6FWM

Structure of the GH99 endo-alpha-mannanase from Bacteroides xylanisolvens in complex with alpha-Glc-1,3-1,2-anhydro-mannose hydrolyzed by enzyme

Summary for 6FWM

• Entry DOI: 10.2210/pdb6fwm/pdb
• Related: 6FWG 6FWI 6FWJ 6FWL 6FWO 6FWP 6FWQ
• Descriptor: Glycosyl hydrolase family 71, ACETATE ION, (1~{S},2~{R},3~{S},4~{R},5~{R})-5-(hydroxymethyl)cyclohexane-1,2,3,4-tetrol, ... (5 entities in total)
• Functional Keywords: hydrolase
• Biological source: Bacteroides xylanisolvens XB1A
• Total number of polymer chains: 1
• Total formula weight: 44350.98

Authors

Sobala, L.F.,Speciale, G.,Hakki, Z.,Fernandes, P.Z.,Raich, L.,Rojas-Cervellera, V.,Bennet, A.,Thompson, A.J.,Bernardo-Seisdedos, G.,Millet, O.,Zhu, S.,Lu, D.,Sollogoub, M.,Rovira, C.,Jimenez-Barbero, J.,Davies, G.J.,Williams, S.J. (deposition date: 2018-03-06, release date: 2019-09-18, Last modification date: 2024-01-17)

Primary citation

Sobala, L.F.,Speciale, G.,Zhu, S.,Raich, L.,Sannikova, N.,Thompson, A.J.,Hakki, Z.,Lu, D.,Shamsi Kazem Abadi, S.,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.
An Epoxide Intermediate in Glycosidase Catalysis.
Acs Cent.Sci., 6:760-770, 2020

PubMed 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 -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 ( ) conformation. Kinetic isotope effects ( / ) for anomeric-H and anomeric-C support an oxocarbenium ion-like transition state, and that for C2-O (1.052 ± 0.006) directly implicates nucleophilic participation by the C2-hydroxyl. Collectively, these data substantiate this unprecedented and long-imagined enzymatic mechanism.

PubMed: 32490192
DOI: 10.1021/acscentsci.0c00111

Experimental method

X-RAY DIFFRACTION (1.28 Å)