BIOSYNTHESIS OF CAPSULAR POLYSACCHARIDES OF PATHOGENIC BACTERIA

病原菌荚膜多糖的生物合成

• 批准号: 3804594
• 负责人: W F VANN
• 金额: --
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3804594
• 关键词: Escherichia coli; bacterial antigens; bacterial capsules; bacterial polysaccharides; bacterial proteins; carbohydrate biosynthesis; chimeric proteins; enzyme mechanism; enzyme structure; ligase; microorganism metabolism; protein purification; protein sequence; sialate; site directed mutagenesis

The polysialic acid of Escherichia coli K1 commonly occurs in invasive E. coli disease. Enzymes involved in its biosynthesis are central to neuraminic acid metabolism in bacteria and mammals. The expression and function of 2 of these enzymes is being investigated, namely, CMP-neuAc synthetase (neuA gene product) and a protein of undefined function, (P7 neuC gene product). CMP-NeuAc synthetase contains two cysteine residues. Site directed mutagenesis of cys-129 or cys-329 to glycine results in loss of activity, suggesting that both residues are important. However, enzyme retains activity when cys-129 is changed to a serine and is therefore not essential for catalysis. Random mutagenesis reveals that cys-329 is essential. The cys-129 to serine (ser-129) results in a less stable enzyme. Both the wild type and the ser-129 enzyme are inactivatable only after partial thermal denaturation with charged sulfhydryl reagents, suggesting a buried residue. Hydrophobic reagents can inactivate both enzymes at 25 degrees C but not in the presence of CTP, suggesting that cys-329 is buried at the active site. To locate common functional domains, monoclonal antibodies were prepared to recombinant CMP-neuAc synthetase. Purification from other sources is underway. Recombinant CMP-neuAc synthetase is being used as a reagent to synthesize sialylated oligosaccharides. The amino terminus of the P7 protein was determined from fusion proteins confirming the suggestion in the previous report that the neuA and neuC genes overlap. Purification of P7 and investigation of its function is underway. Other genes in the K1 cluster: Polysaccharide was isolated from E. coli mutants which do not export polymer. Examination of these polymers by size exclusion, NMR, and TLC suggest kpsT mutants produce polymers similar to wild type but without phosphotidic acid. kpsD mutants produce a polysaccharide with different hydrodynamic behavior.

大肠杆菌K1的聚唾液酸通常存在于侵袭性大肠杆菌中。 大肠杆菌病 参与其生物合成的酶是 细菌和哺乳动物的神经氨酸代谢。 的表达和 正在研究这些酶中的2种的功能，即CMP-neuAc 合成酶（neuA基因产物）和功能不明的蛋白质（P7 neuC基因产物）。 CMP-NeuAc合成酶含有两个半胱氨酸残基。 将cys-129或cys-329定点突变为甘氨酸导致缺失 的活性，这表明这两个残基是重要的。 然而，酶 当Cys-129变为丝氨酸时保留活性，因此不 对催化作用至关重要。 随机突变显示cys-329是 具有本质意义 Cys-129转化为丝氨酸（ser-129）导致较不稳定的 酵素 野生型和ser-129酶都是失活的， 在用带电巯基试剂部分热变性后， 说明有掩埋的残留物 疏水性试剂可同时抑制 酶在25摄氏度，但不存在CTP，这表明， Cys-329被埋在活性位点。 为了定位共同的功能结构域，制备单克隆抗体， 重组CMP-neuAc合成酶。 从其他来源纯化是 正在进行中 重组CMP-neuAc合成酶被用作试剂， 合成唾液酸化寡糖。 从融合蛋白中确定P7蛋白的氨基末端 确认上一次报告中的建议，即neuA和neuC 基因重叠 P7的纯化及其功能研究 正在进行中 K1簇中的其他基因：多糖分离自大肠杆菌。杆菌 不输出聚合物的突变体。 这些聚合物的尺寸检验 排除，NMR和TLC表明kpsT突变体产生类似于 野生型但不含磷脂酸。 kpsD突变体产生一种 多糖具有不同的流体动力学行为。