Universal phosphatase-coupled glycosyltransferase assay

Area of Science:

• Biochemistry
• Enzymology
• Assay Development

Background:

• Glycosyltransferases (GTs) are crucial enzymes in synthesizing complex carbohydrates.
• Accurate measurement of GT activity is essential for understanding biological processes and drug discovery.
• Existing assays often involve radioactivity or are not easily adaptable for high-throughput screening.

Purpose of the Study:

• To develop a nonradioactive, colorimetric assay for quantifying glycosyltransferase activity.
• To enable accurate determination of kinetic parameters for various GTs.
• To facilitate high-throughput screening of GT inhibitors and substrates.

Main Methods:

• Utilizing specific phosphatases to release inorganic phosphate from GT reaction leaving groups.
• Employing colorimetric malachite-based reagents for sensitive phosphate detection.
• Adapting the assay for multiwell plates and quantitation using a plate reader.

Main Results:

• Demonstrated a direct proportionality between released phosphate and transferred sugar molecules.
• Successfully applied the assay to a wide range of GTs, including glucosyltransferases, N-acetylglucosaminyltransferases, N-acetylgalactosyltransferases, galactosyltransferases, fucosyltransferases, and sialyltransferases.
• Validated the assay by characterizing Clostridium difficile toxin B, human KTELC1, and human sialyltransferase ST6GAL1.

Conclusions:

• The developed assay provides a robust, nonradioactive, and high-throughput method for measuring glycosyltransferase activity.
• This assay is versatile and applicable to diverse glycosyltransferase families.
• It offers a valuable tool for biochemical research, drug discovery, and glycobiology studies.