A front-face 'S N i synthase' engineered from a retaining 'double-S N 2' hydrolase.

SN i-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 SN i-like, SN 1 and SN 2 substitution pathways can be energetically comparable, engineered switching could be feasible. Here, engineering of Sulfolobus solfataricus β-glycosidase, which originally catalyzed double SN 2 substitution, changed its mode to SN i-like. Destruction of the first SN 2 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, SN i-like glycosyltransferases. This observation of a front-face mechanism in a β-glycosyltransfer enzyme highlights that SN i-like pathways may be engineered in catalysts with suitable environments and suggests that 'β-SN i' mechanisms may be feasible for natural glycosyltransfer enzymes.