Publication
Article Metrics
Citations
Online attention
An epoxide intermediate in glycosidase catalysis
DOI:
10.1021/acscentsci.0c00111
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

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
Documents:
acscentsci.0c00111.pdf