Article | Published:

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

Nature Chemical Biology volume 13, pages 874881 (2017) | Download Citation

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.

  • Compound

    p-nitrophenyl β-D-galactopyranoside

  • Compound

    p-nitrophenyl β-D-glucopyranoside

  • Compound

    p-nitrophenyl β-D-2-acetamido-2-deoxy-glucopyranoside

  • Compound

    2-methyl-4,5-(2-deoxy-α-D-glucopyrano)[2,1-d]-D2-oxazoline

  • Compound

    methyl β-D-galactopyranoside

  • Compound

    p-nitrophenyl 6-O-(β-D-galactopyranosyl)-β-D-galactopyranoside

  • Compound

    2,4-dinitrophenyl 2-deoxy-2-fluoro-β-D-glucopyranoside

  • Compound

    4-O-(β-D-glucopyranosyl)-D-glucopyranose

  • Compound

    4-O-(β-D-galactopyranosyl)-D-glucopyranose

  • Compound

    methyl β-D-mannopyranoside

  • Compound

    phenyl β-D-glucopyranoside

  • Compound

    phenyl α-D-mannopyranoside

  • Compound

    p-nitrophenyl 3-O-(β-D-galactopyranosyl)-β-D-galactopyranoside

  • Compound

    phenyl 6-O-(β-D-galactopyranosyl)-α-D-mannopyranoside

  • Compound

    phenyl 6-O-(β-D-galactopyranosyl)-β-D-glucopyranoside

  • Compound

    methyl 6-O-(β-D-galactopyranosyl)-β-D-galactopyranoside

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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

Author notes

    • Javier Iglesias-Fernández
    • , Jo Kirkpatrick
    •  & Neil J Oldham

    Present addresses: Institut de Quimica Computacional i Catalisi and Departament de Quimica, Universitat de Girona, Girona, Spain (J.I.-F.), School of Chemistry, University of Nottingham, Nottingham, UK (N.J.O.) and Leibniz Institute on Aging–Fritz Lipmann Institute (FLI), Jena, Germany (J.K.).

    • Anthony Fordham-Skelton

    Deceased.

    • Javier Iglesias-Fernández
    • , Susan M Hancock
    •  & Seung Seo Lee

    These authors contributed equally to this work.

Affiliations

  1. Departament de Química Inorgànica i Orgànica (Secció de Química Orgànica), Universitat de Barcelona, Barcelona, Spain.

    • Javier Iglesias-Fernández
    •  & Carme Rovira
  2. Institut de Quimica Teorica i Computacional (IQTCUB), Universitat de Barcelona, Barcelona, Spain.

    • Javier Iglesias-Fernández
    •  & Carme Rovira
  3. Department of Chemistry, University of Oxford, Oxford, UK.

    • Susan M Hancock
    • , Seung Seo Lee
    • , Maola Khan
    • , Jo Kirkpatrick
    • , Neil J Oldham
    •  & Benjamin G Davis
  4. School of Chemistry, University of Southampton, Southampton, UK.

    • Seung Seo Lee
  5. Diamond Light Source, Didcot, UK.

    • Katherine McAuley
  6. CLRC, Daresbury Laboratory, Warrington, UK.

    • Anthony Fordham-Skelton
  7. Institucio Catalana de Recerca i Estudis Avancats (ICREA), Barcelona, Spain.

    • Carme Rovira

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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.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Carme Rovira or Benjamin G Davis.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Results, Supplementary Tables 1–7 and Supplementary Figures 1–21

  2. 2.

    Supplementary Note

    General synthetic methods

Videos

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    Metadynamics trajectory of the transglycosylation reaction

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DOI

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

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