Brockhausen Inka, Szarek Walter A, Riley John G, Vlahakis Jason Z
Department of Medicine, Queen's University, Kingston, Ontario, Canada.
Methods Mol Biol. 2006;347:253-64. doi: 10.1385/1-59745-167-3:253.
Gram-negative bacteria have lipopolysaccharides terminating in repeating oligosaccharides which comprise the O-antigen. The glycosyltransferases (GTs) assembling the O-chain utilize lipid-linked acceptor substrates and nucleotide sugar donor substrates. The natural undecaprenol-linked acceptor substrates are not readily available, precluding the characterization of O-chain GTs. This chapter describes an assay for a galactosyltransferase (GalT) involved in the synthesis of the O7 antigen of Escherichia coli VW187. The glycolipid GlcNAcalpha-pyrophosphate bound to a phenoxyundecyl moiety, which resembles the natural substrate, was synthesized and employed in GT assays using bacterial membranes as the enzyme source. The assay is simple and allows optimization and characterization of the enzyme reaction. Similar protocols can be used to assay other GTs in the O-chain biosynthesis pathways.
革兰氏阴性菌具有以重复寡糖结尾的脂多糖，这些寡糖构成了O抗原。组装O链的糖基转移酶（GTs）利用脂质连接的受体底物和核苷酸糖供体底物。天然的十一异戊烯醇连接的受体底物不易获得，这妨碍了对O链GTs的表征。本章描述了一种参与大肠杆菌VW187 O7抗原合成的半乳糖基转移酶（GalT）的检测方法。合成了与苯氧基十一烷基部分结合的糖脂GlcNAcalpha-焦磷酸，其类似于天然底物，并在以细菌膜作为酶源的GT检测中使用。该检测方法简单，可用于优化和表征酶反应。类似的方案可用于检测O链生物合成途径中的其他GTs。
