Structure and functional analysis of glycosyltransferases which are involved in the biosynthesis of bacterial polysaccharides

在过去三年中取得了以下结果：对大肠杆菌染色体亚克隆质粒的rfe-rff区进行了分析。我们发现rfe和orf2基因编码的蛋白是膜蛋白。我们还用TnphoA方法分析了这些蛋白的拓扑结构。我们确定了克雷伯菌K2-cps区域的整个核苷酸序列，该区域编码了参与K2荚膜多糖生物合成的酶。在cps区域鉴定出1个sigma^<56 b>的启动子和19个orf，并根据与数据库的同源性搜索结果推测了几个orf的功能。我们还确定了大肠杆菌O9-rfb区域的整个核苷酸序列，该区域参与了O9 LPS的O多糖的生物合成。在该区域鉴定出甘露糖合成酶和3种甘露糖基转移酶。我们从大肠杆菌K12的rfa区将4种糖基转移酶rfaG、rfaB、rfaI和rfaJ亚克隆为表达载体，并进行了表达。我们还用基因靶向的方法分别构建了这些基因的缺陷突变体。利用这些突变体，检测了上述糖基转移酶的表达和酶活性。目前正在进行位点导向突变基因的构建，以确定活性位点。

Following results were obtained in the past three years.1.The rfe-rff region of Escherichia coli chromosome subcloned into a plasmid was analyzed. We found that the proteins encoded by rfe and orf2 genes were membrane proteins. We also analyzed the topology of these proteins with TnphoA method.2.We determined the entire nucleotide sequence of the K2-cps region of Klebsiella which encoded enzymes involved in the biosynthesis of K2 capsular polysaccharide. A sigma^<56> promoter and 19 ORFs were identified in the cps region and the functions of several ORFs were speculated from the results of homology search with the data base.3.We also determined the entire nucleotide sequence of the O9-rfb region of E.coli which was involved in the biosynthesis of the O polysaccharide of O9 LPS.GDPmannose synthetase, and three mannosyltransferases were identified in the region.4.We subloned 4 kinds of glycosyltransferase such as rfaG,rfaB,rfaI and rfaJ from the rfa region of E.coli K12 into a expression vector and expressed these enzymes. We also constructed deficient mutants of these genes, respectively, with a gene targeting method. By using these mutants, the expression and enzymatic activities of above glycosyltransferases were examined. Construction of site-directed mutant genes were currently proceeded for the identification of active sites.