Expression of the Streptococcus pneumoniae type 3 synthase in Escherichia coli. Assembly of type 3 polysaccharide on a lipid primer.
Synthesis of the type 3 capsular polysaccharide of Streptococcus pneumoniae is catalyzed by the membrane-localized type 3 synthase, which utilizes UDP-Glc and UDP-GlcUA to form high molecular mass [3-beta-d-GlcUA-(1-->4)-beta-d-Glc-(1-->](n). Expression of the synthase in Escherichia coli resulted in synthesis of a 40-kDa protein that was reactive with antibody directed against the C terminus of the synthase and was the same size as the native enzyme. Membranes isolated from E. coli contained active synthase, as demonstrated by the ability to incorporate Glc and GlcUA into a high molecular mass polymer that could be degraded by type 3 polysaccharide-specific depolymerase. As in S. pneumoniae, the membrane-bound synthase from E. coli catalyzed a rapid release of enzyme-bound polysaccharide when incubated with either UDP-Glc or UDP-GlcUA alone. The recombinant enzyme expressed in E. coli was capable of releasing all of the polysaccharide from the enzyme, although the chains remained associated with the membrane. The recombinant enzyme was also able to reinitiate polysaccharide synthesis following polymer release by utilizing a lipid primer present in the membranes. At low concentrations of UDP-Glc and UDP-GlcUA (1 microm in the presence of Mg(2+) and 0.2 microm in Mn(2+)), novel glycolipids composed of repeating disaccharides with linkages consistent with type 3 polysaccharide were synthesized. As the concentration of the UDP-sugars was increased, there was a marked transition from glycolipid to polymer formation. At UDP-sugar concentrations of either 5 microm (with Mg(2+)) or 1.5 microm (with Mn(2+)), 80% of the incorporated sugar was in polymer form, and the size of the polymer increased dramatically as the concentration of UDP-sugars was increased. These results suggest a cooperative interaction between the UDP-precursor-binding site(s) and the nascent polysaccharide-binding site, resulting in a non-processive addition of sugars at the lower UDP-sugar concentrations and a processive reaction as the substrate concentrations increase.
Cartee
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RT
Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama 35294.
Forsee
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Jensen
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Yother
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D002462:Cell Membrane / Q000201:enzymology* / Q000378:metabolism; D002845:Chromatography; D002850:Chromatography, Gel; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005944:Glucosamine / Q000031:analogs & derivatives* / Q000378:metabolism; D006017:Glycolipids / Q000096:biosynthesis / Q000378:metabolism; D016695:Glycosyltransferases / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D010739:Phospholipase D / Q000378:metabolism; D011134:Polysaccharides / Q000096:biosynthesis / Q000378:metabolism; D011135:Polysaccharides, Bacterial / Q000096:biosynthesis*; D011994:Recombinant Proteins / Q000378:metabolism; D013296:Streptococcus pneumoniae / Q000201:enzymology* / Q000235:genetics; D014535:Uridine Diphosphate Glucuronic Acid / Q000378:metabolism
D006017:Glycolipids; D011134:Polysaccharides; D011135:Polysaccharides, Bacterial; D011994:Recombinant Proteins; C026712:UDP-glucosamine; D014535:Uridine Diphosphate Glucuronic Acid; D016695:Glycosyltransferases; C413445:type 3 capsular polysaccharide synthase; D010739:Phospholipase D; D005944:Glucosamine