Nature Chemical Biology
Nature Chemical Biology
5 years ago

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

A front-face 'SNi synthase' engineered from a retaining 'double-SN2' hydrolase
Seung Seo Lee, Benjamin G Davis, Jo Kirkpatrick, Javier Iglesias-Fernández, Anthony Fordham-Skelton, Neil J Oldham, Katherine McAuley, Maola Khan, Carme Rovira, Susan M Hancock
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.

Publisher URL: http://dx.doi.org/10.1038/nchembio.2394

DOI: 10.1038/nchembio.2394

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