Sharma Sulav, Creuzenet Carole, Jarrell Kenneth F, Brockhausen Inka
Department of Biomedical and Molecular Sciences, Queen's University, Kingston, ON, Canada.
Department of Microbiology and Immunology, University of Western Ontario, London, ON, Canada.
Methods Mol Biol. 2019;1954:255-268. doi: 10.1007/978-1-4939-9154-9_20.
The donor substrates for the biosynthesis of bacterial polysaccharides include UDP-Glc/Gal and UDP-GlcNAc/GalNAc. The conversion of these nucleotide sugars is catalyzed by 4-epimerases. The wbpP gene of Pseudomonas aeruginosa encodes a 4-epimerase that has a preference for UDP-GlcNAc/GalNAc as substrates. Other 4-epimerases have broad specificities or preference for UDP-Glc/Gal. We have developed coupled assays where the 4-epimerase product is used as a donor substrate for glycosyltransferases that are highly specific for the nucleotide sugar structure. We describe here a method for the study of substrate specificity of WbpP, using coupled assays employing four different glycosyltransferases. These protocols can be applied to the identification and characterization of novel 4-epimerases and to determine their substrate specificities.
细菌多糖生物合成的供体底物包括UDP-葡萄糖/半乳糖和UDP- N -乙酰葡糖胺/ N -乙酰半乳糖胺。这些核苷酸糖的转化由4-表异构酶催化。铜绿假单胞菌的wbpP基因编码一种4-表异构酶，该酶优先选择UDP- N -乙酰葡糖胺/ N -乙酰半乳糖胺作为底物。其他4-表异构酶对UDP-葡萄糖/半乳糖具有广泛的特异性或偏好性。我们开发了偶联测定法，其中4-表异构酶产物用作糖基转移酶的供体底物，这些糖基转移酶对核苷酸糖结构具有高度特异性。我们在此描述一种研究WbpP底物特异性的方法，该方法使用采用四种不同糖基转移酶的偶联测定法。这些方案可应用于新型4-表异构酶的鉴定和表征，并确定它们的底物特异性。
