Mechanism of Type 3 Capsular Polysaccharide Synthesis inStreptococcus pneumoniae

• Published in: The Journal of biological chemistry 2000-02, Vol.275 (6), p.3907-3914
• Main Authors: Cartee, Robert T., Forsee, W.Thomas, Schutzbach, John S., Yother, Janet
• Format: Article
• Language: English
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.275.6.3907

The glycosidic linkages of the type 3 capsular polysaccharide of Streptococcus pneumoniae([3)-β-d-GlcUA-(1→4)-β-d-Glc-(1→]n) are formed by the membrane-associated type 3 synthase (Cps3S), which is capable of synthesizing polymer from UDP sugar precursors. Using membrane preparations of S. pneumoniae in an in vitro assay, we observed type 3 synthase activity in the presence of either Mn2+ or Mg2+ with maximal levels seen with 10–20 mm Mn2+. High molecular weight polymer synthesized in the assay was composed of Glc and glucuronic acid and could be degraded to a low molecular weight product by a type 3-specific depolymerase from Bacillus circulans. Additionally, the polymer bound specifically to an affinity column made with a type 3 polysaccharide-specific monoclonal antibody. The polysaccharide was rapidly synthesized from smaller chains and remained associated with the enzyme-containing membrane fraction throughout its synthesis, indicating a processive mechanism of synthesis. Release of the polysaccharide was observed, however, when the level of one of the substrates became limiting. Finally, addition of sugars to the growing type 3 polysaccharide was shown to occur at the nonreducing end of the polysaccharide chain.