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O-GlcNAc transferase invokes nucleotide sugar pyrophosphate participation in catalysis

Nature Chemical Biology volume 8, pages 969–974 (2012)Cite this article

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Abstract

Protein O-GlcNAcylation is an essential post-translational modification on hundreds of intracellular proteins in metazoa, catalyzed by O-linked β-N-acetylglucosamine (O-GlcNAc) transferase (OGT) using unknown mechanisms of transfer and substrate recognition. Through crystallographic snapshots and mechanism-inspired chemical probes, we define how human OGT recognizes the sugar donor and acceptor peptide and uses a new catalytic mechanism of glycosyl transfer, involving the sugar donor α-phosphate as the catalytic base as well as an essential lysine. This mechanism seems to be a unique evolutionary solution to the spatial constraints imposed by a bulky protein acceptor substrate and explains the unexpected specificity of a recently reported metabolic OGT inhibitor.

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Figure 1: The structure of a ternary hOGT product complex gives insights into the peptide-binding mode and participation of active site residues in O-GlcNAc transfer.
Figure 2: The unusual conformation of the sugar nucleotide in the hOGT pseudo-Michaelis complex suggests substrate-assisted catalysis.
Figure 3: Proposed catalytic mechanism and pH activity profile of hOGT.

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References

  1. Hart, G.W., Housley, M.P. & Slawson, C. Cycling of O-linked β-N-acetylglucosamine on nucleocytoplasmic proteins. Nature 446, 1017–1022 (2007).

    Article  CAS  PubMed  Google Scholar 

  2. Fujiki, R. et al. GlcNAcylation of histone H2B facilitates its monoubiquitination. Nature 480, 557–560 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Hart, G.W., Slawson, C., Ramirez-Correa, G. & Lagerlof, O. Cross talk between O-GlcNAcylation and phosphorylation: roles in signaling, transcription, and chronic disease. Annu. Rev. Biochem. 80, 825–858 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Love, D.C., Krause, M.W. & Hanover, J.A. O-GlcNAc cycling: emerging roles in development and epigenetics. Semin. Cell Dev. Biol. 21, 646–654 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Cantarel, B.L. et al. The Carbohydrate-Active EnZymes database (CAZy): an expert resource for glycogenomics. Nucleic Acids Res. 37, D233–D238 (2009).

    Article  CAS  PubMed  Google Scholar 

  6. Lairson, L.L., Henrissat, B., Davies, G.J. & Withers, S.G. Glycosyltransferases: structures, functions, and mechanisms. Annu. Rev. Biochem. 77, 521–555 (2008).

    Article  CAS  PubMed  Google Scholar 

  7. Iyer, S.P. & Hart, G.W. Roles of the tetratricopeptide repeat domain in O-GlcNAc transferase targeting and protein substrate specificity. J. Biol. Chem. 278, 24608–24616 (2003).

    Article  CAS  PubMed  Google Scholar 

  8. Clarke, A.J. et al. Structural insights into mechanism and specificity of O-GlcNAc transferase. EMBO J. 27, 2780–2788 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Martinez-Fleites, C. et al. Structure of an O-GlcNAc transferase homolog provides insight into intracellular glycosylation. Nat. Struct. Mol. Biol. 15, 764–765 (2008).

    Article  CAS  PubMed  Google Scholar 

  10. Lazarus, M.B., Nam, Y., Jiang, J., Sliz, P. & Walker, S. Structure of human O-GlcNAc transferase and its complex with a peptide substrate. Nature 469, 564–567 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Jiang, J., Lazarus, M.B., Pasquina, L., Sliz, P. & Walker, S. A neutral diphosphate mimic crosslinks the active site of human O-GlcNAc transferase. Nat. Chem. Biol. 8, 72–77 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  12. Gloster, T.M. et al. Hijacking a biosynthetic pathway yields a glycosyltransferase inhibitor within cells. Nat. Chem. Biol. 7, 174–181 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Pathak, S. et al. O-GlcNAcylation of TAB1 modulates TAK1-mediated cytokine release. EMBO J. 31, 1394–1404 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Wang, Z. et al. Extensive crosstalk between O-GlcNAcylation and phosphorylation regulates cytokinesis. Sci. Signal. 3, ra2 (2010).

    PubMed  PubMed Central  Google Scholar 

  15. Vocadlo, D.J., Hang, H.C., Kim, E.J., Hanover, J.A. & Bertozzi, C.R. A chemical approach for identifying O-GlcNAc–modified proteins in cells. Proc. Natl. Acad. Sci. USA 100, 9116–9121 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Macauley, M.S., Whitworth, G.E., Debowski, A.W., Chin, D. & Vocadlo, D.J. O-GlcNAcase uses substrate-assisted catalysis: kinetic analysis and development of highly selective mechanism-inspired inhibitors. J. Biol. Chem. 280, 25313–25322 (2005).

    Article  CAS  PubMed  Google Scholar 

  17. Sala, R.F., MacKinnon, S.L., Palcic, M.M. & Tanner, M.E. UDP-N-trifluoroacetylglucosamine as an alternative substrate in N-acetylglucosaminyltransferase reactions. Carbohydr. Res. 306, 127–136 (1998).

    Article  CAS  PubMed  Google Scholar 

  18. Harris, T.K. & Turner, G.J. Structural basis of perturbed pKa values of catalytic groups in enzyme active sites. IUBMB Life 53, 85–98 (2002).

    Article  CAS  PubMed  Google Scholar 

  19. Hosfield, D.J. et al. Structural basis for bisphosphonate-mediated inhibition of isoprenoid biosynthesis. J. Biol. Chem. 279, 8526–8529 (2004).

    Article  CAS  PubMed  Google Scholar 

  20. Ziegler, M.O., Jank, T., Aktories, K. & Schulz, G.E. Conformational changes and reaction of clostridial glycosylating toxins. J. Mol. Biol. 377, 1346–1356 (2008).

    Article  CAS  PubMed  Google Scholar 

  21. Lee, S.S. et al. Mechanistic evidence for a front-side, SNi-type reaction in a retaining glycosyltransferase. Nat. Chem. Biol. 7, 631–638 (2011).

    Article  CAS  PubMed  Google Scholar 

  22. Lira-Navarrete, E. et al. Structural insights into the mechanism of protein O-fucosylation. PLoS ONE 6, e25365 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Chen, C.I. et al. Structure of human POFUT2: insights into thrombospondin type 1 repeat fold and O-fucosylation. EMBO J. 31, 3183–3197 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Collaborative Computational Project, Number 4. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D Biol. Crystallogr. 50, 760–763 (1994).

  25. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).

    Article  PubMed  Google Scholar 

  26. Schüttelkopf, A.W. & van Aalten, D.M.F. PRODRG: a tool for high-throughput crystallography of protein-ligand complexes. Acta Crystallogr. D Biol. Crystallogr. 60, 1355–1363 (2004).

    Article  PubMed  Google Scholar 

  27. Conner, S.H. et al. TAK1-binding protein 1 is a pseudophosphatase. Biochem. J. 399, 427–434 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Cheung, P.C., Campbell, D.G., Nebreda, A.R. & Cohen, P. Feedback control of the protein kinase TAK1 by SAPK2a/p38α. EMBO J. 22, 5793–5805 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Gold, H. et al. Synthesis of sugar nucleotides by application of phosphoramidites. J. Org. Chem. 73, 9458–9460 (2008).

    Article  CAS  PubMed  Google Scholar 

  30. Meynial, I., Paquet, V. & Combes, D. Simultaneous separation of nucleotides and nucleotide sugars using an ion-pair reversed-phase HPLC: application for assaying glycosyltransferase activity. Anal. Chem. 67, 1627–1631 (1995).

    Article  CAS  Google Scholar 

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Acknowledgements

This work was supported by a Wellcome Trust Senior Research Fellowship (WT087590MA) to D.M.F.v.A.

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  1. Marianne Schimpl, Xiaowei Zheng and Vladimir S Borodkin: These authors contributed equally to this work.

Authors and Affiliations

  1. College of Life Sciences, University of Dundee, Dundee, UK

    Marianne Schimpl, Xiaowei Zheng, Vladimir S Borodkin, David E Blair, Andrew T Ferenbach, Alexander W Schüttelkopf, Iva Navratilova, Tonia Aristotelous, Osama Albarbarawi, David A Robinson & Daan M F van Aalten

  2. Department of Chemistry, Louisiana State University, Baton Rouge, Louisiana, USA

    Megan A Macnaughtan

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Contributions

M.S. and D.M.F.v.A. performed structural biology; M.S. and X.Z. did protein expression and enzyme activity assays; V.S.B. performed synthetic organic chemistry; D.E.B. made enzyme kinetics measurements; I.N., X.Z., D.A.R. and T.A. carried out SPR experiments; A.T.F. performed molecular biology; O.A. performed MS. M.S., X.Z., V.S.B. and D.M.F.v.A. devised the experiments. M.S., X.Z., V.S.B., M.A.M., A.W.S. and D.M.F.v.A. interpreted the data and wrote the manuscript.

Corresponding author

Correspondence to Daan M F van Aalten.

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Schimpl, M., Zheng, X., Borodkin, V. et al. O-GlcNAc transferase invokes nucleotide sugar pyrophosphate participation in catalysis. Nat Chem Biol 8, 969–974 (2012). https://doi.org/10.1038/nchembio.1108

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