WaaP of Pseudomonas aeruginosa is a novel eukaryotic type protein-tyrosine kinase as well as a sugar kinase essential for the biosynthesis of core lipopolysaccharide.
WaaP of P. aeruginosa is a crucial sugar kinase that phosphorylates HepI in the inner core region of lipopolysaccharide (LPS). WaaP shares homology with eukaryotic protein kinases in the conserved functional motifs (I-IX), indicating that it is also a protein kinase. This interpretation is substantiated by several lines of evidence including the following: (i) site-directed mutagenesis on catalytic domain residues abrogated the protein kinase activity; (ii) positive reaction in immunoblotting with anti-phosphotyrosine monoclonal antibody PY20; (iii) matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry and proteolytic peptide mapping showing excess mass equivalent to eight phosphate substituents on the tyrosine residues in WaaP; and (iv) WaaP is capable of catalyzing tyrosine self-phosphorylation as well as phosphorylating an exogenous synthetic co-polymer poly(Glu, Tyr). Thus, WaaP possesses dual kinase functions, and it utilizes a catalytic mechanism similar to that of the eukaryotic protein kinases. WaaP was localized to the cytoplasm, suggesting that phosphorylation of the LPS core occurred prior to translocation to the periplasm and attachment of O-antigen. A chemiluminescence-based enzyme-linked immunosorbent assay (ELISA) was developed to measure the kinetics of the WaaP sugar kinase activity, and the results showed that the K(m) was 0.22 mm for ATP and 14.4 microm for hydrofluoric acid-treated LPS, V(max) was 408.24 pmol min(-1), and k(cat) was 27.23 min(-1).
Zhao
Xin
X
Canadian Bacterial Diseases Network, Department of Microbiology, University of Guelph, Guelph, Ontario N1G 2W1, Canada.
Lam
Joseph S
JS
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D002240:Carbohydrate Sequence; D020134:Catalytic Domain; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005816:Genetic Complementation Test; D007700:Kinetics; D008070:Lipopolysaccharides / Q000096:biosynthesis*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000737:chemistry; D010710:Phosphates / Q000737:chemistry; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D010957:Plasmids / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000737:chemistry*; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013347:Subcellular Fractions / Q000378:metabolism; D014443:Tyrosine / Q000737:chemistry
D000596:Amino Acids; D008070:Lipopolysaccharides; D010455:Peptides; D010710:Phosphates; D014443:Tyrosine; D017853:Phosphotransferases (Alcohol Group Acceptor); D011505:Protein-Tyrosine Kinases