Characterization of the lipid linkage region and chain length of the cellubiuronic acid capsule of Streptococcus pneumoniae.
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.
Forsee
W Thomas
WT
Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA.
Cartee
Robert T
RT
Yother
Janet
J
D016667:Bacterial Capsules / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005965:Glucuronates / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D016695:Glycosyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008055:Lipids / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D011134:Polysaccharides / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D013296:Streptococcus pneumoniae / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D005965:Glucuronates; D008055:Lipids; D011134:Polysaccharides; D016695:Glycosyltransferases