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• W2004677095 abstract "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 kcat of 0.65 ± 0.01 s–1, KmPEP of 6.5 ± 0.4 μm, and Km6-deoxy-AltdiNAc of 9.5 ± 0.7 μm. 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 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 kcat of 0.65 ± 0.01 s–1, KmPEP of 6.5 ± 0.4 μm, and Km6-deoxy-AltdiNAc of 9.5 ± 0.7 μm. 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 (1Black R.E. Levine M.M. Clements M.L. Hughes T.P. Blaser M.J. J. Infect. Dis. 1988; 157: 472-479Crossref PubMed Scopus (832) Google Scholar, 2Butzler J.P. Skirrow M.B. Clin. Gastroenterol. 1979; 8: 737-765Crossref PubMed Google Scholar), and H. pylori causes duodenal ulcers (3Cover T.L. Blaser M.J. Annu. Rev. Med. 1992; 43: 135-145Crossref PubMed Scopus (192) Google Scholar). 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) (4Thibault P. Logan S.M. Kelly J.F. Brisson J.R. Ewing C.P. Trust T.J. Guerry P. J. Biol. Chem. 2001; 276: 34862-34870Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar, 5Logan S.M. Kelly J.F. Thibault P. Ewing C.P. Guerry P. Mol. Microbiol. 2002; 46: 587-597Crossref PubMed Scopus (120) Google Scholar, 6Schirm M. Soo E.C. Aubry A.J. Austin J. Thibault P. Logan S.M. Mol. Microbiol. 2003; 48: 1579-1592Crossref PubMed Scopus (221) Google Scholar, 7Szymanski C.M. Wren B.W. Nat. Rev. Microbiol. 2005; 3: 225-237Crossref PubMed Scopus (333) Google Scholar). The production of pseudaminic acid is important for the development of flagella in these organisms as evidenced by aflagellate C. jejuni (8Linton D. Karlyshev A.V. Hitchen P.G. Morris H.R. Dell A. Gregson N.A. Wren B.W. Mol. Microbiol. 2000; 35: 1120-1134Crossref PubMed Scopus (120) Google Scholar) and H. pylori (6Schirm M. Soo E.C. Aubry A.J. Austin J. Thibault P. Logan S.M. Mol. Microbiol. 2003; 48: 1579-1592Crossref PubMed Scopus (221) Google Scholar) 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 (1Black R.E. Levine M.M. Clements M.L. Hughes T.P. Blaser M.J. J. Infect. Dis. 1988; 157: 472-479Crossref PubMed Scopus (832) Google Scholar, 9Morooka T. Umeda A. Amako K. J. Gen. Microbiol. 1985; 131: 1973-1980PubMed Google Scholar, 10Caldwell M.B. Guerry P. Lee E.C. Burans J.P. Walker R.I. Infect. Immun. 1985; 50: 941-943Crossref PubMed Google Scholar, 11Josenhans C. Labigne A. Suerbaum S. J. Bacteriol. 1995; 177: 3010-3020Crossref PubMed Google Scholar). 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 (12Blacklow R.S. Warren L. J. Biol. Chem. 1962; 237: 3520-3526Abstract Full Text PDF PubMed Google Scholar, 13Holbein B.E. Basson L. J. Bacteriol. 1983; : 728-736PubMed Google Scholar, 14Angata T. Varki A. Chem. Rev. 2002; 102: 439-469Crossref PubMed Scopus (1023) Google Scholar). Recent studies have reported on both the structure of NeuB and its mechanism of action (15Gunawan J. Simard D. Gilbert M. Lovering A.L. Wakarchuk W.W. Tanner M.E. Strynadka N.C.J. J. Biol. Chem. 2005; 280: 3555-3563Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 16Sundaram A.K. Pitts L. Muhammad K. Wu J. Betenbaugh M. Woodard R.W. Vann W.F. Biochem. J. 2004; 383: 83-89Crossref PubMed Scopus (29) Google Scholar). Catalysis was demonstrated to proceed through an overall C-O bond cleavage process where the si-face of phosphoenolpyruvate (PEP) 2The abbreviations used are:PEPphosphoenolpyruvate6-deoxy-AltdiNAc2,4-diacetamido-2,4,6-trideoxy-l-altroseNeuNAcN-acetylneuraminic acidManNAcN-acetyl-d-mannosamineESI-MSelectrospray ionization-mass spectrometryMES4-morpholineethanesulfonic acid 2The abbreviations used are:PEPphosphoenolpyruvate6-deoxy-AltdiNAc2,4-diacetamido-2,4,6-trideoxy-l-altroseNeuNAcN-acetylneuraminic acidManNAcN-acetyl-d-mannosamineESI-MSelectrospray ionization-mass spectrometryMES4-morpholineethanesulfonic acid 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 (17Levin D.H. Racker E. J. Biol. Chem. 1959; 234: 2532-2539Abstract Full Text PDF PubMed Google Scholar, 18Shulami S. Furdui C. Adir N. Shoham Y. Anderson K.S. Baasov T. J. Biol. Chem. 2004; 279: 45110-45120Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar), and 2-keto-3-deoxy-d-arabino-heptulosonate-7-phosphate synthase, which catalyzes the condensation of PEP and d-erythrose-4-phosphate (19DeLeo A.B. Sprinson D.B. Biochem. Biophys. Res. Commun. 1968; 32: 873-877Crossref PubMed Scopus (43) Google Scholar, 20Furdui C. Zhou L. Woodard R.W. Anderson K.S. J. Biol. Chem. 2004; 279: 45618-45625Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). A key mechanistic experiment performed with all three of these synthases was the use of [2-18O]PEP as a substrate during enzymatic incubations (see labeled atoms in Fig. 2). In each case, catalysis resulted in the production of 18O-labeled inorganic phosphate indicating that a C-O bond cleavage process was occurring (15Gunawan J. Simard D. Gilbert M. Lovering A.L. Wakarchuk W.W. Tanner M.E. Strynadka N.C.J. J. Biol. Chem. 2005; 280: 3555-3563Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 19DeLeo A.B. Sprinson D.B. Biochem. Biophys. Res. Commun. 1968; 32: 873-877Crossref PubMed Scopus (43) Google Scholar, 21Hedstrom L. Abeles R. Biochem. Biophys. Res. Commun. 1988; 157: 816-820Crossref PubMed Scopus (82) Google Scholar).FIGURE 2Potential mechanisms for the reaction catalyzed by pseudaminic acid synthase (NeuB3) and sialic acid synthase (NeuB). A, a C-O bond cleavage mechanism. B, a P-O bond cleavage mechanism. 18O isotopic labels are included to highlight the fate of the bridging phosphate oxygen of PEP during catalysis.View Large Image Figure ViewerDownload Hi-res image Download (PPT)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) (8Linton D. Karlyshev A.V. Hitchen P.G. Morris H.R. Dell A. Gregson N.A. Wren B.W. Mol. Microbiol. 2000; 35: 1120-1134Crossref PubMed Scopus (120) Google Scholar, 16Sundaram A.K. Pitts L. Muhammad K. Wu J. Betenbaugh M. Woodard R.W. Vann W.F. Biochem. J. 2004; 383: 83-89Crossref PubMed Scopus (29) Google Scholar). 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 (8Linton D. Karlyshev A.V. Hitchen P.G. Morris H.R. Dell A. Gregson N.A. Wren B.W. Mol. Microbiol. 2000; 35: 1120-1134Crossref PubMed Scopus (120) Google Scholar). 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 aldehyde of the open chain form of 6-deoxy-AltdiNAc producing the open chain form of pseudaminic acid. Precedence for this mechanism comes from pyruvate kinase (22Seeholzer S.H. Jaworowski A. Rose I.A. Biochemistry. 1991; 30: 727-732Crossref PubMed Scopus (22) Google Scholar) and PEP carboxykinase (23Matte A. Tari L.W. Goldie H. Delbaere L.T. J. Biol. Chem. 1997; 272: 8105-8108Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar) where catalysis occurs through formation of the enolate anion of pyruvate via nucleophilic attack at the phosphate group of PEP. In this article we use [2-18O]PEP to show that, like sialic acid synthase, pseudaminic acid synthase also employs a C-O bond cleavage mechanism.EXPERIMENTAL PROCEDURESMaterials and General Methods—Purine nucleoside phosphorylase and phosphoenolpyruvate were purchased from Sigma. 18O-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 (24Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (213377) Google Scholar) 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 μg) 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) (8Linton D. Karlyshev A.V. Hitchen P.G. Morris H.R. Dell A. Gregson N.A. Wren B.W. Mol. Microbiol. 2000; 35: 1120-1134Crossref PubMed Scopus (120) Google Scholar). The primers used were: 5′-CCCCCCCATATGCAAATAGGAAATTTTAAC-3′ (forward sequence) and 5′-CCCCCCGTCGACTCATCATTGGAAATCTCCTTGTTTAAAG-3′ (reverse sequence). The gene was then cloned into the expression plasmid pCWori+ as an NdeI-SalI fragment, and the construct was maintained in Escherichia coli AD202, which was used for enzyme production (25Wakarchuk W.W. Campbell R.L. Sung W.L. Davoodi J. Yaguchi M. Protein Sci. 1994; 3: 467-475Crossref PubMed Scopus (283) Google Scholar). Cells were grown at 37 °C in 500 ml of Luria-Bertani (LB) medium supplemented with 100 μgml–1 ampicillin. Overexpression of NeuB3 was induced through the addition of 0.5 mm isopropyl β-d-thiogalactopyranoside at an A600 of 0.60, with growth at 37 °C for 6 h. Cells were harvested through centrifugation at 5,000 × 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 × g for 1.5 h, passed through a 0.45-μm filter, and loaded directly onto a 5-ml Hi-trap™ Q-Sepharose column (Amersham Biosciences) pre-equilibrated with 20 mm Tris-HCl buffer (pH 7.0). NeuB3 was eluted with a linear gradient of 0to1 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 >90% 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 (26Flowers H.M. Levy A. Sharon N. Carbohydr. Res. 1967; : 189-195Crossref Scopus (50) Google Scholar, 27Liav A.L. Sharon N. Carbohydr. Res. 1973; 30: 109-126Crossref PubMed Scopus (16) Google Scholar). A solution of Tris-DCl buffer prepared in D2O (700 μl, 10 mm, pD 7.4) containing 6-deoxy-AltdiNAc (10 mm) and PEP (20 mm) was placed in an NMR tube. Initial 1H and proton-decoupled 31P 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 MgCl2 to a total volume of 1 ml. After incubation of the reaction mixture for 5 min at 25 °C, Chelex-100 resin (∼20 mg) was added and 1H and 31P NMR spectra were retaken.C-O Versus P-O Bond Cleavage—[2-18O]PEP disodium salt was prepared according to literature reported procedures (28Gore M.P. Nanjappan P. Hoops G.C. Woodard R.W. J. Org. Chem. 1990; 55: 758-760Crossref Scopus (9) Google Scholar, 29Bartlett P.A. Chouinard P.M. J. Org. Chem. 1983; 48: 3854-3855Crossref Scopus (23) Google Scholar). The extent of 18O incorporation at the C-2 position was determined to be 54% by mass spectrometry: ESI-MS (MeOH) m/z 169 (M – H+, 18O, 100), 167 (M – H+, 16O, 85). 31P NMR spectroscopy was used to determine that the position of the label was at the C-2 position: 31P NMR δ –2.999 (s, P-16O, PEP), –3.018 (s, P-18O, PEP).A solution of Tris-DCl buffer prepared in D2O (700 μl, 10 mm, pD 7.4) containing 6-deoxy-AltdiNAc (10 mm) and 20 mm [2-18O]PEP was placed in an NMR tube, and Chelex-100 resin (∼20 mg previously washed with D2O) was added. An initial proton-decoupled 31P 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 μs. 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 MgCl2. The reaction was incubated for 5 min at 25 °C and Chelex-100 resin (∼20 mg previously washed with D2O) was added. After a 1-h time period to allow complete complexation of the metals, another proton-decoupled 31P NMR spectrum was acquired with the same parameters: 31P NMR δ 0.0 (s, Pi-16O), –0.022 (s, Pi-18O), –3.010 (s, P-16O, PEP), –3.029 (s, P-18O, PEP).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 1H, 13C, two-dimensional COSY, HMQC, and one-dimensional TOCSY NMR spectroscopy (in 10 mm deuterated phosphate buffer pD 7.4), and negative ESI-MS mass spectrometry. 1H (D2O) δ 1.0 (d, 3 H, J8,9 6.5 Hz, H-9), 1.95 (dd, 1 H, J3ax,4 12.2 Hz, J3ax,3eq 13.3 Hz, H-3ax), 1.99 (dd, 1 H, J3eq3ax 13.3, J3eq,4 4.6 Hz, H-3eq), 1.86 (s, 3 H, CH3), 1.89 (s, 3 H, CH3), 3.91 (dd, 1 H, J5,6 1.7 Hz, J6,7 10.3 Hz, H-6), 4.01 (dq, 1 H, J7,8 3.7 Hz, J8,9 6.5 Hz, H-8), 4.03 (dd, 1 H, J6,7 10.3, J7,8 3.7, H-7), 4.05 (ddd, 1 H, J3eq,4 4.6, J3ax,4 12.2, J4,5 5.5, H-4), 4.13 (dd, 1 H, J4,5 5.5, J5,6 1.7, H-5). 13C NMR (D2O) δ 15.2 (C-9), 21.8 (CH3-NAc), 22.0 (CH3-NAc), 34.8 (C-3), 48.8 (C-5), 52.9 (C-7), 65.2 (C-4), 66.8 (C-8), 69.9 (C-6), 96.4 (C-2), 173.7, 174.6, 176.3 (C = O). -ve ESI-MS (H2O) m/z 333 (M – H+), m/z 315 (M – H2O).Enzyme Kinetics as Determined by a Continuous Coupled Assay—Enzyme kinetics were measured by a continuous coupled phosphate assay (30Webb M.R. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4884-4887Crossref PubMed Scopus (484) Google Scholar). 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 μm), purine nucleoside phosphorylase (5 units buffer exchanged twice into 100 mm Tris-HCl buffer pH 7.0), and MnCl2 (10 mm) was thermally equilibrated for 5 min at 37 °C. The enzymatic reaction was initiated by the addition of NeuB3 (3.2 μg) for a total assay volume of 500 μl and the enzymatic rate was calculated from the observed increase of absorption at A360 (using ϵ = 11,000 m–1 cm–1). The Km 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 (31Erithacus Software, Ltd.GraFit. Erithacus Software, Ltd., Staines, UK2003Google Scholar).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 (Co2+). Controls included adding 10 mm EDTA or no additives (enzyme as isolated) to the kinetic assay.RESULTS AND DISCUSSIONOverexpression 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 ± 30 using electrospray ionization-mass spectrometry (38,647 predicted).FIGURE 3SDS-PAGE gel showing the expression and purification of recombinant pseudaminic acid synthase (NeuB3) using ion-exchange chromatography. Lane 1 contains molecular mass standards of 66 and 29 kDa, lane 2 shows crude cell extract, lane 3 shows the purified pseudaminic acid synthase.View Large Image Figure ViewerDownload Hi-res image Download (PPT)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 (26Flowers H.M. Levy A. Sharon N. Carbohydr. Res. 1967; : 189-195Crossref Scopus (50) Google Scholar, 27Liav A.L. Sharon N. Carbohydr. Res. 1973; 30: 109-126Crossref PubMed Scopus (16) Google Scholar). 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 Mn2+ and NeuB3, and the reaction was monitored by 1H and 31P NMR spectroscopy. Before the addition of NeuB3, the 31P NMR spectrum shows a single phosphorus peak corresponding to the phosphate group of PEP at –3.01 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.FIGURE 431P NMR spectra monitoring the conversion of PEP to phosphate by NeuB3. The upper panel shows the spectrum before the addition of NeuB3. The lower panel shows the spectrum taken after 5 min of incubation.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The time course as observed by 1H NMR spectroscopy shows the conversion of 6-deoxy-AltdiNAc to pseudaminic acid. The initial 1H NMR spectra shows that 6-deoxy-AltdiNAc exists as a 3:1 ratio of the β to α anomer in solution (Fig. 5, upper panel). The anomeric ratio is apparent from integration of the two acetamido signals of 6-deoxy-AltdiNAc (β-anomer at 1.88 and 1.93 ppm, and α-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 α-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 (J3ax,3eq 13.3 Hz) and strong coupling to the H-4 proton (J3ax,4 12.2 Hz). The large J3ax,4 coupling can only arise if H-3ax and H-4 have a trans-diaxial relationship. This indicates that PEP attacks the si-face of the aldehyde of 6-deoxy-AltdiNAc to generate an (S)-configuration at C-4.FIGURE 51H NMR spectra monitoring the conversion of 6-deoxy-AltdiNAc into pseudaminic acid by NeuB3. The upper panel shows the spectrum taken before the addition of NeuB3. The lower panel shows the spectrum taken after 5 min of incubation.View Large Image Figure ViewerDownload Hi-res image Download (PPT)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 indicates that the stereochemical assignment of pseudaminic acid is correct as the previously assigned l-glycero-l-manno isomer (4Thibault P. Logan S.M. Kelly J.F. Brisson J.R. Ewing C.P. Trust T.J. Guerry P. J. Biol. Chem. 2001; 276: 34862-34870Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar, 32Knirel Y.A. Vinogradov E.V. Lvov V.L. Kocharova N.A. Shashkov A.S. Dmitriev B.A. Kochetkov N.K. Carbohydr. Res. 1984; 133: C5-C8Crossref PubMed Scopus (65) Google Scholar, 33Knirel Y.A. Kocharova N.A. Shashkov A.S. Dmitriev B.A. Kochetkov N.K. Stanislavsky E.S. Mashilova G.M. Eur. J. Biochem. 1987; 163: 639-652Crossref PubMed Scopus (69) Google Scholar, 34Kenne L. Lindberg B. Schweda E. Gustafsson B. Holme T. Carbohydr. Res. 1988; : 285-294Crossref Scopus (37) Google Scholar, 35Kondakova A.N. Perepelov A.V. Bartodziejska B. Shashkov A.S. Senchenkova S.N. Wykrota M. Knirel Y.A. Rozalski A. Carbohydr. Res. 2001; 333: 241-249Crossref PubMed Scopus (15) Google Scholar).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 – H+) and the oxonium ion at m/z 315 (M – H3O+). 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 (4Thibault P. Logan S.M. Kelly J.F. Brisson J.R. Ewing C.P. Trust T.J. Guerry P. J. Biol. Chem. 2001; 276: 34862-34870Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar, 32Knirel Y.A. Vinogradov E.V. Lvov V.L. Kocharova N.A. Shashkov A.S. Dmitriev B.A. Kochetkov N.K. Carbohydr. Res. 1984; 133: C5-C8Crossref PubMed Scopus (65) Google Scholar, 33Knirel Y.A. Kocharova N.A. Shashkov A.S. Dmitriev B.A. Kochetkov N.K. Stanislavsky E.S. Mashilova G.M. Eur. J. Biochem. 1987; 163: 639-652Crossref PubMed Scopus (69) Google Scholar, 34Kenne L. Lindberg B. Schweda E. Gustafsson B. Holme T. Carbohydr. Res. 1988; : 285-294Crossref Scopus (37) Google Scholar, 35Kondakova A.N. Perepelov A.V. Bartodziejska B. Shashkov A.S. Senchenkova S.N. Wykrota M. Knirel Y.A. Rozalski A. Carbohydr. Res. 2001; 333: 241-249Crossref PubMed Scopus (15) Google Scholar). 1H NMR spectroscopy was used to assign the relative configuration of the pyranose ring protons. The large coupling constants of J3a,4 (12.2 Hz) and small coupling constants of J4,5 (5.5 Hz) and J5,6 (1.7 Hz) indicates that H-4 is axial, H-5 is equatorial, and H-6 is axial (TABLE ONE). The large J6,7 of 10.3 Hz indicates that C-6 and C-7 have the erythro configuration as opposed to sialic acid, which has a small J6,7 of 1.2 Hz and the threo configuration (33Knirel Y.A. Kocharova N.A. Shashkov A.S. Dmitriev B.A. Kochetkov N.K. Stanislavsky E.S. Mashilova G.M. Eur. J. Biochem. 1987; 163:" @default.
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