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    An Epoxide Intermediate in Glycosidase Catalysis

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    Author
    Sobala, LF; Speciale, G; Zhu, S; Raich, L; Sannikova, N; Thompson, AJ; Hakki, Z; Lu, D; Abadi, SSK; Lewis, AR; ...
    Date
    2020-05-27
    Source Title
    ACS Central Science
    Publisher
    AMER CHEMICAL SOC
    University of Melbourne Author/s
    Williams, Spencer
    Affiliation
    School of Chemistry
    Metadata
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    Document Type
    Journal Article
    Citations
    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
    Access this item via the Open Access location
    URI
    http://hdl.handle.net/11343/254181
    DOI
    10.1021/acscentsci.0c00111
    Open Access URL
    http://doi.org/10.1021/acscentsci.0c00111
    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.

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