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

Biochemical and structural analysis, combined with metadynamics simulations, illustrate how a single amino acid substitution switches a β-glycosidase from a double S N 2 mechanism to a front-face S N i-like mechanism. S N i-like mechanisms, which involve front-face leaving group departure and nucleo...

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Veröffentlicht in:Nature chemical biology 2017-08, Vol.13 (8), p.874-881
Hauptverfasser: Iglesias-Fernández, Javier, Hancock, Susan M, Lee, Seung Seo, Khan, Maola, Kirkpatrick, Jo, Oldham, Neil J, McAuley, Katherine, Fordham-Skelton, Anthony, Rovira, Carme, Davis, Benjamin G
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Sprache:eng
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Zusammenfassung:Biochemical and structural analysis, combined with metadynamics simulations, illustrate how a single amino acid substitution switches a β-glycosidase from a double S N 2 mechanism to a front-face S N i-like mechanism. S N 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 S N i-like, S N 1 and S N 2 substitution pathways can be energetically comparable, engineered switching could be feasible. Here, engineering of Sulfolobus solfataricus β-glycosidase, which originally catalyzed double S N 2 substitution, changed its mode to S N i-like. Destruction of the first S N 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, S N i-like glycosyltransferases. This observation of a front-face mechanism in a β-glycosyltransfer enzyme highlights that S N i-like pathways may be engineered in catalysts with suitable environments and suggests that 'β-S N i' mechanisms may be feasible for natural glycosyltransfer enzymes.
ISSN:1552-4450
1552-4469
DOI:10.1038/nchembio.2394