An Epoxide Intermediate in Glycosidase Catalysis

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 2hydroxyl 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-2 H and anomeric-13 C support an oxocarbenium ion-like transition state, and that for C2-18 O (1.052 ± 0.006) directly implicates nucleophilic participation by the C2-hydroxyl. Collectively, these data substantiate this unprecedented and long-imagined enzymatic mechanism.

Glycoside hydrolase stabilization of transition state charge: new directions for inhibitor design

“…In contrast to previous KIE/theoretical modeling studies using natural substrates, 32,33,35 our study uses a combination of experimental KIE measurements and QM/MM calculations to demonstrate that the GH a-galactosidase from T. maritima efficiently catalyzes its own covalent labeling by allylic carbasugars through an exploded S N 2 TS in which the enzyme stabilizes positive charge development at C5, a position that is far-removed from the anomeric carbon. Of critical importance to inhibitor design, these results show that GHs are capable of stabilizing positive charge at sites that are remote from those that chemists use to position weakly basic amines -specically, the anomeric carbon, ring oxygen and glycosidic oxygenas exemplied by the deoxynojirimycin, isofagomine and validamine families of inhibitors.…”

“…5,[24][25][26] However, despite the tight binding that many carbasugars exhibit towards GHs, it remains controversial as to whether they are indeed TSA inhibitors. 24,[27][28][29] A more theoretically based approach to the design of TSAs has been used and incorporates the measurement of kinetic isotope effects (KIEs) with computational methods to determine the geometric and electronic structure of enzymatic transition states for the reactions involving natural substrates, [30][31][32] and to use this structural information to design of TSA inhibitors. [33][34][35] In contrast to previous work on non-covalent inhibition of GHs, 36,37 we presented studies of small molecule alkenyl, e.g., 4, 38,39 and cyclopropyl 39,40 carbasugar probes that both covalently label GHs and we provided structural insights into the conformations of the enzyme bound and covalent intermediates that are present during GH inactivation (Fig.…”

Role of metagenomics in prospecting novel endoglucanases, accentuating functional metagenomics approach in second-generation biofuel production: a review

“…Cel01 reported by Heiko nacke [151] has enhanced its activity in the presence of glycerol and the rest of the reported enzymes had a declining effect. Generally, proteins are very stable in an aqueous environment with the help of cosolvents and one such co-solvent is glycerol [195].…”

Illuminating glycoscience: synthetic strategies for FRET-enabled carbohydrate active enzyme probes

“…5,9,10 More recently, evidence for a novel epoxide intermediate in glycosidase catalysis has also been reported. 11 Glycosidases have been organised into more than 120 different families based on amino acid sequence similarity and this classification has led to the generation of the CAZy database (Carbohydrate Active Enzyme, available online at www.cazy.org). [12][13][14] Glycosidases grouped within a particular family share structural similarities and often their hydrolytic mechanisms are identical.…”

“…Starting from tetra-N-acetyl-chitopentaose 11 an optimised ligation method was developed to deliver propargyl modified oligosaccharide 12. By using an excess of propargyl amine ([propargylamine] : [11] : [aniline] stoichiometry = 5 : 1 : 0.5) a yield of 70% for 12 was obtained. The authors proposed that the reaction sequence proceeded via rapid formation of an intermediate aniline-iminium ion which then underwent transimination with propargyl amine followed by reduction with NaCNBH 4 to 12.…”