Abstract
The chain length of Streptococcus pneumoniae type 3 capsular polysaccharide (cellubiuronic acid) is tightly regulated by the cellubiuronic acid synthase through an assembly process involving a catalytic motif that is potentially conserved over a wide range of related processive beta-glucan synthases. Cellubiuronic acid is initiated on a lipid and is composed of alternating beta-1,3-Glc and beta-1,4-glucuronic acid (GlcUA) linkages. The entire assembly process is carried out by a polypeptide synthase thought to contain a single active site, suggesting that the donor specificity is controlled by the terminal nonreducing sugar in the acceptor subsite. Shortly after initiation, the synthase undergoes an allosteric transition accompanied by the tight binding of the nascent chain via its nonreducing oligosaccharide terminal segment to the carbohydrate acceptor recognition site. The chain length of polysaccharide assembled by recombinant synthase in Escherichia coli membranes was determined by an ejection mechanism that appeared to be a reversal of the allosteric transition of the synthase from the transitory to the fully processive state. The rates of both ejection and transition were shown to be highly sensitive to the concentration of UDP-GlcUA. As the concentration of UDP-GlcUA was increased, both the rate of synthesis and the processive turnover time increased. The product of the processive turnover time and the rate of synthesis predicted a marked increase in polysaccharide chain size (from 50 to 1150 kDa) over a relatively narrow concentration range of 1-11.5 microm UDP-GlcUA. The kinetic model chain length predictions were in close agreement with chemically determined sizes of polysaccharides synthesized at the same UDP-sugar concentrations. The model indicates that translocation occurs following the addition of GlcUA to the chain terminus, whereas UDP-Glc drives chain termination when inadequate levels of UDP-GlcUA are present. In sum, type 3 synthase appears to modulate polysaccharide chain length by functioning as a concentration-dependent kinetic timing device.
Highlights
Sis of a wide variety of carbohydrate biopolymers has long been of interest
Raising the concentration of UDP-Glc enhanced the sigmoidal-like character of the polymer formation rate curves, indicative of a positive cooperativity (Fig. 2B). These results are quite similar to the effect of increasing temperature, which resulted in an enhancement in the concavity of the polymer formation rate curves that was coincident with a reduced transition of the cellubiuronan synthase to the fully processive state (13)
There is a correlation between the length of the polysaccharide chain synthesized by cellubiuronan synthase and the concentration of UDP-glucuronic acid (GlcUA) present in the polymerization reaction (35)
Summary
Sis of a wide variety of carbohydrate biopolymers has long been of interest. An early suggestion was that specific sugar modifications might serve as a chain termination signal in proteoglycan biosynthesis (4) or that chain length was determined by the ratio of synthase activity to primer concentration (5). Of particular relevance to the Streptococcus pneumoniae cellubiuronan, which grows by nonreducing end terminal glycosylation (8, 35), is the suggestion that chain length in heparin biosynthesis might be modulated by competition of the two nucleotide sugar substrates, UDP-Nacetylglucosamine and UDP-GlcUA, at the glycosyltransferase binding sites (9). Chain termination is modulated by a UDPsugar-activated ejection process that appears to be an abortive translocation of the polysaccharide that occurs when insufficient levels of the UDP sugar precursors are available to maintain the polymerization reaction (14). UDP-Glc-activated ejection of preformed polysaccharide was almost completely prevented by increasing the concentration of UDP-GlcUA to 10 M. We examined polysaccharide chain length modulation by recombinant synthase in E. coli membranes incubated with 1 mM UDP-Glc and low concentrations of UDP-GlcUA. Polysaccharide assembly has been evaluated under steady state reaction conditions, wherein chain initiation, extension, and ejection were occurring simultaneously
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