Abstract
The processive reaction mechanisms of beta-glycosyl-polymerases are poorly understood. The cellubiuronan synthase of Streptococcus pneumoniae catalyzes the synthesis of the type 3 capsular polysaccharide through the alternate additions of beta-1,3-Glc and beta-1,4-GlcUA. The processive multistep reaction involves the sequential binding of two nucleotide sugar donors in coordination with the extension of a polysaccharide chain associated with the carbohydrate acceptor recognition site. Degradation analysis using cellubiuronan-specific depolymerase demonstrated that the oligosaccharide-lipid and polysaccharide-lipid products synthesized in vitro with recombinant cellubiuronan synthase had a similar oligosaccharyl-lipid at their reducing termini, providing definitive evidence for a precursor-product relationship and also confirming that growth occurred at the nonreducing end following initiation on phosphatidylglycerol. The presence of a lipid marker at the reducing end allowed the quantitative determination of cellubiuronic acid polysaccharide chain lengths. As the UDP-GlcUA concentration was increased from 1 to 11.5 mum, the level of synthase in the transitory processive state decreased, with the predominant oligosaccharide-lipid product containing 3 uronic acid residues, whereas the proportion of synthase in the fully processive state increased and the polysaccharide chain length increased from 320 to 6700 monosaccharide units. In conjunction with other kinetic data, these results suggest that the formation of a complex between a tetrauronosyl oligomer and the carbohydrate acceptor recognition site plays a central role in coordinating the repetitive interaction of the synthase with the nucleotide sugar donors and modulating the chain length of cellubiuronan polysaccharide.
Highlights
Ing GT-2A polymerases that are located in the plasma membrane with their active sites on the cytoplasmic face, and following chain initiation, the synthases are thought to be involved in the extrusion of the nascent chains to the external membrane face [3,4,5,6,7,8]
Studies of the synthase in S. pneumoniae and recombinant Escherichia coli membranes have shown that assembly of the polysaccharide involves two distinct kinetic phases: 1) a transitory processive state wherein the chain is thought to be initiated by the formation of an oligosaccharide-lipid that is loosely associated with the synthase, and 2) a fully processive state in which the polysaccharide is tightly bound to the carbohydrate substrate recognition site, except for a brief period during the translocation stage of each catalytic cycle [5, 12]
Depolymerase Hydrolysis of Cellubiuronan Polysaccharide Products—Cellubiuronic acid polysaccharide products labeled with [14C]GlcUA were subjected to hydrolysis with the cellubiuronan-specific depolymerase
Summary
Ing GT-2A polymerases that are located in the plasma membrane with their active sites on the cytoplasmic face, and following chain initiation, the synthases are thought to be involved in the extrusion of the nascent chains to the external membrane face [3,4,5,6,7,8]. There is relatively little information concerning the initiation process, the facilitation of chain extrusion, the mechanism of translocation, and the regulation of the final chain length during the assembly of these polymers Recent investigations in this laboratory have begun to unravel some of the details of both the early and later stages of the biosynthesis of cellubiuronan. The high degree of specificity of this depolymerase has provided a sensitive analytical tool to further characterize cellubiuronan oligosaccharide- and polysaccharide-lipids, and even more importantly, it has provided a means for determining the chain length of these polysaccharides Both in vivo [18] and in vitro studies [12] indicate that the processivity of cellubiuronan synthase is modulated by the concentration of UDP-GlcUA. The methodology described in this article has allowed a clear demonstration of the relationship between the polysaccharide size and the UDP-GlcUA substrate concentration and in turn has led to the development of a kinetic model [38], which provides both for UDP-sugar modulation of polysaccharide chain length and for the assembly by a single-site synthase of a polysaccharide composed of a repeating heterodisaccharide
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