Glycosyltransferase-Coupled Assays for 4-Epimerase WbpP from Pseudomonas aeruginosa
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.
Key wordsGlycosyltransferases 4-Epimerase WbpP UDP-Glc UDP-Gal UDP-GlcNAc UDP-GalNAc
This work was supported by Natural Sciences and Engineering Research Council of Canada Discovery grants to I.B. and to K.J.
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