|Área de investigación||Química y Ciencia y Tecnología de los Materiales|
|Título||An Epoxide Intermediate in Glycosidase Catalysis|
|Tipo de publicación||Artículo de revista|
|Autores||Sobala, L. F., G. Speciale, S. Zhu, L. Raich, N. Sannikova, A. J. Thompson, Z. Hakki, D. Lu, S. Shamsi Kaz Abadi, A. R. Lewis, V. Rojas-Cervellera, G. Bernardo-Seisdedos, Y. Zhang, O. Millet, J. Jimenez-Barbero, A. J. Bennet, M. Sollogoub, C. Rovira, G. J. Davies, y S. J. Williams|
|Revista||ACS CENTRAL SCIENCE|
|Type of Article||Article|
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-alpha-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 beta-1,2-aziridine and beta-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 (E-3) conformation. Kinetic isotope effects (k(cat)/K-M) for anomeric-H-2 and anomeric-C-1(3) support an oxocarbenium ion-like transition state, and that for C2-O-18 (1.052 +/- 0.006) directly implicates nucleophilic participation by the C2-hydroxyl. Collectively, these data substantiate this unprecedented and long-imagined enzymatic mechanism.