An Epoxide Intermediate in Glycosidase Catalysis
Author
Sobala, LF; Speciale, G; Zhu, S; Raich, L; Sannikova, N; Thompson, AJ; Hakki, Z; Lu, D; Abadi, SSK; Lewis, AR; ...Date
2020-05-27Source Title
ACS Central SciencePublisher
AMER CHEMICAL SOCUniversity of Melbourne Author/s
Williams, SpencerAffiliation
School of ChemistryMetadata
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Journal ArticleCitations
Sobala, L. F., Speciale, G., Zhu, S., Raich, L., Sannikova, N., Thompson, A. J., Hakki, Z., Lu, D., Abadi, S. S. K., Lewis, A. R., Rojas-Cervellera, V., Bernardo-Seisdedos, G., Zhang, Y., Millet, O., Jimenez-Barbero, J., Bennet, A. J., Sollogoub, M., Rovira, C., Davies, G. J. & Williams, S. J. (2020). An Epoxide Intermediate in Glycosidase Catalysis. ACS CENTRAL SCIENCE, 6 (5), pp.760-770. https://doi.org/10.1021/acscentsci.0c00111.Access Status
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http://doi.org/10.1021/acscentsci.0c00111Abstract
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.
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