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


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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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 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)

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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).

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