Universal Phosphatase‐Coupled Glycosyltransferase Assay

A non‐radioactive glycosyltransferase assay is described here. This method takes advantage of coupling phosphatases that release inorganic phosphate quantitatively from the leaving nucleoside phosphates produced during glycosyltransferase reactions. The released phosphate group is then detected using colorimetric malachite‐based reagents. Because the amount of phosphate released is directly proportional to the sugar molecule transferred in a glycosyltransferase reaction, this method can be used to obtain accurate kinetic parameters of the glycosyltransferase. The assay can be performed in multi‐well plates and quantitated by a plate reader, thus making it amenable to high‐throughput screening. It has been successfully applied to all glycosyltransferases available to us, including glucosyltransferases, N‐acetyl‐glucosaminyltransferases, N‐acetylgalactosyltransferases, galactosyltransferases, fucosyltransferases and sialyltransferases. The method is demonstrated using Clostridium difficile toxin B (a protein O‐glucosyltransferase), human KTELC1 (a glucosyltransferase homologue to Rumi from Drosophila) and human sialyltransferase ST6GAL1.