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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.

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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.

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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.

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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.

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Correspondence to Carme Rovira or Benjamin G Davis.

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

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General synthetic methods (PDF 587 kb)

Metadynamics trajectory of the transglycosylation reaction (MOV 5273 kb)

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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

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