Identification and Functional Characterization of the Polymerizing Glycosyltransferase Required for the Transfer of d-Ribose to the d-GalNAc Moiety of the Capsular Polysaccharide of .

is the leading cause of food poisoning in the United States. The exterior surface of this bacterium is coated with a capsular polysaccharide (CPS) that helps protect the organism from the host immune system. In the HS:2 serotype of strain NCTC 11168, the minimal repeating trisaccharide consist of d-ribose, -acetyl-d-galactosamine (GalNAc) and the serinol amide of d-glucuronic acid. Here we demonstrate that the C-terminal domain of Cj1432 (residues 574-914) is responsible for the transfer of d-ribose-5-P from phosphoribosyl pyrophosphate (PRPP) to C5 of the d-GalNAc moiety of the growing polysaccharide chain. In the next step the middle domain of Cj1432 (residues 357-573) catalyzes the hydrolysis of phosphate from this product. The N-terminal domain of Cj1432 (residues 1-356) catalyzes the transfer of d-GlcA from UDP-d-GlcA to C2 of the d-ribose moiety and thus Cj1432 catalyzes three consecutive reactions during the biosynthesis of the capsular polysaccharide of . We have previously shown that the remaining three reactions required for the polymerization of the CPS are catalyzed by the bifunctional enzyme Cj1438 and Cj1435. We have now demonstrated that the minimal repeating trisaccharide of the CPS of NCTC 11168 requires six enzyme-catalyzed reactions with six intermediate structures. This accomplishment will now enable the large-scale cell-free enzyme-catalyzed synthesis of well-defined oligomers of the CPS that can potentially be used in the production of glycoconjugate vaccines for the prevention of infections by .