Vi antigen biosynthesis in Salmonella typhi:: Characterization of UDP-N-acetylglucosamine C-6 dehydrogenase (TviB) and UDP-N-acetylglucosaminuronic acid C-4 epimerase (TviC)

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 alpha-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-G1cNAc) to UDP-N-acetylglucosaminuronic acid (UDP-G1cNAcA) with a k(cat) value of 15.5 +/- 1.0 min(-1). The K-m values of TviB for UDP-G1cNAc and NAD+ are 77 +/- 9 mu M and 276 +/- 52 mu M, respectively. TviC, purified as C-terminal hexahistidine-tagged protein, shows UDP-GlcNAcA 4-epimerase and UDP-N-acetylgalactosamine (UDP-Ga1NAc) 4-epimerase activities. The K-m values of TviC for UDP-G1cNAcA and UDP-N-acetylgalactosaminuronic acid (UDP-Ga1NAcA) are 20 +/- 1 mu M and 42 +/- 2 mu M, respectively. The k(cat) value for the conversion of UDP-G1cNAcA to UDP-Ga1NAcA 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-G1cNAc to UDPGa1NAc, followed by the TviC-catalyzed epimerization at C-4 to form UDP-Ga1NAcA, 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.