| Type 3 capsule of Streptococcus pneumoniae is composed of the repeating disaccharide (GlcA-beta1,4-Glc-beta1,3)n. One of the genes essential for type 3 polysaccharide synthesis is cps3S, which encodes the type 3 polysaccharide synthase. This protein localizes to pneumococcal membranes and utilizes UDP sugar precursors to form both glycosidic linkages of the type 3 polysaccharide. In our initial work characterizing type 3 synthase activity, we established optimal conditions for synthase activity and determined that sugar addition occurred on the nonreducing end of the polysaccharide chain. The growing polysaccharide chain was shown to remain associated with the enzyme throughout polymerization, indicating a processive mechanism of synthesis. A portion of the polysaccharide, however, was released from the enzyme when the level of one of the substrates was depleted.; Further characterization of polysaccharide release showed that the process was actuated by the addition of a single nucleotide sugar substrate and was dependent on time, temperature, and the concentration of substrate. These data indicate that release is enzymatic and likely the result of the normal biosynthetic mechanism. Kinetic comparison between the release and the biosynthetic reactions suggest that there is positive and negative cooperative binding between the nucleotide sugar substrates. Incubation of membranes with a single substrate resulted in a loss of subsequent enzyme activity, suggesting that once the polysaccharide is released the type 3 synthase is not capable of reinitiating synthesis. Characterization of the type 3 synthase expressed in Escherichia coli, however, indicated that the enzyme was capable of reinitiating synthesis after releasing its nascent chain. These data suggest that additional factors may be involved in controlling initiation of type 3 polysaccharide synthesis in S. pneumoniae. |
