An Epoxide Intermediate in Glycosidase Catalysis

Abstract

<div>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, 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 modelling of the reaction coordinate predicted a reaction pathway through a 1,2-anhydro sugar via a transition state in an unprecedented flattened, envelope (<i>E</i><sub>3</sub>) conformation. Kinetic isotope effects for anomeric-<sup>2</sup>H and anomeric-<sup>13</sup>C support an oxocarbenium ion-like transition state and that for C2-<sup>18</sup>O (1.052 ± 0.006) directly implicates nucleophilic participation by the C2-hydroxyl. Collectively, these data substantiate this unprecedented and long-imagined enzymatic mechanism.</div><div><br></div>

Structure of human endo-α-1,2-mannosidase (MANEA), an antiviral host-glycosylation target

“…The catalytic efficiency is similar to that displayed by BtGH99 on the epimeric Man4OMe substrate (KM = 2.6 mM, kcat = 180 min -1 , kcat/KM = 69 mM -1 min -1 ) (31). The E404Q variant of MANEA-Δ97 was inactive on this substrate, consistent with the proposed mechanism (35).…”

Unimolecular, Bimolecular, and Intramolecular Hydrolysis Mechanisms of 4-Nitrophenyl β-d-Glucopyranoside

“…35 Broadly, glycosidases operate at intermediate pH ranges and utilize general catalysis to assist substitution reactions at the anomeric center by water or an enzymatic nucleophile (exceptions occur for outstanding leaving groups such as fluoride and 2,4-dinitrophenolate). 35 However, a mechanism involving neighboring group participation by the 2hydroxyl of an α-mannoside (also likely benefiting from general base catalysis) has been demonstrated for a bacterial endo-α-1,2-mannosidase, 36 which shares obvious similarities to that studied here.…”

Rational Tuning of the Reactivity of Three‐Membered Heterocycle Ring Openings via SN2 Reactions

“…[5] In sharp contrast, aziridine derivatives tend to react faster in enzymatic media, such as in the active sites of glycoside hydrolases, as the NH-group is primed for attack by protonation, [3] with some exceptions. [6] Significantly less is known about the reactivity of phosphiranes and thiiranes, while it has been suggested that thiiranes are more reactive than epoxides. [7] A deeper understanding of the chemistry of nucleophilic ring-opening reactions of these strained three-membered ring systems can guide the design of these irreversible enzyme inhibitors and probes.…”