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Instruments / Technical Area	Publication Details	Published	Actions
I02-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength) I04-Macromolecular Crystallography	An epoxide intermediate in glycosidase catalysis Lukasz F. Sobala , Gaetano Speciale , Sha Zhu , Lluı́s Raich , Natalia Sannikova , Andrew J. Thompson , Zalihe Hakki , Dan Lu , Saeideh Shamsi Kazem Abadi , Andrew R. Lewis , Vı́ctor Rojas-Cervellera , Ganeko Bernardo-Seisdedos , Yongmin Zhang , Oscar Millet , Jesús Jiménez-Barbero , Andrew J. Bennet , Matthieu Sollogoub , Carme Rovira , Gideon J. Davies , Spencer J. Williams Acs Central Science DOI: 10.1021/acscentsci.0c00111 Diamond Proposal Number(s): [9948, 13587] Open Access Show Abstract ▼ 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.	Apr 2020

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I02-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
I04-Macromolecular Crystallography

An epoxide intermediate in glycosidase catalysis