BIOSYNTHESIS OF CAPSULAR POLYSACCHARIDES OF PATHOGENIC BACTERIA

病原菌荚膜多糖的生物合成

• 批准号: 3810962
• 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/3810962
• 关键词: Escherichia coli; Neisseria meningitidis; bacterial antigens; bacterial capsules; bacterial polysaccharides; bacterial proteins; chimeric proteins; enzyme mechanism; enzyme structure; glycoprotein biosynthesis; ligase; microorganism metabolism; peptide chemical synthesis; protein purification; protein sequence; site directed mutagenesis

Some of the enzymes involved in the biosynthesis of the Escherichia coli K1, a pathogen, polysaccharide is central to neuraminic acid metabolism in bacteria and mammals. The expression and function of CMP-neuAc synthetase and a protein of undefined function, P7 is being investigated. The role of the 2 CMP-neuAc synthetase has cysteine residues in function was investigated by chemical modification and site directed mutagenesis. Reducing agents and iodoactate have no effect on the rate of heat inactivation suggesting that a disulfide is not critical for stability. Enzyme can be inactivated by sulfhydryl specific reagents such PCMB and dithiodipyridine. Site directed mutagenesis of either cys-129 or cys-329 to glycine results in a marked decrease in activity, thus both sulfhydryls are important for full activity. Comparison of the nucleotide sequences of CMP-neuAc synthetase and the 2 CMP-KDO synthetase enzymes produced by encapsulated E. coli suggest a consensus sequence in the amino termini. Random mutagensis suggest that some the residues may be important for activity. Of 9 (out of 15 total) arginine residues mutated to glycine only the arg-12 contained in the amino terminal consensus affected activity. Experiments are underway to define the role of arginine-12 in catalysis. The CMP-neuAc synthetase of Neisseria meningitidis has been partially purified. The amino terminus of this enzyme will be sequenced and compared with the current consensus. Analysis of available nucleotide sequence data from our laboratory and the University of Rochester suggest that the genes encoding CMP-neuAc synthetase and P7 are part of an operon ending with P7. The promoter for this operon has not been located. Based on these data and previous experiments, CMP-neuAc and P7 may be translationally coupled. Fusion proteins of P7 and -galactosidase are being constructed to test this hypothesis and to aid in the purification of P7.

一些参与大肠杆菌生物合成的酶 K1是一种病原体，多糖是神经氨酸代谢的核心 在细菌和哺乳动物中。 CMP-neuAc的表达与功能 合成酶和功能不确定的蛋白质，P7被 研究了 2CMP-neuAc合成酶的作用具有半胱氨酸 通过化学修饰和定位研究功能残基 定向诱变 还原剂和碘乙酸盐对 热灭活率表明二硫化物不是 对稳定至关重要。 巯基可使酶失活 特定试剂如PCMB和二硫代二吡啶。 定点 将Cys-129或Cys-329诱变为甘氨酸导致显著的 活性降低，因此两个巯基对于完整的 活动 CMP-neuAc的核苷酸序列比较 合成酶和2种CMP-KDO合成酶， E.大肠杆菌表明氨基末端存在一个共有序列。 随机 诱变表明，一些残留物可能对活性很重要。 在9个（总共15个）精氨酸残基突变为甘氨酸中， 氨基末端共有序列中所含的Arg-12影响活性。 实验正在进行中，以确定精氨酸-12在催化中的作用。 脑膜炎奈瑟氏菌的CMP-neuAc合成酶已被部分 提纯 该酶的氨基末端将被测序， 与目前的共识相比。 可用核苷酸分析 我们实验室和罗切斯特大学的序列数据 提示编码CMP-neuAc合成酶和P7的基因是 以P7结尾的操纵子。 该操纵子的启动子尚未被 所在 基于这些数据和先前的实验，CMP-neuAc和P7 可以被耦合。 P7和P8的融合蛋白 - 半乳糖苷酶正在构建，以测试这一假设，并帮助 纯化P7。