Role of the carbohydrate binding site of the Streptococcus pneumoniae capsular polysaccharide type 3 synthase in the transition from oligosaccharide to polysaccharide synthesis.
The type 3 synthase catalyzes the formation of the Streptococcus pneumoniae type 3 capsular polysaccharide [-3)-beta-D-GlcUA-(1, 4)-beta-D-Glc-(1-]n. Synthesis is comprised of two distinct catalytic phases separated by a transition step whereby an oligosaccharylphosphatidylglycerol primer becomes tightly bound to the carbohydrate acceptor recognition site of the synthase. Using the recombinant synthase in Escherichia coli membranes, we determined that a critical oligosaccharide length of approximately 8 monosaccharides was required for recognition of the growing chain by the synthase. Upon binding of the oligosaccharide-lipid to the carbohydrate recognition site, the polymerization reaction entered a highly processive phase to produce polymer of high molecular weight. The initial oligosaccharide-synthetic phase also appeared to be processive, the duration of which was enhanced by the concentration of UDP-GlcUA and diminished by an increase in temperature. The overall reaction approached a steady state equilibrium between the polymer- and oligosaccharide-forming phases that was shifted toward the former by higher UDP-GlcUA levels or lower temperatures and toward the latter by lower concentrations of UDP-GlcUA or higher temperatures. The transition step between the two enzymatic phases demonstrated cooperative kinetics, which is predicted to reflect a possible reorientation of the oligosaccharide-lipid in conjunction with the formation of a tight binding complex.
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 / Q000096:biosynthesis / Q000378:metabolism*; D001665:Binding Sites / Q000502:physiology; D016695:Glycosyltransferases / Q000737:chemistry* / Q000378:metabolism* / Q000502:physiology; D007700:Kinetics; D050356:Lipid Metabolism; D009844:Oligosaccharides / Q000096:biosynthesis / Q000378:metabolism* / Q000502:physiology; D011135:Polysaccharides, Bacterial / Q000096:biosynthesis / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D013296:Streptococcus pneumoniae / Q000201:enzymology*; D013696:Temperature; D014532:Uridine Diphosphate Glucose / Q000378:metabolism
D009844:Oligosaccharides; D011135:Polysaccharides, Bacterial; C413446:type 3 capsular polysaccharide, Streptococcus pneumoniae; D016695:Glycosyltransferases; C413445:type 3 capsular polysaccharide synthase; D014532:Uridine Diphosphate Glucose