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J. Biol. Chem., Vol. 281, Issue 10, 6283-6289, March 10, 2006

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Role of the Carbohydrate Binding Site of the Streptococcus pneumoniae Capsular Polysaccharide Type 3 Synthase in the Transition from Oligosaccharide to Polysaccharide Synthesis^*

From the Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama 35294

The type 3 synthase catalyzes the formation of the Streptococcus pneumoniae type 3 capsular polysaccharide [-3)--D-GlcUA-(1, 4)--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 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.

Received for publication, October 12, 2005 , and in revised form, January 12, 2006.

^* This work was supported by Public Health Service Grant GM 53017 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Dept. of Microbiology, 845 19th St. S., BBRB 661/12 University of Alabama at Birmingham, Birmingham, AL 35294. Tel.: 205-934-9531; Fax: 205-975-6715; E-mail: jyother{at}uab.edu.

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