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An Epoxide Intermediate in Glycosidase Catalysis

preprint
submitted on 02.09.2019 and posted on 04.09.2019 by Lukasz F. Sobala, Gaetano Speciale, Sha Zhu, Lluís Raich, Natalia Sannikova, Andrew J. Thompson, ZALIHE HAKKI, Dan Lu, Saeideh Shamsi Kazem Abadi, Andrew R. Lewis, Victor Rojas-Cervellera, Ganeko Bernardo-Seisdedos, Yongmin Zhang, Oscar Millet, Jesús Jiménez-Barbero, Andrew J. Bennett, Matthieu Sollogoub, Carme Rovira, Gideon J. Davies, Spencer Williams
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, 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 modelling of the reaction coordinate predicted a reaction pathway through a 1,2-anhydro sugar via a transition state in an unprecedented flattened, envelope (E3) conformation. Kinetic isotope effects 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.

Funding

Dissecting the mechanism and exploring new inhibition strategies for polysaccharide cleaving enzymes

Australian Research Council

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Dissecting catalysis and inhibition of a unique endo-acting mannose-processing glycosidase

Australian Research Council

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Using chemistry to illuminate sulfoglycolysis, a major organosulfur pathway

Australian Research Council

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ERC-2012-AdG-322942

BBSRC

Diamond Light Source

Agency for Management of University and Research Grants of Generalitat de Catalunya

Natural Sciences and Engineering Research Council of Canada

History

Email Address of Submitting Author

sjwill@unimelb.edu.au

Institution

University of Melbourne

Country

Australia

ORCID For Submitting Author

0000-0001-6341-4364

Declaration of Conflict of Interest

No conflict of interest

Version Notes

Version 1.1

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in ACS Central Science

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