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