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An epoxide intermediate in glycosidase catalysis

DOI: 10.1021/acscentsci.0c00111 DOI Help

Authors: Lukasz F. Sobala (University of York) , Gaetano Speciale (University of Melbourne) , Sha Zhu (Sorbonne Universite,́ CNRS) , Lluı́s Raich (Universitat de Barcelona) , Natalia Sannikova (Simon Fraser University) , Andrew J. Thompson (University of York) , Zalihe Hakki (University of Melbourne) , Dan Lu (Sorbonne Universite; CNRS) , Saeideh Shamsi Kazem Abadi (Simon Fraser University) , Andrew R. Lewis (Simon Fraser University) , Vı́ctor Rojas-cervellera (Universitatde Barcelona) , Ganeko Bernardo-seisdedos (CIC bioGUNE, Basque Research Technology Alliance (BRTA)) , Yongmin Zhang (Sorbonne Universite,́ CNRS) , Oscar Millet (CIC bioGUNE, Basque Research Technology Alliance (BRTA)) , Jesús Jiménez-barbero (CIC bioGUNE, Basque Research Technology Alliance (BRTA)) , Andrew J. Bennet (Simon Fraser University) , Matthieu Sollogoub (Sorbonne Universite,́ CNRS) , Carme Rovira (Universitat de Barcelona) , Gideon J. Davies (The University of York) , Spencer J. Williams (University of Melbourne)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Acs Central Science

State: Published (Approved)
Published: April 2020
Diamond Proposal Number(s): 9948 , 13587

Open Access Open Access

Abstract: Retaining glycoside hydrolases cleave their substrates through stereochemical retention at the anomeric position. Typically, this involves two-step mechanisms using either an enzymatic nucleophile via a covalent glycosyl enzyme intermediate or neighboring-group participation by a substrate-borne 2-acetamido neighboring group via an oxazoline intermediate; no enzymatic mechanism with participation of the sugar 2-hydroxyl has been reported. Here, we detail structural, computational, and kinetic evidence for neighboring-group participation by a mannose 2-hydroxyl in glycoside hydrolase family 99 endo-α-1,2-mannanases. We present a series of crystallographic snapshots of key species along the reaction coordinate: a Michaelis complex with a tetrasaccharide substrate; complexes with intermediate mimics, a sugar-shaped cyclitol β-1,2-aziridine and β-1,2-epoxide; and a product complex. The 1,2-epoxide intermediate mimic displayed hydrolytic and transfer reactivity analogous to that expected for the 1,2-anhydro sugar intermediate supporting its catalytic equivalence. Quantum mechanics/molecular mechanics modeling of the reaction coordinate predicted a reaction pathway through a 1,2-anhydro sugar via a transition state in an unusual flattened, envelope (E3) conformation. Kinetic isotope effects (kcat/KM) for anomeric-2H and anomeric-13C support an oxocarbenium ion-like transition state, and that for C2-18O (1.052 ± 0.006) directly implicates nucleophilic participation by the C2-hydroxyl. Collectively, these data substantiate this unprecedented and long-imagined enzymatic mechanism.

Journal Keywords: Peptides and proteins; Reaction mechanisms; Carbohydrates; Transition states; Chemical structure

Subject Areas: Chemistry

Instruments: I02-Macromolecular Crystallography , I04-1-Macromolecular Crystallography (fixed wavelength) , I04-Macromolecular Crystallography