BIOSYNTHESIS OF CAPSULAR POLYSACCHARIDES OF PATHOGENIC BACTERIA

参与大肠杆菌生物合成的几种酶 K1是一种病原体，多糖是神经氨酸代谢的中心 在细菌和哺乳动物身上。CMP-neuAc的表达和功能 合成酶和一种功能不明的蛋白质P7正在 调查过了。Cp-neuAc合成酶2的作用有半胱氨酸 通过化学修饰和定位研究了功能残基 定向诱变。还原剂和碘-乳酸盐对 二硫化物的热失活率表明 对稳定至关重要。酶可被巯基灭活 特定试剂，如多氯联苯和二硫代联吡啶。站点定向 Cys-129或Cys-329对甘氨酸的诱变导致显著的 活性降低，因此这两个巯基对充分 活动。CMP-neuAc的核苷酸序列比较 微囊化产生的合成酶和2种CMP-KDO合成酶 大肠杆菌在氨基末端有一个共同的序列。随机 突变提示一些残基对活性可能是重要的。 在总共15个精氨酸残基中，有9个突变为甘氨酸，只有 氨基末端共识中的Arg-12影响活性。 目前正在进行实验，以确定精氨酸-12在催化中的作用。 脑膜炎奈瑟菌的cMP-neuAc合成酶已部分被 纯净的。该酶的氨基末端将被测序并 与目前的共识相比。可用核苷酸的分析 来自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。这部歌剧的发起人还没有 找到了。根据这些数据和以前的实验，Cp-neuAc和P7 可以是平移耦合的。P7和P7融合蛋白 ，-半乳糖苷酶的构建是为了验证这一假说并帮助 在P7的纯化中。

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.