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

DOI: 10.1038/nchembio.2394 DOI Help

Authors: Javier Iglesias-fernandez (Universitat de Barcelona) , Susan M. Hancock (University of Oxford) , Seung Seo Lee (University of Oxford) , Maola Khan (University of Oxford) , Jo Kirkpatrick (University of Oxford) , Neil J. Oldham (University of Oxford) , Katherine Mcauley (Diamond Light Source) , Anthony Fordham-skelton (CLRC) , Carme Rovira (Institucio Catalana de Recerca i Estudis Avancats (ICREA)) , Benjamin G. Davis (University of Oxford)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Nature Chemical Biology

State: Published (Approved)
Published: June 2017

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.

Journal Keywords: Carbohydrates; Computational chemistry; Enzyme mechanisms; Protein design; X-ray crystallography

Subject Areas: Chemistry

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