Identifcation of reaction center of glycosyl transferase through molecular evolutionary and solid NMR analysis

我们首先利用新引入的PC簇对细菌糖基转移酶基因进行了分子进化分析。我们特别关注了糖基转移酶基因簇，这些基因簇定义了细胞壁外的糖结构，已知这些糖结构在大肠杆菌菌株之间存在差异。杆菌同源性搜索通过使用这些ca。20个糖基转移酶氨基酸序列作为查询，并为每个同源组构建基因同源性。我们发现一组同源基因在进化过程中改变了它们的糖特异性。这表明酶的特异性通过一系列氨基酸的变化而改变。因此，我们选择了大肠杆菌waaO基因。coliK 12菌株，并在大肠杆菌中高效表达其蛋白。使用His-标签亲和柱层析纯化蛋白质，并将适量的纯化蛋白质用于固体NMR分析。使用固体NMR方法观察到几个清晰的天然C13峰。我们还将C13标记的葡萄糖加入到该纯化蛋白中，并发现了新的C13峰，该峰可能对应于葡萄糖-蛋白结合。

We first conducted molecular evolutionary analyses of bacterial glycosyltransferase genes by using newly introduced PC cluster. We particularly focused on cluster of glycosyltransferase genes that define outer cell wall sugar structures that are know to vary from strain to strain of E. coli. Homology search was done by using these ca. 20 gycosyltransferase amino acid sequences as queries, and gene phylogenies were constructed for each homologous group. We found that one group of homologous genes changed their sugar specificities during evolution. This suggests that enzyme specificity was altered through a series of amino acid changes. We therefore chose waaO gene of E. coli K12 strain, and overexpressed its protein. The protein was purified with using His-tag affinity collum chromatography, and appropriate amount of purified protein was used for solid NMR analysis. Several clear native C13 peaks were observed using solid NMR method. We also added C13-labeled glucose to this purified protein, and found new C13 peak that may correspond to glucose-protein binding.