Mechanism of type 3 capsular polysaccharide synthesis in Streptococcus pneumoniae.
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 Mn(2+) or Mg(2+) with maximal levels seen with 10-20 mM Mn(2+). 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.
Cartee
R T
RT
Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA.
Forsee
W T
WT
Schutzbach
J S
JS
Yother
J
J
D016667:Bacterial Capsules / Q000096:biosynthesis*; D002240:Carbohydrate Sequence; D004789:Enzyme Activation / Q000187:drug effects; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D008969:Molecular Sequence Data; D011135:Polysaccharides, Bacterial / Q000096:biosynthesis*; D013296:Streptococcus pneumoniae / Q000201:enzymology / Q000378:metabolism*
D011135:Polysaccharides, Bacterial; D008345:Manganese; D008274:Magnesium