TY  - JOUR
AU  - Sobala, LF
AU  - Speciale, G
AU  - Zhu, S
AU  - Raich, L
AU  - Sannikova, N
AU  - Thompson, AJ
AU  - Hakki, Z
AU  - Lu, D
AU  - Abadi, SSK
AU  - Lewis, AR
AU  - Rojas-Cervellera, V
AU  - Bernardo-Seisdedos, G
AU  - Zhang, Y
AU  - Millet, O
AU  - Jimenez-Barbero, J
AU  - Bennet, AJ
AU  - Sollogoub, M
AU  - Rovira, C
AU  - Davies, GJ
AU  - Williams, SJ
Y2  - 2020/12/14
Y1  - 2020/05/27
SN  - 2374-7943
UR  - http://hdl.handle.net/11343/254181
AB  - 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 (E 3) conformation. Kinetic isotope effects (k cat/K M) 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.
LA  - Chinese
PB  - AMER CHEMICAL SOC
T1  - An Epoxide Intermediate in Glycosidase Catalysis
DO  - 10.1021/acscentsci.0c00111
IS  - ACS Central Science
VL  - 6
IS  - 5
SP  - 760-770
OP  - FT130100103
OP  - DP120101396
OP  - DP180101957
ER  -