Abstract
Visualization of the reaction coordinate undertaken by glycosyltransferases has remained elusive but is critical for understanding this important class of enzyme. Using substrates and substrate mimics, we describe structural snapshots of all species along the kinetic pathway for human O-linked β-N-acetylglucosamine transferase (O-GlcNAc transferase), an intracellular enzyme that catalyzes installation of a dynamic post-translational modification. The structures reveal key features of the mechanism and show that substrate participation is important during catalysis.
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Acknowledgements
This work was supported by the US National Institutes of Health (NIH) grant R01GM094263 to S.W. and the Natural Sciences and Engineering Research Council of Canada and Simon Fraser University. T.M.G. is a Sir Henry Wellcome postdoctoral fellow and a Michael Smith for Health Research (MSFHR) trainee award holder. D.J.V. is an E.W.R. Steacie Memorial Fellow and holds a Canada Research Chair in Chemical Glycobiology. We thank L. Deng for assistance with the chemical synthesis. We thank L. Cai (University of South Carolina, Salkehatchie) for the kind gift of UDP-2-ketoGlc.
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M.B.L. carried out all structural experiments. J.J. helped with X-ray data collection. J.J. and M.B.L. performed kinetics assays. W.F.Z. and T.M.G. performed thiosugar kinetics assays and peptide assays. G.E.W. performed the synthesis of peptide and glycopeptides substrates. M.B.L., J.J., T.M.G., D.J.V. and S.W. designed experiments, analyzed data and wrote the manuscript.
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Lazarus, M., Jiang, J., Gloster, T. et al. Structural snapshots of the reaction coordinate for O-GlcNAc transferase. Nat Chem Biol 8, 966–968 (2012). https://doi.org/10.1038/nchembio.1109
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DOI: https://doi.org/10.1038/nchembio.1109
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