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

Abstract

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...

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

“…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). …”

“…The S. typhi TviB enzyme specifically catalyzed this reaction with kinetic parameters similar to those of the MMP0352 described here [20]. However, the TviB enzyme requires 10 mM DTT to prevent enzyme inactivation, probably due to oxidation of a catalytic cysteine thiol.…”

“…Deproteinized extracts were applied to a CarboPac PA1 column (250 by 4 mm, Dionex) with a guard column (4 by 50 mm) of the same material. The analytes were separated using an ammonium acetate gradient (0.6 ml min -1 ) and detected by a photodiode array [20]. The standard addition method was used to estimate UDP-GlcNAc concentrations from integrated peak areas.…”

“…However, the TviB enzyme requires 10 mM DTT to prevent enzyme inactivation, probably due to oxidation of a catalytic cysteine thiol. Therefore the TviB enzyme cannot be purified by a single affinity chromatographic procedure [20], and it may be susceptible to oxidation by components of the sample matrix. The MMP0352 enzyme is proposed to catalyze the NAD + -dependent oxidation of UDPGlcNAc at the 3″-position to produce the keto-sugar nucleotide UDP-2-acetamido-3-oxo-2,3-dideoxy-α-D-glucopyranose.…”

“…Part C shows a chromatogram after the standard addition of UDP-GlcNAc, indicating a concentration of 0.44 μmol g -1 dry mass. 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].…”