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

Autor: Alteen MG; Department of Chemistry, Simon Fraser University, Burnaby, BC, V5A 1S6, Canada., Gros C; Department of Chemistry, Simon Fraser University, Burnaby, BC, V5A 1S6, Canada., Meek RW; York Structural Biology Laboratory, Department of Chemistry, University of York, York, YO10 5DD, UK., Cardoso DA; Children's Medical Research Institute, The University of Sydney, Sydney, NSW, 2145, Australia., Busmann JA; Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, BC, V5A 1S6, Canada., Sangouard G; Department of Chemistry, Simon Fraser University, Burnaby, BC, V5A 1S6, Canada., Deen MC; Department of Chemistry, Simon Fraser University, Burnaby, BC, V5A 1S6, Canada., Tan HY; Department of Chemistry, Simon Fraser University, Burnaby, BC, V5A 1S6, Canada., Shen DL; Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, BC, V5A 1S6, Canada., Russell CC; Chemistry, School of Environmental and Life Sciences, The University of Newcastle, University Drive, Callaghan, NSW, 2308, Australia., Davies GJ; York Structural Biology Laboratory, Department of Chemistry, University of York, York, YO10 5DD, UK., Robinson PJ; Children's Medical Research Institute, The University of Sydney, Sydney, NSW, 2145, Australia., McCluskey A; Chemistry, School of Environmental and Life Sciences, The University of Newcastle, University Drive, Callaghan, NSW, 2308, Australia., Vocadlo DJ; Department of Chemistry, Simon Fraser University, Burnaby, BC, V5A 1S6, Canada.; Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, BC, V5A 1S6, Canada.
Jazyk: angličtina
Zdroj: Angewandte Chemie (International ed. in English) [Angew Chem Int Ed Engl] 2020 Jun 08; Vol. 59 (24), pp. 9601-9609. Date of Electronic Publication: 2020 Apr 02.
DOI: 10.1002/anie.202000621
Abstrakt: 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.
(© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)
Databáze: MEDLINE
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