5i3d

Publication Abstract from PubMed

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 alpha-glycosyl electrophiles. Since SNi-like, SN1 and SN2 substitution pathways can be energetically comparable, engineered switching could be feasible. Here, engineering of Sulfolobus solfataricus beta-glycosidase, which originally catalyzed double SN2 substitution, changed its mode to SNi-like. Destruction of the first SN2 nucleophile through E387Y mutation created a beta-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 pi-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 beta-glycosyltransfer enzyme highlights that SNi-like pathways may be engineered in catalysts with suitable environments and suggests that 'beta-SNi' mechanisms may be feasible for natural glycosyltransfer enzymes.

A front-face 'SNi synthase' engineered from a retaining 'double-SN2' hydrolase.,Iglesias-Fernandez J, Hancock SM, Lee SS, Khan M, Kirkpatrick J, Oldham NJ, McAuley K, Fordham-Skelton A, Rovira C, Davis BG Nat Chem Biol. 2017 Jun 12. doi: 10.1038/nchembio.2394. PMID:28604696^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.