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.

A front-face 'SNi synthase' engineered from a retaining 'double-SN2' hydrolase

Abstract

SNi-like mechanisms, which involve front-face leaving group departure and nucleophile approach, have been observed experimentally and computationally in chemical and enzymatic substitution at α-glycosyl electrophiles. Since SNi-like, SN1 and SN2 substitution pathways can be energetically comparable, engineered switching could be feasible. Here, engineering of Sulfolobus solfataricus β-glycosidase, which originally catalyzed double SN2 substitution, changed its mode to SNi-like. Destruction of the first SN2 nucleophile through E387Y mutation created a β-stereoselective catalyst for glycoside synthesis from activated substrates, despite lacking a nucleophile. The pH profile, kinetic and mutational analyses, mechanism-based inactivators, X-ray structure and subsequent metadynamics simulations together suggest recruitment of substrates by π–sugar interaction and reveal a quantum mechanics–molecular mechanics (QM/MM) free-energy landscape for the substitution reaction that is similar to those of natural, SNi-like glycosyltransferases. This observation of a front-face mechanism in a β-glycosyltransfer enzyme highlights that SNi-like pathways may be engineered in catalysts with suitable environments and suggests that 'β-SNi' mechanisms may be feasible for natural glycosyltransfer enzymes.

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

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

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

Figure 1: Comparisons of front-face glycosyl transfer.
Figure 2: Mass spectrometric analysis of incubation of SsβG-E387Y with covalent inhibitor DNP-2FGlc.
Figure 3: Structural analysis of SSβG-E387Y.
Figure 4: Analysis of the SNi reaction pathway.

Similar content being viewed by others

Accession codes

Primary accessions

Protein Data Bank

Referenced accessions

Protein Data Bank

References

  1. Sinnott, M.L. & Jencks, W.P. Solvolysis of D-Glucopyranosyl derivatives in mixtures of ethanol and 2,2,2-trifluoroethanol. J. Am. Chem. Soc. 102, 2026–2032 (1980).

    CAS  Google Scholar 

  2. Persson, K. et al. Crystal structure of the retaining galactosyltransferase LgtC from Neisseria meningitidis in complex with donor and acceptor sugar analogs. Nat. Struct. Biol. 8, 166–175 (2001).

    CAS  PubMed  Google Scholar 

  3. Gibson, R.P., Turkenburg, J.P., Charnock, S.J., Lloyd, R. & Davies, G.J. Insights into trehalose synthesis provided by the structure of the retaining glucosyltransferase OtsA. Chem. Biol. 9, 1337–1346 (2002).

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  5. Koshland, D.E. Stereochemistry and the mechanism of enzymatic reactions. Biol. Rev. Camb. Philos. Soc. 28, 416–436 (1953).

    CAS  Google Scholar 

  6. Lewis, E.S. & Boozer, C.E. The kinetics and stereochemistry of the decomposition of secondary alkyl chlorosulfites1. J. Am. Chem. Soc. 74, 308–311 (1952).

    CAS  Google Scholar 

  7. Chan, J., Tang, A. & Bennet, A.J. A stepwise solvent-promoted SNi reaction of α-D-glucopyranosyl fluoride: mechanistic implications for retaining glycosyltransferases. J. Am. Chem. Soc. 134, 1212–1220 (2012).

    CAS  PubMed  Google Scholar 

  8. Vetting, M.W., Frantom, P.A. & Blanchard, J.S. Structural and enzymatic analysis of MshA from Corynebacterium glutamicum: substrate-assisted catalysis. J. Biol. Chem. 283, 15834–15844 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Batt, S.M. et al. Acceptor substrate discrimination in phosphatidyl-myo-inositol mannoside synthesis: structural and mutational analysis of mannosyltransferase Corynebacterium glutamicum PimB′. J. Biol. Chem. 285, 37741–37752 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Chaikuad, A. et al. Conformational plasticity of glycogenin and its maltosaccharide substrate during glycogen biogenesis. Proc. Natl. Acad. Sci. USA 108, 21028–21033 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Yu, H. et al. Notch-modifying xylosyltransferase structures support an SNi-like retaining mechanism. Nat. Chem. Biol. 11, 847–854 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Errey, J.C. et al. Mechanistic insight into enzymatic glycosyl transfer with retention of configuration through analysis of glycomimetic inhibitors. Angew. Chem. Int. Ed. Engl. 49, 1234–1237 (2010).

    CAS  PubMed  Google Scholar 

  13. 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).

    CAS  PubMed  Google Scholar 

  14. Ardèvol, A. & Rovira, C. The molecular mechanism of enzymatic glycosyl transfer with retention of configuration: evidence for a short-lived oxocarbenium-like species. Angew. Chem. Int. Ed. Engl. 50, 10897–10901 (2011).

    PubMed  Google Scholar 

  15. Goedl, C. & Nidetzky, B. Sucrose phosphorylase harbouring a redesigned, glycosyltransferase-like active site exhibits retaining glucosyl transfer in the absence of a covalent intermediate. ChemBioChem 10, 2333–2337 (2009).

    CAS  PubMed  Google Scholar 

  16. Gómez, H., Polyak, I., Thiel, W., Lluch, J.M. & Masgrau, L. Retaining glycosyltransferase mechanism studied by QM/MM methods: lipopolysaccharyl-α-1,4-galactosyltransferase C transfers α-galactose via an oxocarbenium ion-like transition state. J. Am. Chem. Soc. 134, 4743–4752 (2012).

    PubMed  Google Scholar 

  17. Bobovská, A., Tvaroška, I. & Kónňa, J. A theoretical study on the catalytic mechanism of the retaining α-1,2-mannosyltransferase Kre2p/Mnt1p: the impact of different metal ions on catalysis. Org. Biomol. Chem. 12, 4201–4210 (2014).

    PubMed  Google Scholar 

  18. Lira-Navarrete, E. et al. Substrate-guided front-face reaction revealed by combined structural snapshots and metadynamics for the polypeptide N-acetylgalactosaminyltransferase 2. Angew. Chem. Int. Ed. Engl. 53, 8206–8210 (2014).

    CAS  PubMed  Google Scholar 

  19. Gómez, H. et al. A computational and experimental study of O-glycosylation. Catalysis by human UDP-GalNAc polypeptide:GalNAc transferase-T2. Org. Biomol. Chem. 12, 2645–2655 (2014).

    PubMed  PubMed Central  Google Scholar 

  20. Albesa-Jové, D. et al. A native ternary complex trapped in a crystal reveals the catalytic mechanism of a retaining glycosyltransferase. Angew. Chem. Int. Ed. Engl. 54, 9898–9902 (2015).

    PubMed  Google Scholar 

  21. Corbett, K., Fordham-Skelton, A.P., Gatehouse, J.A. & Davis, B.G. Tailoring the substrate specificity of the beta-glycosidase from the thermophilic archaeon Sulfolobus solfataricus. FEBS Lett. 509, 355–360 (2001).

    CAS  PubMed  Google Scholar 

  22. Hancock, S.M., Corbett, K., Fordham-Skelton, A.P., Gatehouse, J.A. & Davis, B.G. Developing promiscuous glycosidases for glycoside synthesis: residues W433 and E432 in Sulfolobus solfataricus beta-glycosidase are important glucoside- and galactoside-specificity determinants. ChemBioChem 6, 866–875 (2005).

    CAS  PubMed  Google Scholar 

  23. Trincone, A., Improta, R. & Gambacorta, G. Enzymatic synthesis of polyol and masked polyol glucosides using b-glycosidase of Sulolobus solfataricus. Biocatal. Biotransformation 12, 77–88 (1995).

    CAS  Google Scholar 

  24. Trincone, A. et al. Enzyme catalyzed synthesis of alkyl beta-D-glycosides with crude homogenate of Sulfolobus solfataricus. Biotechnol. Lett. 13, 235–240 (1991).

    CAS  Google Scholar 

  25. Aguilar, C.F. et al. Crystal structure of the beta-glycosidase from the hyperthermophilic archeon Sulfolobus solfataricus: resilience as a key factor in thermostability. J. Mol. Biol. 271, 789–802 (1997).

    CAS  PubMed  Google Scholar 

  26. Trincone, A., Perugino, G., Rossi, M. & Moracci, M. A novel thermophilic glycosynthase that effects branching glycosylation. Bioorg. Med. Chem. Lett. 10, 365–368 (2000).

    CAS  PubMed  Google Scholar 

  27. Mackenzie, L.F., Wang, Q., Warren, R.A.J. & Withers, S.G. Glycosynthases: mutant glycosidases for oligosaccharide synthesis. J. Am. Chem. Soc. 120, 5583–5584 (1998).

    CAS  Google Scholar 

  28. Zhang, Z. et al. Programmable one-pot oligosaccharide synthesis. J. Am. Chem. Soc. 121, 734–753 (1999).

    CAS  Google Scholar 

  29. Williams, S.J., Mark, B.L., Vocadlo, D.J., James, M.N.G. & Withers, S.G. Aspartate 313 in the Streptomyces plicatus hexosaminidase plays a critical role in substrate-assisted catalysis by orienting the 2-acetamido group and stabilizing the transition state. J. Biol. Chem. 277, 40055–40065 (2002).

    CAS  PubMed  Google Scholar 

  30. Withers, S.G., Street, I.P., Bird, P. & Dolphin, D.H. 2-deoxy-2-fluoroglucosides: a novel class of mechanism-based glucosidase inhibitors. J. Am. Chem. Soc. 109, 7530–7531 (1987).

    CAS  Google Scholar 

  31. Lopez, R. & Fernandez-Mayoralas, A. Enzymatic β-galactosidation of modified monosaccharides: study of the enzyme selectivity for the acceptor and its application to the synthesis of disaccharides. J. Org. Chem. 59, 737–745 (1994).

    CAS  Google Scholar 

  32. Yamamoto, K. & Davis, B.G. Creation of an α-mannosynthase from a broad glycosidase scaffold. Angew. Chem. Int. Ed. Engl. 51, 7449–7453 (2012).

    CAS  PubMed  Google Scholar 

  33. Petzelbauer, I., Reiter, A., Splechtna, B., Kosma, P. & Nidetzky, B. Transgalactosylation by thermostable beta-glycosidases from Pyrococcus furiosus and Sulfolobus solfataricus. Binding interactions of nucleophiles with the galactosylated enzyme intermediate make major contributions to the formation of new beta-glycosides during lactose conversion. Eur. J. Biochem. 267, 5055–5066 (2000).

    CAS  PubMed  Google Scholar 

  34. Reuter, S., Rusborg Nygaard, A. & Zimmermann, W. beta-Galactooligosaccharide synthesis with beta-galactosidases from Sulfolobus solfataricus, Aspergillus oryzae, and Escherichia coli. Enzyme Microb. Technol. 25, 509–516 (1999).

    CAS  Google Scholar 

  35. Crout, D.H.G. & Vic, G. Glycosidases and glycosyl transferases in glycoside and oligosaccharide synthesis. Curr. Opin. Chem. Biol. 2, 98–111 (1998).

    CAS  PubMed  Google Scholar 

  36. Shim, J.-H., Chen, H.M., Rich, J.R., Goddard-Borger, E.D. & Withers, S.G. Directed evolution of a β-glycosidase from Agrobacterium sp. to enhance its glycosynthase activity toward C3-modified donor sugars. Protein Eng. Des. Sel. 25, 465–472 (2012).

    CAS  PubMed  Google Scholar 

  37. Watts, A.G. et al. Trypanosoma cruzi trans-sialidase operates through a covalent sialyl-enzyme intermediate: tyrosine is the catalytic nucleophile. J. Am. Chem. Soc. 125, 7532–7533 (2003).

    CAS  PubMed  Google Scholar 

  38. Lawson, S.L., Warren, R.A.J. & Withers, S.G. Mechanistic consequences of replacing the active-site nucleophile Glu-358 in Agrobacterium sp. beta-glucosidase with a cysteine residue. Biochem. J. 330, 203–209 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Gloster, T.M. et al. Structural studies of the beta-glycosidase from Sulfolobus solfataricus in complex with covalently and noncovalently bound inhibitors. Biochemistry 43, 6101–6109 (2004).

    CAS  PubMed  Google Scholar 

  40. Laio, A. & Parrinello, M. Escaping free-energy minima. Proc. Natl. Acad. Sci. USA 99, 12562–12566 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Asensio, J.L., Ardá, A., Cañada, F.J. & Jiménez-Barbero, J. Carbohydrate-aromatic interactions. Acc. Chem. Res. 46, 946–954 (2013).

    CAS  PubMed  Google Scholar 

  42. Biarnés, X., Nieto, J., Planas, A. & Rovira, C. Substrate distortion in the Michaelis complex of Bacillus 1,3-1,4-β-glucanase. Insight from first principles molecular dynamics simulations. J. Biol. Chem. 281, 1432–1441 (2006).

    PubMed  Google Scholar 

  43. Ardèvol, A. & Rovira, C. Reaction mechanisms in carbohydrate-active enzymes: glycoside hydrolases and glycosyltransferases. Insights from ab Initio quantum mechanics/molecular mechanics dynamic simulations. J. Am. Chem. Soc. 137, 7528–7547 (2015).

    PubMed  Google Scholar 

  44. Davies, G.J., Planas, A. & Rovira, C. Conformational analyses of the reaction coordinate of glycosidases. Acc. Chem. Res. 45, 308–316 (2012).

    CAS  PubMed  Google Scholar 

  45. Speciale, G., Thompson, A.J., Davies, G.J. & Williams, S.J. Dissecting conformational contributions to glycosidase catalysis and inhibition. Curr. Opin. Struct. Biol. 28, 1–13 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Bottoni, A., Miscione, G.P. & De Vivo, M. A theoretical DFT investigation of the lysozyme mechanism: computational evidence for a covalent intermediate pathway. Proteins 59, 118–130 (2005).

    CAS  PubMed  Google Scholar 

  47. Vocadlo, D.J. & Davies, G.J. Mechanistic insights into glycosidase chemistry. Curr. Opin. Chem. Biol. 12, 539–555 (2008).

    CAS  PubMed  Google Scholar 

  48. Biarnés, X., Ardèvol, A., Iglesias-Fernández, J., Planas, A. & Rovira, C. Catalytic itinerary in 1,3-1,4-β-glucanase unraveled by QM/MM metadynamics. Charge is not yet fully developed at the oxocarbenium ion-like transition state. J. Am. Chem. Soc. 133, 20301–20309 (2011).

    PubMed  Google Scholar 

  49. Berrin, J.-G. Substrate (aglycone) specificity of human cytosolic beta-glucosidase. Biochem. J. 373, 41–48 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. Carter, P. & Wells, J.A. Dissecting the catalytic triad of a serine protease. Nature 332, 564–568 (1988).

    CAS  PubMed  Google Scholar 

  51. Janda, K.D. et al. Chemical selection for catalysis in combinatorial antibody libraries. Science 275, 945–948 (1997).

    CAS  PubMed  Google Scholar 

  52. Schreiber, S.L. Rethinking relationships between natural products. Nat. Chem. Biol. 3, 352 (2007).

    CAS  PubMed  Google Scholar 

  53. An, J., Denton, R.M., Lambert, T.H. & Nacsa, E.D. The development of catalytic nucleophilic substitution reactions: challenges, progress and future directions. Org. Biomol. Chem. 12, 2993–3003 (2014).

    CAS  PubMed  Google Scholar 

  54. Bradford, M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254 (1976).

    CAS  PubMed  Google Scholar 

  55. Dixon, M. The determination of enzyme inhibitor constants. Biochem. J. 55, 170–171 (1953).

    CAS  PubMed  PubMed Central  Google Scholar 

  56. Joshi, M.D. et al. Hydrogen bonding and catalysis: a novel explanation for how a single amino acid substitution can change the pH optimum of a glycosidase. J. Mol. Biol. 299, 255–279 (2000).

    CAS  PubMed  Google Scholar 

  57. Vagin, A. & Teplyakov, A. MOLREP: an automated program for molecular replacement. J. Appl. Crystallogr. 30, 1022–1025 (1997).

    CAS  Google Scholar 

  58. Murshudov, G.N., Vagin, A.A. & Dodson, E.J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D Biol. Crystallogr. 53, 240–255 (1997).

    CAS  PubMed  Google Scholar 

  59. McRee, D.E. XtalView/Xfit--A versatile program for manipulating atomic coordinates and electron density. J. Struct. Biol. 125, 156–165 (1999).

    CAS  PubMed  Google Scholar 

  60. Lamzin, V.S. & Wilson, K.S. Automated refinement of protein models. Acta Crystallogr. D Biol. Crystallogr. 49, 129–147 (1993).

    CAS  PubMed  Google Scholar 

  61. Laskowski, R.A., MacArthur, M.W., Moss, D.S. & Thornton, J.M. PROCHECK: a program to check the stereochemical quality of protein structures. J. Appl. Crystallogr. 26, 283–291 (1993).

    CAS  Google Scholar 

  62. Hooft, R.W.W., Vriend, G., Sander, C. & Abola, E.E. Errors in protein structures. Nature 381, 272 (1996).

    CAS  PubMed  Google Scholar 

  63. Evans, P. Scaling and assessment of data quality. Acta Crystallogr. D Biol. Crystallogr. 62, 72–82 (2006).

    PubMed  Google Scholar 

  64. McCoy, A.J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  65. Emsley, P., Lohkamp, B., Scott, W.G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  66. Afonine, P.V. et al. Towards automated crystallographic structure refinement with phenix.refine. Acta Crystallogr. D Biol. Crystallogr. 68, 352–367 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  67. Davis, I.W. et al. MolProbity: all-atom contacts and structure validation for proteins and nucleic acids. Nucleic Acids Res. 35, W375–W383 (2007).

    PubMed  PubMed Central  Google Scholar 

  68. Pearlman, D.A. et al. AMBER, a package of computer programs for applying molecular mechanics, normal mode analysis, molecular dynamics and free energy calculations to simulate the structural and energetic properties of molecules. Comput. Phys. Commun. 91, 1–41 (1995).

    CAS  Google Scholar 

  69. Cornell, W.D. et al. A second generation force field for the simulation of proteins, nucleic acids, and organic molecules. J. Am. Chem. Soc. 117, 5179–5197 (1995).

    CAS  Google Scholar 

  70. Kirschner, K.N. et al. GLYCAM06: a generalizable biomolecular force field. Carbohydrates. J. Comput. Chem. 29, 622–655 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  71. Barducci, A., Bonomi, M. & Parrinello, M. Metadynamics. Wiley Interdiscip. Rev. Comput. Mol. Sci. 1, 826–843 (2011).

    CAS  Google Scholar 

  72. Phillips, J.C. et al. Scalable molecular dynamics with NAMD. J. Comput. Chem. 26, 1781–1802 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  73. Laio, A., VandeVondele, J. & Rothlisberger, U. A Hamiltonian electrostatic coupling scheme for hybrid Car–Parrinello molecular dynamics simulations. J. Chem. Phys. 116, 6941–6947 (2002).

    CAS  Google Scholar 

  74. Car, R. & Parrinello, M. Unified approach for molecular dynamics and density-functional theory. Phys. Rev. Lett. 55, 2471–2474 (1985).

    CAS  PubMed  Google Scholar 

  75. Lin, I.-C. Library of dispersion-corrected atom-centered potentials for generalized gradient approximation functionals: Elements H, C, N, O, He, Ne, Ar, and Kr. Phys. Rev. B 75, 205131 (2007).

    Google Scholar 

  76. Nosé, S. A molecular dynamics method for simulations in the canonical ensemble. Mol. Phys. 52, 255–268 (1984).

    Google Scholar 

  77. Iannuzzi, M., Laio, A. & Parrinello, M. Efficient exploration of reactive potential energy surfaces using Car-Parrinello molecular dynamics. Phys. Rev. Lett. 90, 238302 (2003).

    PubMed  Google Scholar 

Download references

Acknowledgements

We thank the Engineering and Physical Sciences Research Council (EPSRC) and High Force Research (S.M.H.), the BBSRC (S.S.L., BB/E004350/1), MINECO (grant CTQ2014-55174-P to C.R.) and AGAUR (grant and 2014SGR-987 to C.R.) for funding. B.G.D. was a Royal Society Wolfson Research Merit Award recipient during the course of this work. The authors gratefully acknowledge the computer resources at MareNostrum and the technical support provided by BSC-CNS (RES-QCM-2013-3-0011). We would like to thank the referee who suggested possibly similar roles of aromatic side chains in glycosyl- and terpenyl-processing enzymes that we note in the discussion. This paper is dedicated to the memory of Tony Fordham-Skelton, a friend, mentor and comrade who is still very much missed.

Author information

Authors and Affiliations

Authors

Contributions

J.I.-F. designed and performed calculations. S.M.H., S.S.L., M.K. performed biochemical experiments. S.M.H., M.K., J.K., N.J.O. performed mass spectrometric experiments. S.M.H., K.M., A.F.-S. determined X-ray structures. All authors analyzed results. C.R., S.S.L., B.G.D. wrote the manuscript. All authors except A.F.-S. read and commented on the manuscript.

Corresponding authors

Correspondence to Carme Rovira or Benjamin G Davis.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Tables 1–7 and Supplementary Figures 1–21 (PDF 19515 kb)

Supplementary Note

General synthetic methods (PDF 587 kb)

Metadynamics trajectory of the transglycosylation reaction (MOV 5273 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Iglesias-Fernández, J., Hancock, S., Lee, S. et al. A front-face 'SNi synthase' engineered from a retaining 'double-SN2' hydrolase. Nat Chem Biol 13, 874–881 (2017). https://doi.org/10.1038/nchembio.2394

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchembio.2394

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