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

Robert T Cartee,W Thomas Forsee,Janet Yother

doi:10.1074/jbc.m511124200

Robert T Cartee, W Thomas Forsee + Show 1 more

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https://doi.org/10.1074/jbc.m511124200

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Abstract

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.

Highlights

We previously suggested that these concave rate curves might be due to polysaccharide ejection from the carbohydrate acceptor binding site of the synthase [5], since the highest rates of ejection occur at low concentrations of UDP-GlcUA and high levels of UDP-Glc
Carbohydrate acceptor binding sites are thought to play an important role in the biosynthesis of numerous polysaccharides, but very little data is available on this part of the assembly process
Under most conditions the polysaccharide chain remains tightly associated with the enzyme, and virtually no dissociation occurs during various experimental manipulations, including centrifugation, freezing, and thawing, and incubation at room temperature

Summary

Introduction

Based on hydrophobic cluster analysis, sequence homology, and x-ray crystallographic data, a fairly detailed picture is emerging of the binding and catalytic interaction of some glycosyltransferases with their nucleotide diphosphate sugar donor (10 –12) This information has led to the development of a rather strong argument for the existence of only a single nucleotide-sugar binding site in polymerases, such as type 3 synthase, that require two distinct nucleotide sugar donors [11]. Enzymes similar to the type 3 synthase have been postulated to undergo a translocation step after the addition of every two sugars to avoid the necessity of rotating either the polymer or the synthase [2, 13] Previous work from this laboratory has shown that polysaccharide chains are ejected from the type 3 synthase when one UDP-sugar is present at high concentration and the other is limiting or absent, suggesting that an abortive translocation process might serve to terminate chain growth [5]. The current investigation indicates that the carbohydrate recognition site may form a complex with an approximate eight-sugar segment of the growing polysaccharide chain, and that this interaction may provide for the high degree of processivity of chain assembly as well as for controlling the ultimate size of the polymer

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