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J. Biol. Chem., Vol. 276, Issue 8, 5498-5504, February 23, 2001
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From the Department of Microbiology, University of Guelph, Guelph,
Ontario N1G 2W1, Canada
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 [
Purification and Characterization of WaaP from Escherichia
coli, a Lipopolysaccharide Kinase Essential for Outer Membrane
Stability*
and
-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 µM 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.
*
This work was supported in part through funding by the
Canadian Bacterial Diseases Network (Network of Centers of Excellence) (to C. W.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Recipient of a Doctoral Research Award from the Canadian
Institutes of Health Research.
§
A Canadian Institutes of Health Research Senior Scientist. To whom
correspondence should be addressed. Tel.: 519-824-4120 (ext. 3478);
Fax: 519-837-1802; E-mail: cwhitfie@uoguelph.ca.
Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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