Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 275, Issue 6, Pages 3907-3914Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.275.6.3907
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- NHLBI NIH HHS [T32HL07553] Funding Source: Medline
- NIGMS NIH HHS [GM53017] Funding Source: Medline
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The glycosidic linkages of the type 3 capsular polysaccharide of Streptococcus pneumoniae ([3)-beta-D-GlcUA-(1-->4)-beta-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.
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