5I3D

Sulfolobus solfataricus beta-glycosidase - E387Y mutant

5I3D の概要

• エントリーDOI: 10.2210/pdb5i3d/pdb
• 分子名称: Beta-galactosidase, 3,6,9,12,15,18,21-HEPTAOXATRICOSANE-1,23-DIOL, ACETATE ION, ... (4 entities in total)
• 機能のキーワード: glycosidase, hydrolase
• 由来する生物種: Sulfolobus solfataricus (strain ATCC 35092 / DSM 1617 / JCM 11322 / P2)
• タンパク質・核酸の鎖数: 4
• 化学式量合計: 228429.89

構造登録者

Iglesias-Fernandez, J.,Hancock, S.M.,Lee, S.S.,McAuley, K.E.,Fordham-Skelton, A.,Rovira, C.,Davis, B.D. (登録日: 2016-02-10, 公開日: 2017-02-01, 最終更新日: 2024-01-10)

主引用文献

Iglesias-Fernandez, J.,Hancock, S.M.,Lee, S.S.,Khan, M.,Kirkpatrick, J.,Oldham, N.J.,McAuley, K.,Fordham-Skelton, A.,Rovira, C.,Davis, B.G.
A front-face 'SNi synthase' engineered from a retaining 'double-SN2' hydrolase.
Nat. Chem. Biol., 13:874-881, 2017

PubMed Abstract: Si-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 Si-like, S1 and S2 substitution pathways can be energetically comparable, engineered switching could be feasible. Here, engineering of Sulfolobus solfataricus β-glycosidase, which originally catalyzed double S2 substitution, changed its mode to Si-like. Destruction of the first S2 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, Si-like glycosyltransferases. This observation of a front-face mechanism in a β-glycosyltransfer enzyme highlights that Si-like pathways may be engineered in catalysts with suitable environments and suggests that 'β-Si' mechanisms may be feasible for natural glycosyltransfer enzymes.

PubMed: 28604696
DOI: 10.1038/nchembio.2394

実験手法

X-RAY DIFFRACTION (2.16 Å)