Mechanism of Type 3 Capsular Polysaccharide Synthesis inStreptococcus pneumoniae

Abstract

The glycosidic linkages of the type 3 capsular polysaccharide of Streptococcus pneumoniae ([3)-␤-D-GlcUA-(134)-␤-D-Glc-(13] 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.

Hyaluronan Molecular Weight Is Controlled by UDP-N-acetylglucosamine Concentration in Streptococcus zooepidemicus

“…In this study, we explored the commonly stated but unproven hypothesis that the molecular weight of polysaccharides produced by processive synthases depends on the availability of activated sugar substrates (23,43). We cloned and overexpressed the five genes of the has operon in S. zooepidemicus (Table 3), to manipulate the levels of UDP-GlcUA and UDP-GlcNAc.…”

“…Although changed culture conditions affect the physiochemical environment of the HA synthase, a more likely explanation is that molecular weight is affected by the availability of activated sugar substrates (UDP-GlcUA and UDP-GlcNAc) as well as the concentration of possible effector molecules, such as free UDP (21). Although such a mechanism has been suggested for several processive synthases (22,23), there has never been any direct evidence linking molecular weight to the concentration of a substrate.…”

Biosynthesis of Type 3 Capsular Polysaccharide inStreptococcus pneumoniae

“…Preparation and Assay of Type 3 Synthase-Membranes containing type 3 synthase were isolated from the encapsulated WU2 S. pneumoniae strain (16) as described previously (3). Membranes were stored at Ϫ80°C in a solution containing 100 mM Hepes (pH 8.0), 10% glycerol, and 10 mM sodium thioglycolate, at a protein concentration of 3 mg/ml.…”

“…The glucan backbone of type 3 polysaccharide is identical to that for hyaluronic acid; however, the overall structure differs in having the carboxyl groups on the 1-4-linked rather than the 1-3-linked glucosyl units, and there are no acetamido constituents. In vitro studies have shown that the biosynthesis of type 3 polysaccharide occurs by the alternate addition of Glc and GlcUA 1 from UDP-Glc and UDP-GlcUA (2), and work in our laboratory has recently demonstrated that growth of the polymer occurs at the nonreducing end (3). Genetic analysis has established that a single open reading frame (cps3S) encodes the protein that catalyzes the formation of the glycosidic linkages in the type 3 polysaccharide and that the predicted Cps3S protein has significant homology to a number of polysaccharide synthases which form polymers composed of ␤ (1-4)linked repeating disaccharide units (4).…”

Functional Characteristics and Catalytic Mechanisms of the Bacterial Hyaluronan Synthases

“…These findings suggest that, as for hyaluronan synthases, different types of cellulose synthases might exist among bacterial species. Chain elongation for the type 3 capsular polysaccharide of Streptococcus pneumoniae also occurs at the nonreducing end (41). Although synthesis of the type 3 polysaccharide does not involve polyprenol intermediates, other lipid intermediates appear to be involved in the early stages of polymerization (42).…”