Jorasch P, Wolter F P, Zähringer U, Heinz E
Institut für Allgemeine Botanik, Universität Hamburg, Germany.
Mol Microbiol. 1998 Jul;29(2):419-30. doi: 10.1046/j.1365-2958.1998.00930.x.
We have isolated the ypfP gene (accession number P54166) from genomic DNA of Bacillus subtilis Marburg strain 60015 (Freese and Fortnagel, 1967) using PCR. After cloning and expression in E. coli, SDS-PAGE showed strong expression of a protein that had the predicted size of 43.6 kDa. Chromatographic analysis of the lipids extracted from the transformed E. coli revealed several new glycolipids. These glycolipids were isolated and their structures determined by nuclear magnetic resonance (NMR) and mass spectrometry. They were identified as 3-[O-beta-D-glucopyranosyl-(1-->6)-O-beta-D-glucopyranosyl]-1,2-diacylgl ycerol, 3-[O-beta-D-glucopyranosyl-(1-->6)-O-beta-D-glucopyranosyl-(1-->6)-O-bet a-D-glucopyranosyl]-1,2-diacylglycerol and 3-[O-beta-D-glucopyranosyl-(1-->6)-O-beta-D-glucopyranosyl-(1-->6)-O-bet a-D-glucopyranosyl-(1-->6)-O-beta-D-glucopyranosyl]-1,2-diacylglycerol. The enzymatic activity expected to catalyse the synthesis of these compounds was confirmed by in vitro assays with radioactive substrates. In these assays, one additional glycolipid was formed and tentatively identified as 3-[O-beta-D-glucopyranosyl]-1,2-diacylglycerol, which was not detected in the lipid extract of transformed cells. Experiments with some of the above-described glycolipids as 14C-labelled sugar acceptors and unlabelled UDP-glucose as glucose donor suggest that the ypfP gene codes for a new processive UDP-glucose: 1,2-diacylglycerol-3-beta-D-glucosyl transferase. This glucosyltransferase can use diacylglycerol, monoglucosyl-diacylglycerol, diglucosyl diacylglycerol or triglucosyl diacylglycerol as sugar acceptor, which, apart from the first member, are formed by repetitive addition of a glucopyranosyl residue in beta (1-->6) linkage to the product of the preceding reaction.
我们使用聚合酶链式反应（PCR）从枯草芽孢杆菌马尔堡菌株60015（Freese和Fortnagel，1967）的基因组DNA中分离出ypfP基因（登录号P54166）。在大肠杆菌中克隆并表达后，十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳（SDS - PAGE）显示出一种预测大小为43.6 kDa的蛋白质的强表达。对从转化的大肠杆菌中提取的脂质进行色谱分析，发现了几种新的糖脂。这些糖脂被分离出来，并通过核磁共振（NMR）和质谱法确定了它们的结构。它们被鉴定为3 - [O - β - D - 吡喃葡萄糖基 - （1→6） - O - β - D - 吡喃葡萄糖基] - 1,2 - 二酰基甘油、3 - [O - β - D - 吡喃葡萄糖基 - （1→6） - O - β - D - 吡喃葡萄糖基 - （1→6） - O - β - D - 吡喃葡萄糖基] - 1,2 - 二酰基甘油和3 - [O - β - D - 吡喃葡萄糖基 - （1→6） - O - β - D - 吡喃葡萄糖基 - （1→6） - O - β - D - 吡喃葡萄糖基 - （1→6） - O - β - D - 吡喃葡萄糖基] - 1,2 - 二酰基甘油。通过使用放射性底物的体外测定，证实了预期催化这些化合物合成的酶活性。在这些测定中，形成了一种额外的糖脂，并初步鉴定为3 - [O - β - D - 吡喃葡萄糖基] - 1,2 - 二酰基甘油，在转化细胞的脂质提取物中未检测到。以上述一些糖脂作为14C标记的糖受体，未标记的UDP - 葡萄糖作为葡萄糖供体进行的实验表明，ypfP基因编码一种新的持续性UDP - 葡萄糖：1,2 - 二酰基甘油 - 3 - β - D - 葡萄糖基转移酶。这种葡萄糖基转移酶可以使用二酰基甘油、单葡萄糖基二酰基甘油、双葡萄糖基二酰基甘油或三葡萄糖基二酰基甘油作为糖受体，除了第一个成员外，它们是通过将β（1→6）连接的吡喃葡萄糖基残基重复添加到前一反应的产物上形成的。
