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 μm for hydrofluoric acid-treated LPS,V max was 408.24 pmol min−1, andk cat was 27.23 min−1.

In eukaryotes, protein-tyrosine kinases play important roles in biological regulation, i.e. signal transduction and growth control. Crystallography studies of protein kinases provided an insight into molecular recognition at the substrate and ATP binding sites as well as the mechanisms of action of these enzymes. At present, little is known about tyrosine kinases in prokaryotes, since they are regarded as rare and poorly defined (1)(2)(3). Recent reports that described a number of protein-phos-photyrosine kinases (PTKs) 1 involved in polysaccharide biosynthesis include Wzc cps in Escherichia coli isolates with group 1 capsules (4), Wzc in E. coli K-12 (5,6), Etk in E. coli (1), PTK in Acinetobacter johnsonii (7,8), and CpsD in Streptococcus pneumoniae (9). Most of these enzymes are either proposed or identified to be involved in the transportation or regulation of the production of exopolysaccharides required for virulence (1,8). Interestingly, none of them showed significant homology to the typical tyrosine kinases from eukaryotes (10). Also, no proteintyrosine kinase has been reported to date to phosphorylate the core lipopolysaccharide of Gram-negative bacteria.
Pseudomonas aeruginosa is an opportunistic pathogen that can cause life-threatening infections in compromised patients including those with burn wounds or cystic fibrosis and individuals receiving chemotherapy (11). Lipopolysaccharide (LPS) located in the outer membrane of P. aeruginosa is one of the major virulent factors. It is composed of lipid A, core oligosaccharide (including inner core and outer core regions), and Oantigen (Fig. 1). The inner core LPS is composed of L-glycero-D-manno-heptose and 3-deoxy-D-manno-octulosonic acid. LPS of P. aeruginosa is known to be the most highly phosphorylated among Gram-negative bacteria (12,13). The multiple phosphoryl substituents in this region are essential for the outer membrane stability (14). Its inner core possesses three phosphate groups located on C-2, C-4, and C-6 of HepI ( Fig. 1), respectively. These phosphate substituents contribute negative charges that are crucial in forming ionic bridges with divalent cations to stabilize the outer membrane.
The involvement of waaP in the phosphorylation of HepI of P. aeruginosa LPS has been investigated at the genetic and LPS structural levels by our laboratory (14). Mutation of this gene is lethal to the bacterium, and the knockout of the chromosomal waaP gene was accomplished only when another copy of waaP was added in trans (14), indicating that the presence of phosphate(s) on HepI is essential for the viability of P. aeruginosa. Furthermore, waaP P. aeruginosa (waaP Pa ) can complement a Salmonella typhimurium waaP mutant and restore resistance to SDS and novobiocin in this mutant. By performing two-dimensional 1 H/ 31 P NMR analysis, our group also demonstrated that waaP Pa can reconstitute the phosphate on C-4 of HepI. These data enabled us to conclude that waaP encodes a sugar kinase to phosphorylate C-4 on HepI (14). Importantly, since WaaP is crucial to P. aeruginosa, inhibitors of the kinase * This work was supported by funding from the Canadian Bacterial Disease Network (to J. S. L.). The MALDI-TOF mass spectrometry equipment at the University of Guelph was acquired through a grant jointly funded by the Canadian Foundation of Innovation and the Ontario Research and Development Challenge Fund (to Krassimir Yankulov (principal investigator), J. S. L., and others (co-recipients)). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18  function may have therapeutic value. Therefore, this protein is an attractive target for the development of novel drugs to control infection by P. aeruginosa, which is intrinsically resistant to a wide range of antibiotics. This requires an in depth understanding of the biochemical properties of this enzyme and development of an assay that can be automated for screening large numbers of potential inhibitors.
WaaP of E. coli (WaaP Ec ) shares 52% homology with WaaP Pa . The kinase activity of WaaP Ec was determined by an assay using [ 33 P]ATP to phosphorylate the LPS from the waaP knockout mutant of E. coli (15). In that study, the authors focused on the purification of the enzyme and characterization of the enzyme kinetics. However, a k cat value was not obtained, and there were no data to link WaaP Ec to the family of tyrosine kinases.
In this paper, we report the overexpression and purification of WaaP and also provide evidence to show that this protein is a eukaryotic-type protein tyrosine kinase. We also developed an enzyme-linked immunosorbent assay (ELISA) based method for measuring the activity of WaaP in phosphorylating HepI in the LPS core of P. aeruginosa strain PAO1.

Amino Acid Alignment Analysis of WaaP with WaaP Ec and Protein
Kinases-Amino acid sequence of WaaP was aligned with the protein kinases in the subdomains stated in the nomenclature of Hanks and Quinn (10). The alignment of WaaP Pa and WaaP Ec was accomplished by using the Basic Local Alignment Searching Tool (BLAST) and the nonredundant GenBank TM CDS data base (16).
Site-directed Mutagenesis and in Vivo Complementation Assay-waaP was amplified by PCR from pCOREc1 (17) with the flanking forward and reverse primers 5Ј-ATAATAGGATCCATGAGGCTGGTGCTGG-3Ј and 5Ј-TATATTAAGCTTCAGAGCAGGTCTCCG-3Ј containing BamHI and HindIII, respectively. The PCR product was cloned into pUCP26 (18) as a positive control for complementation assay. Mutations of waaP were constructed by the method of "overlapping extension" as described by Horton (19) using PCR with the flanking primers as well as the primers shown below. The K69A mutation was introduced into the gene with the primer 5Ј-GCTCACCGCCGCGCTCCCGGTG-3Ј; K69R was introduced with 5Ј-GCTCACCGCCAGGCTCCCGGTGCTCGGC-3Ј; D163A was introduced with 5Ј-CAACCATCGCGCCTGCTACATCTGTC-3Ј; and D163E was introduced with 5Ј-CAACCATCGCGAGTGCTACATCTGTC-3Ј. The underlined nucleotides indicate the mutations. The PCR products were then cloned into pUCP26 at BamHI and HindIII sites, respectively, and transformed into E. coli F470 waaP Ϫ (20). Constructs containing the mutations of waaP were confirmed by nucleotide sequencing (performed at the Laboratory Services Division, University of Guelph, Ontario, Canada). In vivo complementation was tested by assessing the minimum inhibitory concentration (MIC) of SDS and novobiocin, respectively, according to Walsh et al. (14). E. coli F470 waaP Ϫ (15) was used as the negative control.
Cloning of waaP into an Expression Vector-waaP was amplified by PCR using pCOREc1, as the template, which contains the core gene cluster of P. aeruginosa (14). The forward and reverse primers were 5Ј-TATATATCATATGAGGCTGGTGCTGG-3Ј and 5Ј-TATATAAGCT-TAGAGAGCAGGTCTCCG-3Ј, containing NdeI and HindIII restriction endonuclease sites, respectively. The reverse primer also contains the mutation (underlined) to change the stop codon of waaP from TGA to TCT. This PCR product was cloned into pET30a expression vector (Novagen, Madison, WI) at NdeI and HindIII sites to be in frame with the His 6 tag at the C terminus of the protein. The construct was then introduced into E. coli JM109 by CaCl 2 transformation (21), and the transformants were selected on Luria agar (Fisher) containing 30 mg/ liter kanamycin. Both strands of DNA were sequenced to verify the sequence of the cloned waaP and the in frame His 6 tag. The resultant construct waaP was overexpressed in E. coli BL21(DE3)pLysS (Novagen). All of the chemicals used in this paper were from Sigma unless stated.
Overexpression of the Plasmid-encoded waaP-Terrific broth (22) supplemented with 3 mg/liter kanamycin and 3.4 mg/liter chloramphenicol was used for the overexpression of WaaP. The cells were first cultivated with shaking at 37°C to 0.6 at A 600 . The overexpression of recombinant protein was induced with 1 mM isopropyl-␤-O-thiogalactopyranoside for 3.5 h. Cells were harvested by centrifugation at 5000 ϫ g and pellets were frozen at Ϫ20°C. pET30a/E. coli BL21(DE3)plysS (Novagen) was used as the control for comparison with the overexpression of WaaP.
Purification of WaaP-Two grams of frozen cell pellet was suspended in 20 ml Tris buffer (20 mM Tris-HCl, 0.5 M NaCl, pH 8.0) containing 5 mM imidazole and 10 mM ␤-mercaptoethanol. A protease inhibitor mixture of 20 l that contains 4-(2-aminoethyl)benzenesulfonyl fluoride, bestatin, pepstatin A, trans-epoxysuccinyl-L-leucylamido(4-guanidino)butane (E-64), and N-(␣-rhamnopyranosyloxyhydroxyphosphiny)-Leu-Trp(phosphoramidon) was added. Cells were broken by sonication on ice with a macroprobe at a power setting of 4 for 2 min (Ultrasonic Processor XL 2020; MANDEL Scientific Company Ltd., Guelph, Ontario, Canada) followed by centrifugation at 10,000 ϫ g at 4°C for 20 min. The supernatant containing the soluble WaaP protein was mixed with 3 ml of cobalt-based immobilized metal ion affinity chromatography (IMAC) resin (TALON metal affinity resin; CLONTECH Laboratories, Palo Alto, CA) and incubated at 4°C for 1 h with gentle shaking. Then the mixture was loaded onto a 1.6-cm diameter column and washed with 20 bed volumes of 5 mM imidazole/Tris buffer. The column was further washed with 10 bed volumes of 50 mM imidazole/Tris buffer, and WaaP was eluted with 1 M imidazole/Tris buffer.
The eluted protein was dialyzed extensively at 4°C against 20 mM Tris-HCl, pH 8, using dialysis tubing with a 3500 molecular weight cut-off (Spectrum Laboratories, Inc., Rancho Dominguez, CA), and concentrated with polyethylene glycol 8000.
Protein Assay-Protein concentration was determined by the BCA method (23) following the procedure described by the manufacturer (Pierce). Bovine serum albumin (BSA) was used as the standard.
Peptide Mapping on Proteolytic Digested WaaP and MALDI-TOF Mass Spectrometry-IMAC purified WaaP was digested with proteases including trypsin and chymotrypsin (sequence grade; Roche Molecular Biochemicals), separately, at 10 g of protein/g of protease in a 20-l solution by incubating at 30°C for 24 h. After mixing with trifluoroacetic acid and guanidine hydrochloric acid to a final concentration of 1% and 2 M, respectively, the digested peptides were loaded onto a Zip-Tip C18 pipette tip (bed volume was 0.6 l) (Millipore Corp., Bedford, MA) that was prewetted with 50% acetonitrile and equilibrated with 0.1% trifluoroacetic acid for purification. Then the ZipTip C18 was washed with 20 bed volumes of 0.1% trifluoroacetic acid. Finally, the purified peptides were eluted with 5 l of 50% acetonitrile in 0.1% trifluoroacetic acid, and 0.5 l was used for MALDI-TOF analysis. MALDI-TOF mass spectrometry was performed locally with a Bruker-Relex (Bruker-Franzen Analytik, Bremen, Germany) in reflector configuration at an acceleration voltage of 20 kV and delayed ion extraction. Mass spectrum was recorded in the negative ion mode. For determination of the molecular mass of WaaP, cytochrome c and carbonic anhydrase were used as standards to calibrate the molecular mass.
The phosphorylated tyrosine residues in WaaP were identified by comparing the actual mass of the individual peptides (from MALDI-TOF analysis) with the predicted mass of the corresponding peptides that were obtained from the on-line analysis tool "Peptide Mass" program.
Self-phosphorylation and Phosphorylation of Exogenous Substrate Using a Chemiluminescence-based ELISA-in opaque, high binding, 96-well microtiter plates (Corning), 5 g of purified WaaP in 100 l of 20 mM Tris-HCl, pH 7.5, were precoated in each well by incubating at 37°C for 3 h. Then the wells were washed with 200 l of washing buffer (100 mM NaCl, 0.1% Tween 20 in 20 mM Tris-HCl, pH 7.5) five times for 10 s each and blocked with blocking buffer (1% BSA, 100 mM NaCl, and 0.1% Tween 20 in 20 mM Tris-HCl, pH 7.5) by incubating at 37°C for 1 h. The plates were washed again as above. Self-phosphorylation of WaaP was performed in a 100-l solution in the microtiter plate well. The reactions were started by adding ATP mixture containing 250 M ATP, 10 mM dithiothreitol, 10 mM MgCl 2 , 10 mM MnCl 2 in 20 mM Tris-HCl, pH 7.5, and incubated at 37°C for 1 h. The reactions were stopped by washing the wells and blocked again as described above. After another washing step, 100 l of monoclonal antibody (mAb) PY20 (anti-phosphotyrosine antibody) diluted 1:3000 in blocking buffer was added to the well as the primary antibody and incubated at 37°C for 2 h. Then the wells were washed again, and 100 l of alkaline phosphatase-conjugated goat anti-mouse F(abЈ) 2 (Jackson ImmunoResearch Laboratory), diluted at 1:2000 in blocking buffer, was added as the secondary antibody and incubated at 37°C for 1 h. After another washing step, the ELISA was developed by adding 100 l of chemiluminescence substrate CDP-Star® Ready-to-Use with Emerald-II™ (CDP*) (Applied Biosystems, Bedford, MA), diluted 1:5 (v/v) in diethanolamine buffer (9.6% (v/v) and 0.01% (w/v), MgCl 2 , pH 9.8). After incubating at room temperature for 20 min, the level of chemiluminescence was measured on a 1420-VICTOR 2 Multilabel Counter (Wallac, Montreal, Quebec, Canada).
For determining unequivocally that phosphorylation in WaaP involved the phosphotyrosine residues, WaaP was bound on cobalt-based IMAC column, dephosphorylated in situ in the column with 10 units of protein-tyrosine phosphatase (CEDARLANE Laboratories, Hornby, Ontario, Canada) over 1 h at 30°C, eluted with 1 M imidazole in 20 mM Tris-HCl, pH 8, and dialyzed against 100 column volumes of 20 mM Tris-HCl, pH 8. A comparison of the phosphate contents of the dephosphorylated WaaP and untreated WaaP was made by the chemiluminescence-based ELISA as described above.
For self-phosphorylation reactions using additional WaaP in solution, 5 g of IMAC-purified WaaP and the ATP mixture were added to each well that was precoated with WaaP and incubated at 37°C for 1 h before proceeding with the chemiluminescence detection as described above.
For determining protein kinase activity of WaaP on exogenous tyrosine-containing substrate, 10 g of poly(Glu, Tyr) 4:1 (Sigma) in 100 l of 20 mM Tris-HCl, pH 7.5, was coated on the opaque 96-well microtiter plate as described above by incubating at 37°C for 3 h. Then the plates were washed and blocked with the same washing and blocking buffers as described above. The kinase reactions were performed in a 100-l solution including 5 g of IMAC-purified WaaP, 250 M ATP, 10 mM dithiothreitol, 10 mM MgCl 2 , 10 mM MnCl 2 in 20 mM Tris-HCl, pH 7.5. After incubating at 37°C for 1 h, the reactions were stopped by washing the plates followed by blocking the wells again. Then ELISA was performed and developed as described above.
P. aeruginosa Cell Fractionation-P. aeruginosa PAO1 cells were fractionated according to the method described by Morona (26) with minor modifications. Cells were cultivated in 100 ml of LB broth to a density of 0.6 at A 600 and then sedimented by centrifugation at 6000 ϫ g for 10 min at 4°C. The cell pellet was resuspended in 2 ml of cold 20% sucrose in 30 mM Tris-HCl, pH 8.1, and 200 l of lysozyme at 1 mg/ml in 100 mM EDTA was added. The mixture was incubated for 20 min on ice and centrifuged at 6000 ϫ g for 10 min at 4°C, and the pellet was suspended in 6 ml of 3 mM EDTA, pH 7.3, followed by sonication for 2 min on ice with the power setting at 4 using a macroprobe. The mixture was centrifuged again at 6000 ϫ g for 10 min at 4°C, and the supernatant was subjected to ultracentrifugation (Ti80 rotor, Beckman Instruments, Palo Alto, CA) at 85,000 ϫ g for 90 min at 10°C. The supernatant was the cytoplasmic fraction. The pellet was resuspended in 2 ml of cold H 2 O, and an equal volume of 4% Triton X-100, 2 mM MgCl 2 in 50 mM Tris-HCl, pH 7.5 was added. The mixture was vortexed intermittently for 30 min at room temperature and ultracentrifuged again at 85,000 ϫ g for 90 min at 10°C. The supernatant was the cytoplasmic membrane fraction, and the pellet was the outer membrane fraction. The periplasmic fraction was isolated by osmotic shock using the method modified from Kessler and Safrin (27). Briefly, cells from 3 ml of P. aeruginosa culture (grown to a density of 0.6 at A 600 ) were sedimented, resuspended in 2 ml of 200 mM cold MgCl 2 in 10 mM Tris-HCl, pH 8.4, and kept on ice for 20 min. After centrifugation at 6000 ϫ g for 20 min, the supernatant was kept as the periplasmic fraction.
Preparation of Polyclonal Antibody against WaaP-Rabbit anti-WaaP antiserum was raised against purified WaaP using protocols described by our group (28). The polyclonal antibodies were purified by immunoaffinity adsorption according to the method of Olmsted (29) with modifications. Briefly, purified WaaP was electrophoresed and transferred on to a nitrocellulose membrane. The WaaP protein band was stained with Ponceau S (0.1% Ponceau S in 5% acetic acid) and cut out. The membrane was then blocked with 5% skim milk, 0.1% Tween 20 in PBS (phosphate-buffered saline, containing 0.8% NaCl, 0.02% KH 2 PO 4 , 0.29% Na 2 HPO 4 , 0.05% KCl, pH 7.4) at 37°C for 1 h. After three washes for 10 min each in PBS plus 0.1% Tween 20, the membrane was incubated with 4 ml of antiserum at room temperature for 4 h on a rocking platform. Then the membrane was washed three times in PBS-Tween 20 and one time with PBS and cut into small pieces. The antibody was eluted by incubating with 0.7 ml of 0.2 M HCl-glycine buffer (pH 2.2) at room temperature for 15 min. The pH of the eluate was brought up to 7 with 0.3 ml of 1 M K 2 HPO 4 , and the antibody was dialyzed against PBS at 4°C. For Western immunoblotting, this antibody was used at a 1:500 dilution.
Preparation of PAO1-LPS and HF-LPS-P. aeruginosa PAO1 cells were cultivated in 300 ml of LB overnight and harvested by centrifugation at 6000 ϫ g for 10 min. After the cells were washed two times with PBS, LPS was extracted by the standard hot water-phenol method of Westphal and Jann (30), lyophilized, and stored at room temperature. Wild type LPS was dephosphorylated with 48% hydrofluoric acid (HF) at 4°C for 48 h and dialyzed extensively against H 2 O in the fume hood, and the HF-LPS was recovered by lyophilization (31). Phosphate analysis was performed according to the method of Ames et al. (32).
Enzymatic Reconstitution of HF-LPS by WaaP and Enzyme Activity Assay Using ELISA-Enzymatic phosphorylation of HF-LPS was performed in 50 l of solution containing 100 ng of HF-LPS, 20 mM MgCl 2 , 50 mM dithiothreitol, 250 M ATP in 20 mM Tris-HCl buffer, pH 7.8, and the reaction was started by the addition of 5 g of enzyme (WaaP purified by IMAC). The reaction mixture was incubated at 37°C for 30 min (15 min for kinetics experiments) and quenched by the addition of 60 l of chloroform/ethanol (1:10) solution. The mixtures were then centrifuged at 13,000 ϫ g for 20 min, and 100 l of the supernatant was transferred into opaque, polystyrene, high binding, 96-well microtiter plates (Corning). The plates were then dried at room temperature overnight in the fume hood. ELISA was performed according to Bantroch et al. (33) to detect the phosphorylated LPS using the primary antibody mAb 7-4 (inner core-specific) generated in our laboratory (34). mAb 7-4 had been shown to specifically recognize phosphorylated LPS. 2 The chemiluminescence-based ELISA was as described earlier under "Self-phosphorylation and Phosphorylation of Exogenous Substrate Using a Chemiluminescence-based ELISA." PAO1-LPS was used to establish a standard curve to quantify phosphorylated LPS.

WaaP Has Features Consistent with Eukaryotic Type Protein
Kinases-Our group (14) has previously provided genetic evidence to show that WaaP is a sugar (heptose) kinase. To further investigate its kinase function and compare it with other kinases including protein kinases, alignment comparisons between the amino acid sequence of WaaP and those of a number of the well characterized protein kinases from eukaryotes were performed (Fig. 2). Since WaaP Pa and WaaP Ec share 52% identity at the amino acid sequence level, both sequences were also aligned and compared with the protein kinases. Two members of protein kinases (protein kinase C-␣ and SNF1) from serine/ threonine kinase (protein kinase C) family and two (Src and epidermal growth factor receptor) from tyrosine kinase family (Src) were selected, respectively, for the alignment comparisons to WaaP Pa (Fig. 2). The sequences of these protein kinases can be divided into 12 subdomains (I-XII) according to the nomenclature of Hanks et al. (10,36). Only subdomains I-IX are shown in Fig. 2. The results indicated that WaaP has significant identity on the conserved, functional residues of the protein kinases. Subdomain I is rich in glycine residues, and the 45 GXG or 55 GXGXG (in which X can be any amino acid) (41) is the signature of the nucleotide binding. Lys 69 in subdomain II is the well characterized catalytic domain residue that is involved in the proton transfer in the phosphotransfer reaction (42). In the central core of the catalytic domain VI through IX, the invariant residues Asp 163 , Asp 181 , and Phe 182 have been implicated in ATP binding, and this is also the feature of other bacterial phosphotransferases that use ATP as the phosphate donor (36). Furthermore, Asp 163 and Asp 181 may interact with the phosphate groups of ATP through Mg 2ϩ salt bridges (10,36,44,45). The presence of these protein kinase-like conserved motifs suggested that WaaP might contain the activity of a protein kinase in addition to being a sugar kinase.
In contrast, the protein sequence of WaaP Ec (from E. coli F470) (20) did not align well with the functional motifs of the protein kinases. It did not contain the signature of the nucleotide binding site (GXG) in subdomain I, and therefore, the catalytic lysine in subdomain II, which is corresponding to Lys 69 in WaaP Pa , is difficult to localize. As well, the Glu residue in subdomain III of WaaP Ec cannot be aligned with the other protein kinases. The only region of WaaP Ec that aligns well with the protein kinases is the catalytic domain HRD (corresponding to 161 HRD in WaaP Pa ) in subdomain VI. Then again, it lost the alignment with Asp 181 -Phe 182 of WaaP Pa . Thus, these results showed that the sequence of WaaP Ec is not consistent with the typical pattern of functional motifs that are characteristic of tyrosine kinases.
To validate the accuracy of the alignment comparisons in Fig. 2, site-directed mutagenesis of waaP Pa was performed targeting Lys 69 and Asp 181 , respectively. The effect of the sitedirected mutation was evaluated by testing whether the mutant constructs could complement waaP Ϫ Ec . It is noteworthy that the complementation assay was performed using a waaP Ϫ Ec mutant as a recipient, since waaP mutation is lethal to P. aeruginosa. The complementation of waaP Ϫ Ec by wild type waaP Pa increased the MICs of waaP Ϫ Ec by 3 and 30 times to novobiocin and SDS, respectively. However, the MICs of waaP Ϫ Ec complemented with the mutants of waaP Pa did not show any difference when compared with those of the waaP Ϫ Ec mutant (Table I). This indicated that Lys 69 or Asp 181 are essential residues for the kinase function of waaP in P. aeruginosa. Therefore, these results substantiated the significance of the alignment of WaaP Pa with the protein kinases shown in Fig. 2.
Purification of WaaP by IMAC-Results from SDS-PAGE and the corresponding Western immunoblotting with Penta-His TM antibody showed that the His 6 tag was expressed as part of WaaP, and a band with an apparent molecular mass of 33 kDa was observed. This is very close to the predicted molecular mass of 32.9 kDa (i.e. the mass of WaaP plus His 6 tag), and over 90% of this protein was expressed in the soluble form (data not 2 X. Zhao and J. S. Lam, submitted for publication.

FIG. 2. Alignment analysis of the amino acid sequence of WaaP from P. aeruginosa with WaaP E. coli and protein kinases from eukaryotic cells.
The subdomains I-IX were defined based on the nomenclature of Hanks (10). PKC-␣, protein kinase C, ␣ form from bovine brain (37); SNF1, "sucrose nonfermenting" mutant wild type gene product from Saccharomyces cerevisiae (38); Src, cellular homolog of oncogene product from Rous avian sarcoma virus from human fetal liver (39); EGFR, epidermal growth factor receptor from human placenta and A431 cell line (40). Conserved functional amino acids are labeled on a dark background.
shown). The IMAC purification of WaaP has been optimized, and the yield obtained was 0.5 mg of protein/liter of culture with over 95% purity (Fig. 3, A and B).
Determining the Presence of Phosphotyrosine Residues in WaaP-To investigate if WaaP is a self-phosphorylated kinase, purified WaaP was examined by Western immunoblotting using anti-phosphotyrosine mAb PY20, and a single band was observed (Fig. 3C). This showed that WaaP contains phosphotyrosine, which is probably the result of self-phosphorylation. WaaP contains eight tyrosine residues; therefore, we proceeded to determine the number of tyrosine residues that are phosphorylated.

Assessment of the State of Phosphorylation among the Eight Tyrosine Residues of WaaP-Full-length
WaaP protein with a C-terminal His tag (WaaPHisC) was subjected to MALDI-TOF mass spectrometry to determine the accurate molecular mass. The actual mass of WaaP from the MALDI-TOF analysis was m/z 33544.618 (Fig. 4), which is larger than the predicted (nonphosphorylated) molecular mass of 32897.38 Da. The extra mass of 647.328 matched the value of 8.094 phosphate substituents (HPO 3 ; mass ϭ 79.969). This result provided evidence that all eight tyrosine residues in WaaP may be phosphorylated. We further performed ELISA using anti-phosphotyrosine mAb PY20 to interact with WaaP that had been dephosphorylated with phosphotyrosine-specific protease (protein-tyrosine phosphatase). The results showed that over 10% lower signal was detected from the protein-tyrosine phosphatase-treated WaaP than that of the nontreated WaaP (data not shown). This indicates that WaaP can be dephosphorylated by protein-tyrosine phosphatase, and therefore the extra mass of WaaP is due to the phosphorylation and not sulfation; the latter would have resulted in contributing approximately the same extra mass.
To identify the location of the phosphorylated amino acid residues in WaaP, purified WaaP was digested with trypsin and chymotrypsin, respectively. The peptides generated were analyzed by MALDI-TOF and compared with the predicted peptide map of WaaP digested by these two proteases, respectively. Each tyrosine-containing peptide from digested WaaP had extra mass corresponding to the addition of a phosphate group m/z 80 (Table II). This indicated that all eight tyrosine residues in WaaP can be phosphorylated.
Self-phosphorylation Activity of WaaP-To determine whether WaaP catalyzes tyrosine self-phosphorylation, purified WaaP was used in the self-phosphorylation assay with a sensitive, chemiluminescence-based ELISA using anti-phosphotyrosine PY20 antibody. The chemiluminescence signal of the self-phosphorylation of WaaP was 407.8 chemiluminescence units⅐g Ϫ1 ⅐min Ϫ1 , which was 21% higher than that of the control, 337.1 chemiluminescence units⅐g Ϫ1 ⅐min Ϫ1 (Table III), indicating that WaaP exhibits self-phosphorylation activities. To further examine the mechanism of the self-phosphorylation (i.e. if the self-phosphorylation occurred within one molecule or between molecules), purified WaaP protein was coated on the 96well plates, and self-phosphorylation assays were performed in two distinct ways. One approach was by the addition of an ATP mixture, and the other was by the addition of exogenous WaaP plus the ATP mixture (Table III). The use of exogenous WaaP did not improve the phosphorylation level of the coated WaaP (Table  III), which indicated that the phosphorylation of WaaP probably occurs intramolecularly and not intermolecularly.
Determining the Kinase Activity of WaaP in Interaction with Exogenous Tyrosine-containing Substrate, Poly(Glu, Tyr)-We further investigated the ability of WaaP to phosphorylate in vitro an exogenous substrate, poly(Glu, Tyr) copolymer, which was used to precoat the 96-well microtiter plates. Phosphorylation was monitored by the chemiluminescence-based ELISA. The phosphorylation of poly(Glu, Tyr) gave a chemiluminescence response of 782 units/g/min (Table III). This showed that WaaP could catalyze the phosphorylation of exogenous tyrosine substrates.
Cellular Localization of WaaP and Its State of Phosphorylation in the Cell Fractions-Cell fractionation was performed to localize WaaP in P. aeruginosa and the fractions were examined by SDS-PAGE and Western immunoblotting with purified polyclonal antibody against WaaP. WaaP can only be found in the cytoplasmic fraction of P. aeruginosa (Fig. 5B, lane 5). The overexpressed WaaP with the His 6 tag exhibited higher molecular mass at about 33 kDa, which is close to the expected mass at 32.9 kDa (Fig. 5B, lane 2), and WaaP from P. aeruginosa (Fig. 5B, lane 5) showed smaller size at 31-kDa and is also close to the expected molecular mass at about 31.3 kDa. In Western immunoblotting of the different fractions using anti-phosphotyrosine mAb PY20, WaaP from P. aeruginosa (Fig. 5C, lane 5) and the overexpressed WaaP in E. coli (Fig. 5C, lane 2) were both strongly reactive with this antibody.
Determining the Sugar Kinase Activity of WaaP-To identify the reconstitution of phosphate on HF-LPS by WaaP enzymatic reaction, phosphate analysis was performed on the reconstituted HF-LPS as well as on wild type PAO1-LPS using the method of Ames and Dubin (32) and Zhao and Lam. 2 Approximately 10% of phosphate was reconstituted on HF-LPS (20 nmol of P i /ng of LPS) compared with 190 nmol of P i /ng of LPS  for PAO1-LPS. This indicated that the incorporation of the phosphate to the HF-LPS occurred due to the enzyme reaction; therefore, WaaP is also a sugar kinase in addition to a selfphosphorylated protein-tyrosine kinase. To develop a nonradiolabeling assay for determining the activity of WaaP, mAb 7-4 that specifically recognizes the phosphorylated LPS was used as the primary antibody. mAb 7-4 reacted with wild type LPS from strain PAO1 and did not react with HF-LPS that had been dephosphorylated. This antibody allowed the development of a highly sensitive, chemiluminescence-based ELISA (the details of the ELISA development are described elsewhere). 2 ELISAs on the time course of WaaP reactions indicated that enzyme activities increased sharply in the initial 20 min and slowed down afterward (data not shown). Therefore, the reactions for the kinetic studies measured the phosphorylation within the initial 15 min. The enzyme reactions of WaaP were  a Only two peptides were listed in this table to localize each phosphotyrosine. Trypsin cleaves on the carboxyl side of arginine and lysine residues, and chymotrypsin is selective for peptide bonds on the carboxyl side of the aromatic side chains of tyrosine, tryptophan, and phenylalanine and large hydrophobic residues such as methionine (43).  (Table IV). Approximately 70% of enzyme activity remained after storage at Ϫ20°C for 7 days.

DISCUSSION
Carbohydrates are probably the least understood of all classes of biologically important molecules (47), and much less is known about the properties of the sugar kinases involved in the biosynthetic pathway.
It is intriguing to observe that WaaP, being a sugar kinase, showed significant amino acid identities in most of the functional motifs (subdomains I-IX) with the eukaryotic type protein kinases including members in both protein-tyrosine kinase and Ser/Thr kinase families. Importantly, we were able to validate this prediction on the conserved motifs by site-directed mutagenesis and the subsequent complementation assay. In subdomain I, WaaP has two glycine-rich regions, 45 GXG and 55 GXGXG (where X can be any amino acid), but so far no mutations have been made to show the importance of the space between the functional glycine region and the invariant lysine that lies 14 -23 residues downstream (36). Therefore, in WaaP, either of these two glycine regions could be the nucleotidebinding site.
In prokaryotes, several protein-tyrosine kinases such as Wzc in E. coli (5,6), PTK in A. johnsonii (7,8), and CpsD in Streptococcus pneumoniae (9) were reported to be involved in the transportation or regulation of LPS or capsule biosynthesis in bacteria. But they do not share sequence identities with WaaP or with the eukaryotic protein-tyrosine kinases in most of the functional motifs (36). Those proteins share the Walker A and Walker B consensus among their sequences, and the tyrosine residues in these sequences form a tyrosine-rich region and localize downstream of Walker B. However, in this study, we have shown that WaaP is a eukaryotic type proteintyrosine kinase, and the tyrosine residues were found to scatter throughout the sequence of the protein.
Both WaaP from P. aeruginosa PAO1 and the overexpressed WaaP were found to be phosphorylated. This is different from most of the reported tyrosine kinases from bacteria in which the self-phosphorylation could only be detected in the overexpressed protein (1). The self-phosphorylation of WaaP in P. aeruginosa may contribute to its role as a dual functional kinase.
It is intriguing to observe that WaaP is a sugar kinase in addition to a protein kinase. Since the kinase functional domain in WaaP spanned over 200 amino acids, which is about 72% of the total WaaP sequence (276 amino acids), the enzyme probably utilizes the same functional domain to perform sugar phosphorylation and self-phosphorylation. Furthermore, our amino acid alignment analysis strongly suggested that WaaP might utilize a catalytic mechanism similar to that of the eukaryotic type PTKs. Crystallization of WaaP is under way to solve the mechanisms of the kinase activities of WaaP.
The localization of WaaP to the cytoplasmic cell fraction has shed some light on the events of core LPS substitutions during the biosynthesis of this region of the LPS. It is evident that the phosphorylation of HepI in P. aeruginosa LPS occurs before O-antigen units are attached to the core in the periplasm.
In contrast to WaaP Pa , the amino acid sequence of WaaP Ec aligned rather poorly in regions corresponding to the conserved functional motifs in eukaryotic type protein kinases. Also, in the complementation assay (Table I), wild type waaP Pa could only partially complement the E. coli F470waaP Ϫ mutant, and the MIC values to the novobiocin and SDS were higher than in the waaP mutant but lower than in the wild type E. coli F470. These results indicated that WaaP Pa and WaaP Ec might be structurally different although they both have heptose kinase activity and similar kinetic properties. Importantly, in this study we were able to demonstrate that WaaP Pa is a selfphosphorylated tyrosine kinase, whereas such a function has not been reported for WaaP Ec . This implies that WaaP Pa is an enzyme with dual functions, and it may also be involved in other functions such as transportation in the LPS biosynthesis like other tyrosine kinases (5,7,8,9). This may account for the reason that waaP that encodes this enzyme is essential, since the mutation in this gene is lethal to P. aeruginosa (14), whereas mutation in waaP Ec was not lethal to E. coli (15).
As a crucial enzyme to P. aeruginosa, WaaP is a rational drug target for developing new antibiotics. In recent years, enormous efforts have been made to develop protein-tyrosine kinase inhibitors for treatment of diseases such as cancer, psoriasis, and osteoporosis. Several new high throughput PTK assay technologies have been described, and a number of inhibitors have already been put through clinical trials (35). Most of the inhibitors (e.g. members of the 4-amilinoquinozolinones family) are small molecules that are competitive at the ATP binding site (46). Since WaaP held such good identity with the typical protein kinases, the screening of inhibitors could begin by using these ATP competitors or analogues.
To develop a nonradiolabeling assay for the LPS phosphorylation, the identification of the phosphate as the epitope for mAb 7-4 was critical. Since mAb 7-4 only recognizes the phosphate substituents in the core region of large LPS molecules, it is also critical that a much more sensitive, chemiluminescence-based method was used to develop the ELISA.
Another advantage of the WaaP-ELISA using mAb 7-4 is the capability to quantitatively determine the enzyme activity of WaaP with high sensitivity compared with traditional colorimetric methods. Yethon and Whitfield (15) recently determined the enzyme activity of WaaP Ec using the LPS isolated from an E. coli waaP mutant as the substrate. In that report, [␥-33 P]ATP was used to assay the enzyme activity, and the K m on ATP was 0.13 mM, which is quite close to that determined by us at 0.22 mM for WaaP Pa . In our study, the K m of WaaP Pa for HF-LPS, at 14.4 M, was 5 times lower than that of WaaP Ec , at 76 M, reported by Yethon and Whitfield (15). The lower K m of WaaP from P. aeruginosa reflects a higher binding of WaaP with LPS, or it could be due to the nature of the HF-LPS used in this study. The k cat of WaaP Pa was 27.23 min Ϫ1 ; however, no k cat value was reported for WaaP Ec (15). These two proteins share 52% identity at the amino acid level, and the differences in the sequences may result in the variations in the catalytic characters. WaaP Ec has the His tag at the N terminus of the protein, while the WaaP described in this paper contains the C-terminal His tag. This might also account for the differences in the kinetic parameters between the two proteins. Also, the LPSs used in both reactions were from two distinct bacterial species and therefore possessed different physical properties (i.e., the shorter O-antigen chain length of LPS Pa and the differences in LPS sugar compositions of the two bacteria).
In conclusion, we have provided the evidence to show that WaaP Pa possesses dual kinase functions. It is a novel eukaryotic type, self-phosphorylating PTK as well as a heptose kinase associated with the biosynthesis of the LPS core. We also demonstrated that the phosphorylation of LPS in P. aeruginosa occurred before the O-antigen was assembled onto the core. The sensitive chemiluminescence-based ELISA was successfully applied to elucidate the kinetic parameters of WaaP. This assay is appropriate for the screening of novel antibiotics to control infection from P. aeruginosa and other Gram-negative bacteria. Kinetic studies were performed with various concentrations of enzyme (0 -15 g), ATP (0 -500 M), and HF-LPS (0 -5 ng). The data were collected within the initial 15 min of the enzyme reaction. The kinetic parameters were determined by the Michaelis-Menten equation. Note that although 150 pmol of enzyme was added to the enzyme-substrate reaction mixture, because of the tendency on the enzyme to precipitate, only 10% (15 pmol) of the enzyme was left in the supernatant after centrifugation.