Glycosyltransferase activity assay using colorimetric methods

Abstract

The glycosyltransferases (GTs) are an important subclass of enzymes that catalyze the biosynthesis of glycosidic bonds in oligosaccharides, polysaccharides and glycoconjugates by transferring a sugar residue from a donor substrate to an acceptor substrate. The membrane-associated GTs play a vital role in the biosynthesis of bacterial cell-wall polysaccharides. Characterization and quantification of GT activities is important for studies of biosynthesis of polysaccharides, drug target development, and production of bacterial products. In this chapter, colorimetric assays for the measurement of GT activities will be presented. Assays for GTs acting on monosaccharide-derivatives are based on the cleavage of unreacted glycosyl-p-nitrophenol acceptors followed by detection of p-nitrophenolate. GT reactions coupled with phosphatases and detection of inorganic phosphate are suitable for most GTs. These assays permit convenient quantification of GT activities and kinetics without the use of radioactive sugars.