Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 281, Issue 10, Pages 6283-6289Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M511124200
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- NIGMS NIH HHS [GM 53017] Funding Source: Medline
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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 similar to 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.
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