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A direct fluorescent activity assay for glycosyltransferases enables convenient high-throughput screening: Application to O-GlcNAc Transferase

DOI: 10.1002/anie.202000621 DOI Help

Authors: David Jaro Vocadlo (Simon Fraser University) , Matthew G. Alteen (Simon Fraser University) , Christina Gros (Simon Fraser University) , Richard Meek (University of York) , David A. Cardoso (The University of Sydney) , Jil Busmann (Simon Fraser University) , Gontran Sangouard (Ecole Polytechnique Federale de Lausanne) , Matthew C. Deen (Simon Fraser University) , Hong-Yee Tan (Simon Fraser University) , David L. Shen (Simon Fraser University) , Cecilia C. Russell (University of Newcastle, Australia) , Gideon J. Davies (University of York) , Phillip J. Robinson (University of Sydney) , Adam Mccluskey (University of Newcastle, Australia)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Angewandte Chemie International Edition

State: Published (Approved)
Published: February 2020
Diamond Proposal Number(s): 18598

Abstract: Glycosyltransferases carry out important cellular functions in species ranging from bacteria to humans. Despite their essential roles in biology, simple and robust activity assays that can be easily applied to high‐throughput screening for inhibitors of these enzymes have been challenging to develop. Here we report a bead‐based strategy to sensitively measure the group transfer activity of glycosyltransferases using simple fluorescence measurements, without the need for coupled enzymes or secondary reactions. We validate the performance and accuracy of the assay using O ‐GlcNAc Transferase (OGT) as a model system through detailed Michaelis‐Menten kinetic analysis of various substrates and inhibitors. Optimization of this assay and application to high‐throughput screening enabled screening for inhibitors of OGT, leading to a novel inhibitory scaffold. We believe this assay will prove valuable not only for the study of OGT, but also more widely as a general approach for the screening of glycosyltransferases and other group transfer enzymes.

Journal Keywords: Glycosyltransferase; enzyme assay; high-throughput screening; O-GlcNAc; glycoprotein; fluorescent substrates

Diamond Keywords: Enzymes

Subject Areas: Chemistry, Biology and Bio-materials

Instruments: I04-1-Macromolecular Crystallography (fixed wavelength)

Added On: 04/03/2020 10:30

Discipline Tags:

Catalysis Life Sciences & Biotech Structural biology Chemistry Biochemistry

Technical Tags:

Diffraction Macromolecular Crystallography (MX)