Role of the carbohydrate binding site of the Streptococcus pneumoniae capsular polysaccharide type 3 synthase in the transition from oligosaccharide to polysaccharide synthesis

Academic Article

Abstract

  • The type 3 synthase catalyzes the formation of the Streptococcus pneumoniae type 3 capsular polysaccharide [-3)-β-D-GlcUA-(1, 4)-β-D-Glc-(1-] n. Synthesis is comprised of two distinct catalytic phases separated by a transition step whereby an oligosaccharyl-phosphatidylglycerol 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 ∼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. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.
  • Authors

    Published In

    Digital Object Identifier (doi)

    Author List

  • Forsee WT; Cartee RT; Yother J
  • Start Page

  • 6283
  • End Page

  • 6289
  • Volume

  • 281
  • Issue

  • 10