A front-face 'S_Ni synthase' engineered from a retaining 'double-S_N2' hydrolase

S_Ni-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 S_Ni-like, S_N1 and S_N2 substitution pathways can be energetically comparable, engineered switching could be feasible. Here, engineering of Sulfolobus solfataricus β-glycosidase, which originally catalyzed double S_N2 substitution, changed its mode to S_Ni-like. Destruction of the first S_N2 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, S_Ni-like glycosyltransferases. This observation of a front-face mechanism in a β-glycosyltransfer enzyme highlights that S_Ni-like pathways may be engineered in catalysts with suitable environments and suggests that 'β-S_Ni' mechanisms may be feasible for natural glycosyltransfer enzymes.