shows low levels of similarity (10-15% identity) to eu karyotic protein kinases, but its kinase activity has never been tested. He re me report the purification of WaaP and the reconstitution of its enzymat ic activity in vitro. The purified enzyme catalyzes the incorporation of P- 33 from [gamma-P-33]ATP into acceptor LPS purified from a defined E. coli w aaP mutant. Enzymatic activity is dependent upon the presence of Mg2+ and i s maximal from pH 8.0 to 9.6. The apparent K-m (determined at saturating co ncentrations of the second substrate) is 0.13 mM for ATP and 76 muM for LPS . These data are the first; proof that WaaP is indeed an LPS kinase. Furthe r, site-directed mutagenesis of a predicted catalytic residue suggests that WaaP shares a common mechanism of action with eukaryotic protein kinases.