- Title
- A direct fluorescent activity assay for glycosyltransferases enables convenient high-throughput screening: application to O-GlcNAc transferase
- Creator
- Alteen, Matthew G.; Gros, Christina; Davies, Gideon J.; Robinson, Phillip J.; McCluskey, Adam; Vocadlo, David J.; Meek, Richard W.; Cardoso, David A.; Busmann, Jil A.; Sangouard, Gontran; Deen, Matthew C.; Tan, Hong-Yee; Shen, David L.; Russell, Cecilia C.
- Relation
- NHMRC.GNT1162515 http://purl.org/au-research/grants/nhmrc/GNT1162515
- Relation
- Angewandte Chemie - International Edition Vol. 59, Issue 24, p. 9601-9609
- Publisher Link
- http://dx.doi.org/10.1002/anie.202000621
- Publisher
- Wiley
- Resource Type
- journal article
- Date
- 2020
- Description
- 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. Herein, we report a bead-based strategy to measure the group-transfer activity of glycosyltransferases sensitively 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.
- Subject
- enzymes; fluorescent probes; glycosylation; high-throughput screening; inhibitors
- Identifier
- http://hdl.handle.net/1959.13/1424751
- Identifier
- uon:38143
- Identifier
- ISSN:1433-7851
- Rights
- This is the peer reviewed version of the following article: Alteen, Matthew G.; Gros, Christina; Davies, Gideon J.; Robinson, Phillip J.; McCluskey, Adam; Vocadlo, David J.; Meek, Richard W.; Cardoso, David A.; Busmann, Jil A.; Sangouard, Gontran; Deen, Matthew C.; Tan, Hong-Yee; Shen, David L.; Russell, Cecilia C. “A direct fluorescent activity assay for glycosyltransferases enables convenient high-throughput screening: application to O-GlcNAc transferase”. Angewandte Chemie - International Edition Vol. 59, Issue 24, p. 9601-9609 (2020), which has been published in final form athttp://dx.doi.org/10.1002/anie.202000621. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions. This article may not be enhanced, enriched or otherwise transformed into a derivative work, without express permission from Wiley or by statutory rights under applicable legislation. Copyright notices must not be removed, obscured or modified. The article must be linked to Wiley’s version of record on Wiley Online Library and any embedding, framing or otherwise making available the article or pages thereof by third parties from platforms, services and websites other than Wiley Online Library must be prohibited.
- Language
- eng
- Full Text
- Reviewed
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