Biochemical Characterization of WbpA, a UDP-N-acetyl-d-glucosamine 6-Dehydrogenase Involved in O-antigen Biosynthesis in Pseudomonas aeruginosa PAO1

Miller, Wayne L.; Wenzel, Cory Q.; Daniels, Craig; Larocque, Suzon; Brisson, Jean‐Robert; Lam, Joseph S.

doi:10.1074/jbc.m404749200

Cited by 44 publications

(58 citation statements)

References 50 publications

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“…The K m values determined for UDP-GlcNAc (2) and NAD + , respectively, are comparable to those reported for WbpA (K m of 94 μM for 2 and 220 μM for NAD + ). 16 In contrast, the k cat value for TviB reaction was found to be nearly 6-fold lower than that determined for WbpA (15.5 versus 86 min −1 ). It is possible that TviBcatalyzed dehydrogenation is the rate-limiting step in the Vi antigen biosynthesis.…”

Section: Discussionmentioning

confidence: 62%

Vi Antigen Biosynthesis inSalmonella typhi: Characterization of UDP-N-acetylglucosamine C-6 Dehydrogenase (TviB) and UDP-N-acetylglucosaminuronic Acid C-4 Epimerase (TviC)

et al. 2006

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Vi antigen, the virulence factor of Salmonella typhi, has been used clinically as a molecular vaccine. TviB and TviC are two enzymes involved in the formation of Vi antigen, a linear polymer consisting of α-1,4-linked N-acetylgalactosaminuronate. Protein sequence analysis suggests that TviB is a dehydrogenase and TviC is an epimerase. Both enzymes are expected to be NAD + dependent. In order to verify their functions, TviB and TviC were cloned, expressed in Escherichia coli, and characterized. The C-terminal His 6 -tagged TviB protein, purified from soluble cell fractions in the presence of 10 mM DTT, shows UDP-N-acetylglucosamine 6-dehydrogenase activity, and is capable of catalyzing the conversion of UDP-N-acetylglucosamine (UDP-GlcNAc) to UDP-Nacetylglucosaminuronic acid (UDP-GlcNAcA) with a k cat value of 15.5 ± 1.0 min −1 . The K m values of TviB for UDP-GlcNAc and NAD + are 77 ± 9 μM and 276 ± 52 μM, respectively. TviC, purified as C-terminal hexahistidine-tagged protein, shows UDP-GlcNAcA 4-epimerase and UDP-Nacetylgalactosamine (UDP-GalNAc) 4-epimerase activities. The K m values of TviC for UDPGlcNAcA and UDP-N-acetylgalactosaminuronic acid (UDP-GalNAcA) are 20 ± 1 μM and 42 ± 2 μM, respectively. The k cat value for the conversion of UDP-GlcNAcA to UDP-GalNAcA is 56.8 ± 0.5 min −1 , while that for the reverse reaction is 39.1 ± 0.6 min −1 . These results show that the biosynthesis of Vi antigen is initiated by the TviB-catalyzed oxidation of UDP-GlcNAc to UDPGalNAc, followed by the TviC-catalyzed epimerization at C-4 to form UDP-GalNAcA, which serves as the building block for the formation of Vi polymer. These results set the stage for future in vitro biosynthesis of Vi antigen. These enzymes may also be drug targets to inhibit Vi antigen production.Typhoid fever (TF), caused by Salmonella typhi and Salmonella paratyphi C, is a serious public health problem in many developing countries. 1 These Salmonella strains, similar to many pathogenic bacteria implicated in severe invasive infections of humans, such as Neisseria meningitides, which causes meningitis, Streptococcus pneumoniae, which causes pneumonia, and Haemophilus influenzae type b, which causes septicaemia, are encapsulated by polysaccharides. The capsules in S. typhi and S. praratyhi are linear homopolymers made up of α-1,4-linked N-acetylgalactosaminuronate (GalNAcA), with 60-70% of the monomeric units O-acetylated at the C-3 position. 2,3 This capsular polysaccharide, commonly referred as Vi antigen (1), has a molecular mass typically over 200 kDa. It is a virulence antigen which enhances S. typhi virulence in mice and induces immune response in rabbits. 3,4 Purified Vi † This work was supported in part by a National Institutes of Health Grant (GM35906 Sequence analysis showed that protein (TviB) encoded by the tviB gene belongs to the nucleotidyldiphosphohexose dehydrogenase superfamily. 12,13,14 TviB exhibits high amino acid sequence identity (65% identity and 83% similarity) with WbpO, which is an UDPGalNAc 6-dehydrogenase from Pseu...

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Section: Discussionmentioning

confidence: 62%

Vi Antigen Biosynthesis inSalmonella typhi: Characterization of UDP-N-acetylglucosamine C-6 Dehydrogenase (TviB) and UDP-N-acetylglucosaminuronic Acid C-4 Epimerase (TviC)

et al. 2006

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“…This analysis showed the MMP0352 enzyme efficiently catalyzed UDP-GlcNAc oxidation with apparent K m and turnover values comparable to parameters reported for the UDP-GlcNAc 6″-dehydrogenases from Pseudomonas aeruginosa [22] and Salmonella typhi [20] (Table 1). …”

Section: Oxidoreductase Activity Of Mmp0352mentioning

confidence: 54%

“…Table 1 Apparent kinetic parameters for the UDP-GlcNAc oxidoreductases a c The parameters for S. typhi TviB UDP-GlcNAc 6″-dehydrogenase were reported by Zhang et al [20]. d The K 0.5 values and turnover number for P. aeruginosa WbpA UDP-GlcNAc 6″-dehydrogenase were reported by Miller et al [22]. e N.D., not determined.…”

Section: Potential Applications and Enhancementsmentioning

confidence: 99%

Enzymatic analysis of uridine diphosphate N-acetyl-d -glucosamine

Namboori

Graham

2008

Analytical Biochemistry

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The Methanococcus maripaludis MMP0352 protein belongs to an oxidoreductase family that has been proposed to catalyze the NAD + -dependent oxidation of the 3″-position of uridine diphosphate N-acetyl-D-glucosamine (UDP-GlcNAc), forming a 3-hexulose sugar nucleotide. The heterologously expressed MMP0352 protein was purified and shown to efficiently catalyze UDP-GlcNAc oxidation forming one NADH equivalent. This enzyme was used to develop a fixed endpoint fluorometric method to analyze UDP-GlcNAc. The enzyme is highly specific for this acetamido sugar nucleotide, and the procedure had a detection limit of 0.2 μM UDP-GlcNAc in a 1-ml sample. Using the method of standard addition, UDP-GlcNAc concentrations were measured in deproteinized extracts of Escherichia coli, Saccharomyces cerevisiae and HeLa carcinoma cells. Equivalent concentrations were determined by both enzymatic and chromatographic analyses, validating this method. This procedure can be adapted for the high-throughput analysis of changes in cellular UDP-GlcNAc concentrations during time series experiments or inhibitor screens.

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“…Cells were harvested by centrifugation (Sorvall Evolution RC, Mandel Scientific, Guelph, Ontario, Canada) at 5,000 ϫ g for 15 min at 4°C, and pellets were stored at Ϫ20°C. WbpP and WbpA O5 were expressed as described previously (19,26). WbpA O5 from P. aeruginosa PAO1 (serotype O5) has been shown to be a UDP-D-GlcNAc 6-dehydrogenase (26).…”

Section: Materials-udp-d-glcnac Udp-d-galnac Nadmentioning

confidence: 99%

“…Capillary electrophoresis (CE) analysis was performed using a P/ACE MDQ glycoprotein system with UV detection (Beckman Coulter, Inc., Fullerton, CA) as described previously (26). …”

Section: Materials-udp-d-glcnac Udp-d-galnac Nadmentioning

confidence: 99%

Flagellin Glycosylation in Pseudomonas aeruginosa PAK Requires the O-antigen Biosynthesis Enzyme WbpO

Miller

Matewish

McNally

et al. 2008

Journal of Biological Chemistry

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Pseudomonas aeruginosa PAK (serotype O6) produces a single polar, glycosylated flagellum composed of a-type flagellin. To determine whether or not flagellin glycosylation in this serotype requires O-antigen genes, flagellin was isolated from the wild type, three O-antigen-deficient mutants wbpL, wbpO, and wbpP, and a wbpO mutant complemented with a plasmid containing a wild-type copy of wbpO. Flagellin from the wbpO mutant was smaller (42 kDa) than that of the wild type (45 kDa), or other mutants strains, and exhibited an altered isoelectric point (pI 4.8) when compared with PAK flagellin (pI 4.6). These differences were because of the truncation of the glycan moiety in the wbpO-flagellin. Thus, flagellin glycosylation in P. aeruginosa PAK apparently requires a functional WbpO but not WbpP. Because WbpP was previously proposed to catalyze a metabolic step in the biosynthesis of B-band O-antigen that precedes the action of WbpO, these results prompted us to reevaluate the two-step pathway catalyzed by WbpO and WbpP. Results from WbpO-WbpP-coupled enzymatic assays showed that either WbpO or WbpP is capable of initiating the two-step pathway; however, the kinetic parameters favored the WbpO reaction to occur first, converting UDP-N-acetyl-D-glucosamine to UDP-N-acetyl-D-glucuronic acid prior to the conversion to UDP-N-acetyl-D-galacturonic acid by WbpP. This is the first report to show that a C4 epimerase could utilize UDP-N-acetylhexuronic acid as a substrate.

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Biochemical Characterization of WbpA, a UDP-N-acetyl-d-glucosamine 6-Dehydrogenase Involved in O-antigen Biosynthesis in Pseudomonas aeruginosa PAO1

Cited by 44 publications

References 50 publications

Vi Antigen Biosynthesis inSalmonella typhi: Characterization of UDP-N-acetylglucosamine C-6 Dehydrogenase (TviB) and UDP-N-acetylglucosaminuronic Acid C-4 Epimerase (TviC)

Vi Antigen Biosynthesis inSalmonella typhi: Characterization of UDP-N-acetylglucosamine C-6 Dehydrogenase (TviB) and UDP-N-acetylglucosaminuronic Acid C-4 Epimerase (TviC)

Enzymatic analysis of uridine diphosphate N-acetyl-d -glucosamine

Flagellin Glycosylation in Pseudomonas aeruginosa PAK Requires the O-antigen Biosynthesis Enzyme WbpO

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