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Article Dans Une Revue ACS Central Science Année : 2020

An Epoxide Intermediate in Glycosidase Catalysis

Lukasz Sobala
  • Fonction : Auteur
Gaetano Speciale
  • Fonction : Auteur
Sha Zhu
  • Fonction : Auteur
Lluı́s Raich
  • Fonction : Auteur
Natalia Sannikova
  • Fonction : Auteur
Andrew Thompson
Zalihe Hakki
  • Fonction : Auteur
Dan Lu
  • Fonction : Auteur
Saeideh Shamsi Kazem Abadi
  • Fonction : Auteur
Andrew Lewis
  • Fonction : Auteur
Vı́ctor Rojas-Cervellera
  • Fonction : Auteur
Ganeko Bernardo-Seisdedos
  • Fonction : Auteur
Oscar Millet
Jesús Jiménez-Barbero
Andrew Bennet
Carme Rovira
Gideon Davies
Spencer Williams

Résumé

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.

Domaines

Chimie

Dates et versions

hal-04049786 , version 1 (28-03-2023)

Identifiants

Citer

Lukasz Sobala, Gaetano Speciale, Sha Zhu, Lluı́s Raich, Natalia Sannikova, et al.. An Epoxide Intermediate in Glycosidase Catalysis. ACS Central Science, 2020, 6 (5), pp.760-770. ⟨10.1021/acscentsci.0c00111⟩. ⟨hal-04049786⟩
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