A Direct Fluorescent Activity Assay for Glycosyltransferases Enables Convenient High‐Throughput Screening: Application to O‐GlcNAc Transferase

Peer reviewed: 
Yes, item is peer reviewed.
Scholarly level: 
Faculty/Staff
Final version published as: 

Alteen, M. G., Gros, C., Meek, R. W., Cardoso, D. A., Busmann, J. A., Sangouard, G., Deen, M. C., Tan, H.-Y., Shen, D. L., Russell, C. C., Davies, G. J., Robinson, P. J., McCluskey, A., & Vocadlo, D. J. (2020). A Direct Fluorescent Activity Assay for Glycosyltransferases Enables Convenient High-Throughput Screening: Application to O-GlcNAc Transferase. Angewandte Chemie International Edition, 59(24), 9601–9609. https://doi.org/10.1002/anie.202000621.

Date created: 
2020-02-24
Identifier: 
DOI: 10.1002/anie.202000621
Keywords: 
Glycosyltransferase
Enzyme assay
High-throughput screening
O-GlcNAc
Glycoprotein
Fluorescent substrates
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.

Description: 

The full text of this paper will be available in Feb 2021 due to the embargo policies of Angewandte Chemie. Contact summit@sfu.ca to enquire if the full text of the accepted manuscript can be made available to you.

Language: 
English
Document type: 
Article
Rights: 
Rights remain with the authors.
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