Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Using simple donors to drive the equilibria of glycosyltransferase-catalyzed reactions

Nature Chemical Biology volume 7, pages 685–691 (2011)Cite this article

Subjects

Abstract

We report that simple glycoside donors can drastically shift the equilibria of glycosyltransferase-catalyzed reactions, transforming NDP-sugar formation from an endothermic to an exothermic process. To demonstrate the utility of this thermodynamic adaptability, we highlight the glycosyltransferase-catalyzed synthesis of 22 sugar nucleotides from simple aromatic sugar donors, as well as the corresponding in situ formation of sugar nucleotides as a driving force in the context of glycosyltransferase-catalyzed reactions for small-molecule glycodiversification. These simple aromatic donors also enabled a general colorimetric assay for glycosyltransfer, applicable to drug discovery, protein engineering and other fundamental sugar nucleotide–dependent investigations. This study directly challenges the general notion that NDP-sugars are 'high-energy' sugar donors when taken out of their traditional biological context.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Representative glycosyltransferase-catalyzed reactions.
Figure 2: Evaluation of putative donors for sugar nucleotide synthesis.
Figure 3: The synthesis of sugar nucleotides from 2-chloro-4-nitrophenyl glucosides.
Figure 4: Evaluation of 2-chloro-4-nitrophenyl glycosides as sugar donors in coupled glycosyltransferase-catalyzed transglycosylation reactions.
Figure 5: Use of a colorimetric screen for glycosyl transfer.

Similar content being viewed by others

References

  1. Varki, A. et al. (eds.). Essentials of Glycobiology 2nd edn. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA, 2009).

  2. Thibodeaux, C.J., Melançon, C.E. III & Liu, H. Unusual sugar biosynthesis and natural product glycodiversification. Nature 446, 1008–1016 (2007).

    Article  CAS  PubMed  Google Scholar 

  3. Lairson, L.L., Wakarchuk, W.W. & Withers, S.G. Alternative donor substrates for inverting and retaining glycosyltransferases. Chem. Commun. (Camb.), 365–367 (2007).

  4. Lougheed, B., Ly, H.D., Wakarchuk, W.W. & Withers, S.G. Glycosyl fluorides as substrates for nucleotide phosphosugar-dependent glycosyltransferases. J. Biol. Chem. 274, 37717–37722 (1999).

    Article  CAS  PubMed  Google Scholar 

  5. Zhang, C. et al. Exploiting the reversibility of natural product glycosyltransferase-catalyzed reactions. Science 313, 1291–1294 (2006).

    Article  CAS  PubMed  Google Scholar 

  6. Zhang, C., Albermann, C., Fu, X. & Thorson, J.S. The in vitro characterization of the iterative avermectin glycosyltransferase AveBI reveals reaction reversibility and sugar nucleotide flexibility. J. Am. Chem. Soc. 128, 16420–16421 (2006).

    Article  CAS  PubMed  Google Scholar 

  7. Minami, A., Kakinuma, K. & Eguchi, T. Algycon switch approach toward unnatural glycosides from natural glycoside with glycosyltransferase VinC. Tetrahedron Lett. 46, 6187–6190 (2005).

    Article  CAS  Google Scholar 

  8. Modolo, L.V., Escamilla-Treviño, L.L., Dixon, R.A. & Wang, X. Single amino acid mutations of Medicago glycosyltransferase UGT85H2 enhance activity and impart reversibility. FEBS Lett. 583, 2131–2135 (2009).

    Article  CAS  PubMed  Google Scholar 

  9. Zhang, C., Moretti, R., Jiang, J. & Thorson, J.S. The in vitro characterization of polyene glycosyltransferases AmphDI and NysDI. ChemBioChem 9, 2506–2514 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Quirós, L.M., Carbajo, R.J., Braña, A.F. & Salas, J.A. Glycosylation of macrolide antibiotics: purification and kinetic studies of a macrolide glycosyltransferase from Streptomyces antibioticus. J. Biol. Chem. 275, 11713–11720 (2000).

    Article  PubMed  Google Scholar 

  11. Okada, T. et al. Bidirectional N-acetylglucosamine transfer mediated by β-1,4-N-acetylglucosaminyltransferase III. Glycobiology 19, 368–374 (2009).

    Article  CAS  PubMed  Google Scholar 

  12. Bolam, D.N. et al. The crystal structure of two macrolide glycosyltransferases provides a blueprint for host cell antibiotic immunity. Proc. Natl. Acad. Sci. USA 104, 5336–5341 (2007).

    Article  CAS  PubMed  Google Scholar 

  13. Williams, G.J., Zhang, C. & Thorson, J.S. Expanding the promiscuity of a natural-product glycosyltransferase by directed evolution. Nat. Chem. Biol. 3, 657–662 (2007).

    Article  CAS  PubMed  Google Scholar 

  14. Williams, G.J., Goff, R.D., Zhang, C. & Thorson, J.S. Optimizing glycosyltransferase specificity via “hot spot” saturation mutagenesis presents a catalyst for novobiocin glycorandomization. Chem. Biol. 15, 393–401 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Williams, G.J. & Thorson, J.S. A high-throughput fluorescence-based glycosyltransferase screen and its application in directed evolution. Nat. Protoc. 3, 357–362 (2008).

    Article  CAS  PubMed  Google Scholar 

  16. Gantt, R.W., Goff, R.D., Williams, G.J. & Thorson, J.S. Probing the aglycon promiscuity of an engineered glycosyltransferase. Angew. Chem. Int. Ed. 47, 8889–8892 (2008).

    Article  CAS  Google Scholar 

  17. Williams, G.J., Yang, J., Zhang, C. & Thorson, J.S. Recombinant E. coli prototype strains for in vivo glycorandomization. ACS Chem. Biol. 6, 95–100 (2011).

    Article  CAS  PubMed  Google Scholar 

  18. Yang, M. et al. Probing the breadth of macrolide glycosyltransferases: in vitro remodeling of a polyketide antibiotic creates active bacterial uptake and enhances potency. J. Am. Chem. Soc. 127, 9336–9337 (2005).

    Article  CAS  PubMed  Google Scholar 

  19. Rexach, J.E., Clark, P.M. & Hsieh-Wilson, L.C. Chemical approaches to understanding O-GlcNAc glycosylation in the brain. Nat. Chem. Biol. 4, 97–106 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Sakabe, K. & Hart, G.W. O-GlcNAc transferase regulates mitotic chromatin dynamics. J. Biol. Chem. 285, 34460–34468 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Hanson, S., Best, M., Bryan, M.C. & Wong, C. Chemoenzymatic synthesis of oligosaccharides and glycoproteins. Trends Biochem. Sci. 29, 656–663 (2007).

    Article  Google Scholar 

  22. Weigel, P.H. & DeAngelis, P.L. Hyaluronan synthases: a decade-plus of novel glycosyltransferases. J. Biol. Chem. 282, 36777–36781 (2007).

    Article  CAS  PubMed  Google Scholar 

  23. Bojarová, P. et al. Synthesis of LacdiNAc-terminated glyconjugates by mutant galactosyltransferase—a way to new glycodrugs and materials. Glycobiology 19, 509–517 (2009).

    Article  PubMed  Google Scholar 

  24. Liu, R. et al. Chemoenzymatic design of heparin sulfate oligosaccharides. J. Biol. Chem. 285, 34240–34249 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Lau, K. et al. Highly efficient chemoenzymatic synthesis of β1–4-linked galasctosides with promiscuous bacterial β1–4-galactosyltransferases. Chem. Commun. (Camb.) 46, 6066–6068 (2010).

    Article  CAS  Google Scholar 

  26. Maccioni, H.J.F., Quiroga, R. & Ferrari, M.L. Cellular and molecular biology of glycosphingolipid glycosylation. J. Neurochem. 117, 589–602 (2011).

    CAS  PubMed  Google Scholar 

  27. Rupprath, C., Schumacher, T. & Elling, L. Nucleotide deoxysugars: essential tools for the glycosylation engineering of novel bioactive compounds. Curr. Med. Chem. 12, 1637–1675 (2005).

    Article  CAS  PubMed  Google Scholar 

  28. Thibodeaux, C.J., Melançon, C.E. III & Liu, H. Natural-product sugar biosynthesis and enzymatic glycodiversification. Angew. Chem. Int. Ed. 47, 9814–9859 (2008).

    Article  CAS  Google Scholar 

  29. Yang, J., Hoffmeister, D., Liu, L., Fu, X. & Thorson, J.S. Natural product glycorandomization. Bioorg. Med. Chem. 12, 1577–1584 (2004).

    Article  CAS  PubMed  Google Scholar 

  30. Takahashi, H., Liu, Y. & Liu, H. A two-stage one-pot enzymatic synthesis of TDP-l-mycarose from thymidine and glucose-1-phosphate. J. Am. Chem. Soc. 128, 1432–1433 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Rupprath, C., Kopp, M., Hirtz, D., Muller, R. & Elling, L. An enzyme module system for in situ regeneration of deoxythymidine 5′-diphosphate (dTDP)-activated deoxy sugars. Adv. Synth. Catal. 349, 1489–1496 (2007).

    Article  CAS  Google Scholar 

  32. Timmons, S.C., Mosher, R.H., Knowles, S.A. & Jakeman, D.L. Exploiting nucleotidylyltransferases to prepare sugar nucleotides. Org. Lett. 9, 857–860 (2007).

    Article  CAS  PubMed  Google Scholar 

  33. Wagner, G.K., Pesnot, T. & Field, R.A. A survey of chemical methods for sugar-nucleotide synthesis. Nat. Prod. Rep. 26, 1172–1194 (2009).

    Article  CAS  PubMed  Google Scholar 

  34. Ko, H. et al. Molecular recognition in the P2Y14 receptor: probing the structurally permissive terminal sugar moiety of uridine-5′-diphosphoglucose. Bioorg. Med. Chem. 17, 5298–5311 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Losey, H.C. et al. Incorporation of glucose analogs by GtfE and GtfD from the vancomycin biosynthetic pathway to generate variant glycopeptides. Chem. Biol. 9, 1305–1314 (2002).

    Article  CAS  PubMed  Google Scholar 

  36. Fu, X. et al. Antibiotic optimization via in vitro glycorandomization. Nat. Biotechnol. 21, 1467–1469 (2003).

    Article  CAS  PubMed  Google Scholar 

  37. Kahne, D., Leimkuhler, C., Lu, W. & Walsh, C. Glycopeptide and lipoglycopeptide antibiotics. Chem. Rev. 105, 425–448 (2005).

    Article  CAS  PubMed  Google Scholar 

  38. Schitter, G. & Wrodnigg, T.M. Update on carbohydrate-containing antibacterial agents. Expert Opin. Drug Discov. 4, 315–356 (2009).

    Article  CAS  PubMed  Google Scholar 

  39. Wagner, G.K. & Pesnot, T. Glycosyltransferases and their assays. ChemBioChem 11, 1939–1949 (2010).

    Article  CAS  PubMed  Google Scholar 

  40. Kittl, R. & Withers, S.G. New approaches to enzymatic glycoside synthesis through directed evolution. Carbohydr. Res. 345, 1272–1279 (2010).

    Article  CAS  PubMed  Google Scholar 

  41. Pesnot, T., Palcic, M.M. & Wagner, G.K. A novel fluorescent probe for retaining galactosyltransferases. ChemBioChem 11, 1392–1398 (2010).

    Article  CAS  PubMed  Google Scholar 

  42. Romero, P.A. & Arnold, F.H. Exploring protein fitness landscapes by directed evolution. Nat. Rev. Mol. Cell Biol. 10, 866–876 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Williams, G.J., Gantt, R.W. & Thorson, J.S. The impact of enzyme engineering upon natural product glycodiversfication. Curr. Opin. Chem. Biol. 12, 556–564 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Hancock, S.M., Vaughan, M.D. & Withers, S.G. Engineering of glycosidases and glycosyltransferases. Curr. Opin. Chem. Biol. 10, 509–519 (2006).

    Article  CAS  PubMed  Google Scholar 

  45. Zhang, B. et al. Golgi nucleotide sugar transporter modulates cell wall biosynthesis and plant growth in rice. Proc. Natl. Acad. Sci. USA 108, 5110–5115 (2011).

    Article  CAS  PubMed  Google Scholar 

  46. Dickmanns, A. et al. Structural basis for the broad substrate range of the UDP-sugar pyrophosphorylase from Leishmania major. J. Mol. Biol. 405, 461–478 (2011).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This manuscript is dedicated to the late C. Richard Hutchinson for his pioneering contributions to engineered natural product glycosylation. We thank the School of Pharmacy Analytical Instrumentation Center (University of Wisconsin–Madison) for analytical support and G.J. Williams (North Carolina State University) for materials and helpful discussion. R.W.G. is an American Foundation for Pharmaceutical Education Pre-Doctoral Fellow. J.S.T. is a University of Wisconsin H.I. Romnes Fellow and holds the Laura and Edward Kremers Chair in Natural Products. This work was supported by US National Institutes of Health grant AI52218.

Author information

Authors and Affiliations

  1. Pharmaceutical Sciences Division, School of Pharmacy, Wisconsin Center for Natural Products Research, University of Wisconsin–Madison, Madison, Wisconsin, USA

    Richard W Gantt, Pauline Peltier-Pain, William J Cournoyer & Jon S Thorson

Authors
  1. Richard W Gantt
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Pauline Peltier-Pain
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. William J Cournoyer
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jon S Thorson
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

R.W.G., P.P.-P. and J.S.T. contributed to the experimental design. R.W.G., P.P.-P. and W.J.C. performed all experimental work. R.W.G., P.P.-P. and J.S.T. wrote and edited the manuscript.

Corresponding author

Correspondence to Jon S Thorson.

Ethics declarations

Competing interests

J.S.T. is a co-founder of Centrose (Madison, Wisconsin, USA).

Supplementary information

Supplementary Text and Figures

Supplementary Methods and Supplementary Results (PDF 15098 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Gantt, R., Peltier-Pain, P., Cournoyer, W. et al. Using simple donors to drive the equilibria of glycosyltransferase-catalyzed reactions. Nat Chem Biol 7, 685–691 (2011). https://doi.org/10.1038/nchembio.638

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchembio.638

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing