Williams Danielle M, Ovchinnikova Olga G, Whitfield Chris
Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, Canada.
Methods Mol Biol. 2019;1954:245-253. doi: 10.1007/978-1-4939-9154-9_19.
In vitro assays using fluorescently tagged sugar residues can facilitate the characterization of glycosyltransferase function. Here we describe the use of in vitro assays to characterize the three glycosyltransferase modules of the protein designated WbbB from Klebsiella pneumoniae O12. This protein combines key activities necessary to synthesize the O antigenic polysaccharide portion of lipopolysaccharide. The specificities of the three glycosyltransferases were investigated in vitro, using purified proteins, the activated donor sugars (dTDP-Rha, UDP-GlcNAc and CMP-β-Kdo) and synthetic acceptors terminating in either α1,3-linked Rha or β1,4-linked GlcNAc. The reaction products were verified by mass spectrometry and nuclear magnetic resonance methods.
使用荧光标记糖残基的体外测定法有助于对糖基转移酶功能进行表征。在此，我们描述了使用体外测定法来表征来自肺炎克雷伯菌O12的名为WbbB的蛋白质的三个糖基转移酶模块。该蛋白质结合了合成脂多糖O抗原多糖部分所需的关键活性。使用纯化的蛋白质、活化的供体糖（dTDP-Rha、UDP-GlcNAc和CMP-β-Kdo）以及以α1,3-连接的Rha或β1,4-连接的GlcNAc结尾的合成受体，在体外研究了这三种糖基转移酶的特异性。通过质谱和核磁共振方法对反应产物进行了验证。
