Identification and characterization of NeuB3 from Campylobacter jejuni as a pseudaminic acid synthase.

Campylobacter jejuni and Campylobacter coli are the main causes of bacterial diarrhea worldwide, and Helicobacter pylori is known to cause duodenal ulcers. In all of these pathogenic organisms, the flagellin proteins are heavily glycosylated with a 2-keto-3-deoxy acid, pseudaminic acid (5,7-diacetamido-3,5,7,9-tetradeoxy-L-glycero-L-manno-nonulosonic acid). The presence of pseudaminic acid is required for the proper development of the flagella and is thereby necessary for motility in, and invasion of, the host. In this study we report the first characterization of NeuB3 from C. jejuni as a pseudaminic acid synthase; the enzyme directly responsible for the biosynthesis of pseudaminic acid. Pseudaminic acid synthase catalyzes the condensation of phosphoenolpyruvate (PEP) with the hexose, 2,4-diacetamido-2,4,6-trideoxy-L-altrose (6-deoxy-AltdiNAc), to form pseudaminic acid and phosphate. The enzymatic activity was monitored using 1H and 31P NMR spectroscopy, and the product was isolated and characterized. Kinetic analysis reveals that pseudaminic acid synthase requires the presence of a divalent metal ion for catalysis and that optimal catalysis occurs at pH 7.0. A coupled enzymatic assay gave the values for k(cat) of 0.65 +/- 0.01 s(-1), K(m)PEP of 6.5 +/- 0.4 microM, and K(m)6-deoxy-AltdiNAc of 9.5 +/- 0.7 microM. A mechanistic study on pseudaminic acid synthase, using [2-18O]PEP, shows that catalysis proceeds through a C-O bond cleavage mechanism similar to other PEP condensing synthases such as sialic acid synthase.

Campylobacter jejuni, Campylobacter coli, and Helicobacter pylori are flagellated, motile, Gram-negative bacteria that colonize the gastrointestinal tract of people worldwide. C. jejuni and C. coli are the main causative agents of bacterial diarrhea (1,2), and H. pylori causes duodenal ulcers (3). Their flagellin proteins are heavily glycosylated with a 9-carbon 2-keto-3-deoxy acid, pseudaminic acid (5,7-diacetamido-3,5,7,9-tetradeoxy-L-glycero-L-manno-nonulosonic acid) (Fig. 1A) (4 -7). The production of pseudaminic acid is important for the development of flagella in these organisms as evidenced by aflagellate C. jejuni (8) and H. pylori (6) mutants unable to produce pseudaminic acid. Motility is a key factor implicated in the colonization of the viscous gastrointestinal tract of the host and therefore flagella are necessary for their invasiveness (1, 9 -11). By studying the enzymes involved in the biosynthesis of pseudaminic acid, methods can be developed for combating these pathogenic organisms.
Pseudaminic acid shares great structural similarity with another 9-carbon 2-keto-3-deoxy acid, N-acetylneuraminic acid (NeuNAc or sialic acid) (Fig. 1B). The biosynthesis of sialic acid has been well studied and the enzyme directly responsible for forming sialic acid is known as sialic acid synthase or N-acetylneuraminic acid synthase (SAS or NeuB). Sialic acid synthase catalyzes the condensation of phosphoenolpyruvate with N-acetyl-D-mannosamine (ManNAc) to form sialic acid and phosphate (12)(13)(14). Recent studies have reported on both the structure of NeuB and its mechanism of action (15,16). Catalysis was demonstrated to proceed through an overall C-O bond cleavage process where the si-face of phosphoenolpyruvate (PEP) 2 initially attacks the aldehyde of the open chain form of ManNAc and water adds to the C-2 position forming a tetrahedral intermediate (Fig. 2a). The tetrahedral intermediate then collapses to form orthophosphate and the open chain form of sialic acid that cyclizes to the pyranose form in solution. Formation of the tetrahedral intermediate was proposed to proceed in a stepwise fashion through an oxocarbenium ion intermediate. Two other well studied PEP condensing enzymes that utilize very similar mechanisms are 2-keto-3-deoxy-D-manno-octulosonate-8-phosphate synthase, which catalyzes the condensation of PEP and D-arabino-5-phosphate (17,18), and 2-keto-3-deoxy-D-arabino-heptulosonate-7-phosphate synthase, which catalyzes the condensation of PEP and D-erythrose-4phosphate (19,20). A key mechanistic experiment performed with all three of these synthases was the use of [2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18] O]PEP as a substrate during enzymatic incubations (see labeled atoms in Fig. 2). In each case, catalysis resulted in the production of 18 O-labeled inorganic phosphate indicating that a C-O bond cleavage process was occurring (15,19,21).
To date none of the enzymes of pseudaminic acid biosynthesis have been characterized, however; it is reasonable to assume that a pseudaminic acid synthase exists that would catalyze the condensation of PEP with the hexose, 2,4-diacetamido-2,4,6-trideoxy-L-altrose (6-deoxy-AltdiNAc) (Fig. 1A). Three genes that show homology to neuB were identified in C. jejuni, where one encoded a sialic acid synthase (neuB1) and the other two genes were thought to encode proteins that modify the flagellin proteins (neuB2 and neuB3) (8,16). NeuB3 shows 35.0% identity to the NeuB from Neisseria meningitidis and was found to be essential for the formation of flagella because an insertional mutation in the gene resulted in aflagellate C. jejuni (8). In this study we report the first identification and characterization of the activity of NeuB3 as a pseudaminic acid synthase that catalyzes the condensation of PEP with 6-deoxy-AltdiNAc to form pseudaminic acid.
There are two potential mechanisms proposed for pseudaminic acid synthase. The first and most likely mechanism is a C-O bond cleavage mechanism similar to that of sialic acid synthase ( Fig. 2A). The second potential mechanism involves P-O bond cleavage whereby a direct attack of water onto the phosphate group of PEP generates phosphate and the enolate anion of pyruvate (Fig. 2B). The enolate then attacks the * This work was supported by the Canadian Institutes of Health Research and the Natural Sciences and Engineering Research Council of Canada. 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 U.S.C. Section 1734 solely to indicate this fact. 1 To whom correspondence may be addressed.

EXPERIMENTAL PROCEDURES
Materials and General Methods-Purine nucleoside phosphorylase and phosphoenolpyruvate were purchased from Sigma. 18 O-Enriched water (95%) was purchased from Icon Isotopes. 2-Amino-6-mercapto-7-methylpurine ribonucleoside was purchased from Berry and Associates. Protein concentrations were determined by the method of Bradford (24) using bovine serum albumin as the standard. Concentrations of stock substrate solutions were determined enzymatically using the reported assay conditions with high concentrations of NeuB3 (64 g) and excess co-substrate. NMR spectroscopy was performed on a Bruker AV300 or AV400 spectrometer. ESI-MS was performed on a Bruker Esquire LC mass spectrometer.
Cloning, Overexpression, and Purification of NeuB3-The neuB3 gene, Cj1317, was obtained from the genome strain C. jejuni 11168 by PCR amplification with Pwo polymerase according to manufacturer's conditions (Roche) (8). The primers used were: 5Ј-CCCCCCCATAT-GCAAATAGGAAATTTTAAC-3Ј (forward sequence) and 5Ј-CCC-CCCGTCGACTCATCATTGGAAATCTCCTTGTTTAAAG-3Ј (reverse sequence). The gene was then cloned into the expression plasmid pCWoriϩ as an NdeI-SalI fragment, and the construct was maintained in Escherichia coli AD202, which was used for enzyme production (25). Cells were grown at 37°C in 500 ml of Luria-Bertani (LB) medium supplemented with 100 g ml Ϫ1 ampicillin. Overexpression of NeuB3 was induced through the addition of 0.5 mM isopropyl ␤-D-thiogalactopyranoside at an A 600 of 0.60, with growth at 37°C for 6 h. Cells were harvested through centrifugation at 5,000 ϫ g for 30 min, resuspended  in Tris-HCl buffer (20 mM, pH 7.0) containing pepstatin A (1 mg/liter) and aprotonin (1 mg/liter), and lysed with two passes through a French press. The cell lysate was centrifuged at 7,000 ϫ g for 1.5 h, passed through a 0.45-m filter, and loaded directly onto a 5-ml Hi-trap TM Q-Sepharose column (Amersham Biosciences) pre-equilibrated with 20 mM Tris-HCl buffer (pH 7.0). NeuB3 was eluted with a linear gradient of 0 to 1 M NaCl in 20 mM Tris-HCl buffer (pH 7.0). Fractions containing active enzyme were desalted 2 times by concentration and reconstitution with 20 mM Tris-HCl buffer (pH 7.0) using Amicon Ultra Centricons (Millipore), and then flash frozen with 10% glycerol. Protein samples were determined to be Ͼ90% pure by SDS-PAGE. The molecular mass was determined to be 38,670 using ESI-MS (38,647 predicted).
NMR Incubation Studies and Tests for Activity-6-Deoxy-AltdiNAc was synthesized according to literature procedures (26,27). A solution of Tris-DCl buffer prepared in D 2 O (700 l, 10 mM, pD 7.4) containing 6-deoxy-AltdiNAc (10 mM) and PEP (20 mM) was placed in an NMR tube. Initial 1 H and proton-decoupled 31 P NMR spectra were taken. The solution was removed from the tube and mixed with 50 mg of NeuB3 (buffer exchanged with the deuterated Tris-HCl buffer (pD 7.4)) and 1 mM MgCl 2 to a total volume of 1 ml. After incubation of the reaction mixture for 5 min at 25°C, Chelex-100 resin (ϳ20 mg) was added and 1 H and 31 P NMR spectra were retaken.

C-O Versus P-O Bond Cleavage-[2-
18 O]PEP disodium salt was prepared according to literature reported procedures (28,29). The extent of 18 O incorporation at the C-2 position was determined to be 54% by mass spectrometry: 16 O, 85). 31 P NMR spectroscopy was used to determine that the position of the label was at the C-2 position: 31 A solution of Tris-DCl buffer prepared in D 2 O (700 l, 10 mM, pD 7.4) containing 6-deoxy-AltdiNAc (10 mM) and 20 mM [2-18 O]PEP was placed in an NMR tube, and Chelex-100 resin (ϳ20 mg previously washed with D 2 O) was added. An initial proton-decoupled 31 P NMR spectrum was obtained with the following parameters: spectral frequency of 121.5 MHz, sweep width of 2437 Hz, acquisition time of 13.4 s, pulse delay of 2 s, and pulse width of 10 s. The solution was decanted from the Chelex resin and mixed with 50 mg of NeuB3 (buffer exchanged with the deuterated Tris-HCl buffer (pD 7.4)) and 1 mM MgCl 2 . The reaction was incubated for 5 min at 25°C and Chelex-100 resin (ϳ20 mg previously washed with D 2 O) was added. After a 1-h time period to allow complete complexation of the metals, another protondecoupled 31 P NMR spectrum was acquired with the same parameters: 31  Isolation and Characterization of Pseudaminic Acid-Enzyme was removed from the reactions by centrifugal ultrafiltration and the resulting filtrate was loaded onto a 10-ml column of Dowex AG1-X8 resin (formate form) pre-equilibrated with water. A stepwise gradient of 0 -1.0 M formic acid in water with 0.2 M increments (50 ml per increment) was used to elute the pseudaminic acid from the column. Pseudaminic acid eluted from the column in the 0.2 and 0.4 M fractions that were concentrated in vacuo and then lyophilized. Pseudaminic acid was characterized using 1 H, 13  Enzyme Kinetics as Determined by a Continuous Coupled Assay-Enzyme kinetics were measured by a continuous coupled phosphate assay (30). A cuvette containing 100 mM Tris-HCl buffer (pH 7.0), 6-deoxy-AltdiNAc (variable), PEP (variable), 2-amino-6-mercapto-7-methylpurine ribonucleoside (200 M), purine nucleoside phosphorylase (5 units buffer exchanged twice into 100 mM Tris-HCl buffer pH 7.0), and MnCl 2 (10 mM) was thermally equilibrated for 5 min at 37°C. The enzymatic reaction was initiated by the addition of NeuB3 (3.2 g) for a total assay volume of 500 l and the enzymatic rate was calculated from the observed increase of absorption at A 360 (using ⑀ ϭ 11,000 M Ϫ1 cm Ϫ1 ). The K m value for 6-deoxy-AltdiNAc was measured in the presence of 1 mM PEP (saturating), and that for PEP was measured in the presence of 1 mM 6-deoxy-AltdiNAc (saturating). Kinetic parameters were determined from initial velocities fit to Michaelis-Menten kinetics using the program Grafit (31).
pH Versus Rate Profile Experiment-The pH versus rate profile was constructed using a mixture of 50 mM Tris-HCl and 50 mM MES buffer at pH 6 -9. Purine nucleoside phosphorylase was buffer exchanged twice in the same buffer at the different pH values. Saturating 6-deoxy-AltdiNAc (1 mM) and saturating PEP (1 mM) were used with the kinetic assay above. Initial rates were plotted against pH.
Metal Dependence Experiment-The metal dependence experiment was carried out with saturating 6-deoxy-AltdiNAc (1 mM) and saturating PEP (1 mM) at pH 7.0 using the kinetic assay above. Different divalent metal cations were independently added at a concentration of 10 mM and the initial velocities were determined. The rates were normalized to the fastest rate (Co 2ϩ ). Controls included adding 10 mM EDTA or no additives (enzyme as isolated) to the kinetic assay.

RESULTS AND DISCUSSION
Overexpression and Purification of NeuB3-C. jejuni-The neuB3 gene, Cj1317, was obtained from the genome strain C. jejuni 11168 by PCR amplification and overexpressed in E. coli. NeuB3 was isolated using a single anion exchange chromatographic step and the resulting protein was found to be greater than 90% pure as determined by SDS-PAGE (Fig. 3). The molecular mass of NeuB3 was determined to be 38,670 Ϯ 30 using electrospray ionization-mass spectrometry (38,647 predicted).
Test for Activity of NeuB3-One of the main hurdles in studying pseudaminic acid synthase is that the putative substrate 6-deoxy-Altdi-NAc is not commercially available. To obtain a sample it was necessary to chemically synthesize the compound in 12 steps from L-fucose using a protocol described in the literature (26,27). To test whether NeuB3 showed pseudaminic acid synthase activity, 1 eq of 6-deoxy-AltdiNAc and 2 eq of PEP were incubated in deuterated buffer containing Mn 2ϩ and NeuB3, and the reaction was monitored by 1 H and 31 P NMR spectroscopy. Before the addition of NeuB3, the 31 P NMR spectrum shows a single phosphorus peak corresponding to the phosphate group of PEP at Ϫ3.01 ppm (Fig. 4, upper panel). After the addition, a new peak appears at 0 ppm corresponding to inorganic phosphate released during catalysis (Fig. 4, lower panel). The identities of the phosphorus peaks were verified by spiking with PEP and phosphate standards (data not shown). Control incubations lacking either enzyme or 6-deoxy-AltdiNAc did not show the formation of orthophosphate under identical conditions, indicating that the reaction was not because of either background hydrolysis or a phosphatase impurity. In addition, ManNAc could not replace 6-deoxy-AltdiNAc as a substrate indicating that NeuB3 does not possess detectable sialic acid synthase activity.
The time course as observed by 1 H NMR spectroscopy shows the conversion of 6-deoxy-AltdiNAc to pseudaminic acid. The initial 1 H NMR spectra shows that 6-deoxy-AltdiNAc exists as a 3:1 ratio of the ␤ to ␣ anomer in solution (Fig. 5, upper panel). The anomeric ratio is apparent from integration of the two acetamido signals of 6-deoxy-AltdiNAc (␤-anomer at 1.88 and 1.93 ppm, and ␣-anomer at 1.89 and 1.90 ppm) and also the anomeric protons (data not shown). After the addition of NeuB3, the complete conversion of the substrate into largely a single anomer of a new product is observed (acetamido peaks at 1.81 and 1.85 ppm). This would be expected for the pyranose form of a nonulosonic acid that would strongly favor the ␣-anomer in solution. Further support is seen in the appearance of the key characteristic peaks of pseudaminic acid because of the signals of the C-3 methylene protons (Fig. 5, lower panel). The H-3ax (axial) proton signal is a doublet of doublets that appears at 1.62 ppm and the H-3eq (equatorial) proton signal is a doublet of doublets that appears at 1.77 ppm (slightly obscured by the methyl N-acetyl proton signals) under these conditions. The fact that the H-3ax proton signal appears as a triplet is because of strong geminal coupling to the H-3eq proton (J 3ax,3eq 13.3 Hz) and strong coupling to the H-4 proton (J 3ax, 4  Whereas previous studies have demonstrated that the absolute stereochemistry of pseudaminic acid from Pseudomonas aeruginosa is of the L-glycero-L-manno configuration, this has not been unequivocally shown in the case of C. jejuni. One can postulate reasonable biosynthetic routes to either enantiomer using very similar enzymatic pathways and therefore it was conceivable that either the D-or L-enantiomer of 6-deoxy-AltdiNAc could be the correct substrate for NeuB3. The fact that the L-isomer of 6-deoxy-AltdiNAc is the substrate of NeuB3 indi-  cates that the stereochemical assignment of pseudaminic acid is correct as the previously assigned L-glycero-L-manno isomer (4,(32)(33)(34)(35).
Isolation and Characterization of Pseudaminic Acid-The pseudaminic acid produced from the NeuB3 reaction was isolated using anion exchange chromatography and fully characterized. Mass spectral analysis (-ve ESI-MS) showed signals for the expected parental ion at m/z 333 (M Ϫ H ϩ ) and the oxonium ion at m/z 315 (M Ϫ H 3 O ϩ ). The NMR spectra were similar to those previously reported in the literature for pseudaminic acid derivatives characterized from the lipopolysaccharide of P. aeruginosa, Shigella boydii, Vibrio cholerae, Proteus vulgaris, and the flagellin proteins of C. jejuni (4,(32)(33)(34)(35). 1 H NMR spectroscopy was used to assign the relative configuration of the pyranose ring protons. The large coupling constants of J 3a,4 (12.2 Hz) and small coupling constants of J 4,5 (5.5 Hz) and J 5,6 (1.7 Hz) indicates that H-4 is axial, H-5 is equatorial, and H-6 is axial (TABLE ONE). The large J 6,7 of 10.3 Hz indicates that C-6 and C-7 have the erythro configuration as opposed to sialic acid, which has a small J 6,7 of 1.2 Hz and the threo configuration (33). In the 13 C NMR spectrum (TABLE TWO), the C-9 chemical shift is 15.3 ppm, which is similar to the chemical shift of the methyl group for N-acetyl L-allothreonine (17.5 ppm) indicating that C-7 and C-8 has the erythro conformation (versus 19.5 ppm with N-acetyl L-threonine) (33). The difference in the 1 H chemical shifts of H-3ax and H-3eq is small (0.14 ppm), indicating that the ␣-anomer is the predominant anomer because the ␤-anomer has a larger difference in chemical shift between the two proton signals (0.77 ppm) as seen in C-2 glycosylated pseudaminic acid that is ␤-linked (4,(32)(33)(34)(35).
Enzyme Kinetics of NeuB3 as Determined by a Continuous Coupled Assay-The kinetic pararmeters of the NeuB3 reaction were determined using a continuous coupled assay for phosphate release (30). Initial reaction velocities ( o ) were determined for each substrate by varying its concentration in the presence of saturating amounts of the other substrate. The kinetic data obeyed Michaelis-Menten kinetics (Fig. 6) and gave kinetic constants of k cat ϭ 0.65 Ϯ 0.01 s Ϫ1 , K m PEP ϭ 6.5 Ϯ 0.4 M and K m 6-deoxy-AltdiNAc of 9.5 Ϯ 0.7 M (100 mM Tris-HCl, pH 7.0, 10 mM Mn 2ϩ and 37°C). Whereas the turnover number is similar to that observed with sialic acid synthase, the K m values were considerably lower, potentially reflecting the lower concentration of 6-deoxy-AltdiNAc that is available in the cell. A qualitative screen of maximal rate versus pH gave a bell-shaped profile and indicated that optimal activity was observed at pH 7. Finally, an assessment of the metal ion requirements for pseudaminic acid synthase was performed (TABLE THREE). When the enzyme was assayed directly as isolated, only low levels of activity were observed. Activity was gained upon the addition of various divalent metal ions with the highest values observed in the presence of Mn 2ϩ and Co 2ϩ . The addition of EDTA completely abolished the activity of the synthase. These observations indicate that pseudaminic acid synthase requires a divalent cation for activity and that the bound metal is largely lost during isolation of the enzyme. Similar observations have been made with sialic acid synthase, NeuB, which apparently uses the divalent cation to increase the electrophilicity of the substrate aldehyde (15). A recent x-ray crystal structure of NeuB complexed with Mn 2ϩ , PEP, and N-acetylmannosaminitol (a reduced form of ManNAc bearing a hydroxyl group at C-1) showed that the divalent metal ion was in close proximity (2.5 Å) to the C-1 hydroxyl group of the substrate analog (Protein Data Bank file 1XUZ). This indi-   cates that in the Michaelis complex the Mn 2ϩ would be in a favorable position for activation of the aldehyde (15). Mechanistic Studies on Pseudaminic Acid Synthase-To probe the mechanism of pseudaminic acid synthase, [2-18 O]PEP was synthesized with a 54% incorporation of the label (28,29). The location of the 18 O label was determined using 31 P NMR spectroscopy because the substitution of 16 (Fig. 7,  upper panel). After addition of NeuB3 and incubation for 5 min, two new peaks appear corresponding to inorganic phosphate (0 ppm) and 18 O-labeled inorganic phosphate (Ϫ0.02 ppm) (Fig. 7, lower panel). The ratio of 16 O to 18 O in the inorganic phosphate produced was the same as that of the starting material indicating that the label was fully retained and that C-O bond cleavage was occurring during catalysis.
PEP-condensing enzymes that catalyze C-O bond cleavage processes must inherently proceed through a tetrahedral intermediate. Therefore the proposed catalytic mechanism of pseudaminic acid synthase is the attack of the C-3 of PEP to the aldehyde of the open chain form of 6-deoxy-AltdiNAc and an attack of water to the C-2 of PEP to form a tetrahedral intermediate (Fig. 2A) Fig. 2A) that is subsequently attacked by water to form the tetrahedral intermediate. The most convincing evidence for the oxocarbenium ion intermediate comes from the work done on 2-keto-3-deoxy-D-mannooctulosonate-8-phosphate synthase where a cationic mimic of the oxocarbenium ion intermediate was found to be a potent inhibitor (37)(38)(39).
Conclusion-This is the first identification and kinetic characterization of NeuB3 from C. jejuni as a metal-dependent pseudaminic acid synthase that catalyzes the condensation of PEP with 6-deoxy-Altdi-NAc. The characterization of the enzymatically produced pseudaminic acid verifies that the stereochemical assignment of pseudaminic acid is correct as the L-glycero-L-manno configuration. A mechanistic 18 O labeling study shows that catalysis proceeds through a C-O bond cleavage mechanism similar to other PEP condensing enzymes.