Theoretical study on the deglycosylation mechanism of rice BGlu1 β‐glucosidase

Abstract

It is proposed that the catalysis of GH1 enzymes follows a double‐displacement mechanism involving a glycosylation and a deglycosylation steps. In this article, the deglycosylation step was studied using quantum mechanical/molecular mechanical (QM/MM) approach. The calculation results reveal that the nucleophilic water (Wat1) attacks to the anomeric C1, and the deglycosylation step experiences a barrier of 21.4 kcal/mol from the glycosyl‐enzyme intermediate to the hydrolysis product, in which an oxocarbenium cation‐like transition state (TS) is formed. At the TS, the covalent glycosyl‐enzyme bond is almost broken (distance of 2.45 Å), and the new covalent bond between the attacking oxygen of the water molecule and C1 is basically established (length of 2.14 Å). In addition, a short hydrogen bridge is observed between the nucleophilic E386 and the C2OH of sugar ring (distance of 1.94 Å) at the TS, which facilitates the ring changing from a chair form to half‐chair form, and stabilizes the oxocarbenium cation‐like TS. © 2013 Wiley Periodicals, Inc.

Investigation of the rescue mechanism catalyzed by a nucleophile mutant of rice BGlu1

“…5d. Two Figures indicate that the initial conformation of C1 atom of pNP-cellotriose is reversed after the reaction, in which the conformation of C1 changes from S to R type, similar to previous study [9,10]. Analyses of the superpositions of nucleophiles (formate and E386) in two systems reveal that orientations of nucleophiles change slightly in the reactants and transition states, but adjust largely in the products.…”

“…The catalytic groups are the carboxylic acid of these key residues, with the reaction proceeding via a covalent glycosyl-enzyme intermediate. The glycosylation and deglycosylation steps of this mechanism have been studied extensively by the quantum mechanical/molecular mechanical (QM/MM) method [9,10]. Mutations of ␤-glucosidase at catalytic glutamate residue in which the carboxylate nucleophile is replaced by a nonnucleophilic residue make the hydrolytic activity decrease to a very low level.…”

Glycosynthesis in a waterworld: new insight into the molecular basis of transglycosylation in retaining glycoside hydrolases

“…step [63,64]. Accordingly, based on the findings of QM/MM it has been postulated that TS2 is more dissociative, meaning that the (C1-nucleophile) bond is almost broken before the nascent C1-OR bond (with OR from acceptor HOR, with R = H for water) is formed [65].…”

Comparing Hydrolysis and Transglycosylation Reactions Catalyzed by Thermus thermophilus β-Glycosidase. A Combined MD and QM/MM Study

“…Computational studies on retaining GHs have mainly been focused on the glycosylation step, fewer to the hydrolysis one and very few to transglycosylation. For enzymes belonging to CAZy family GH1, the one investigated in this work, quantum mechanical molecular mechanical (QM/MM) studies on glycosylation and hydrolysis steps catalyzed by Oryza sativa (rice) β-glucosidase (Osβ-gly) acting on glucose disaccharides have been reported (Wang et al, 2011, 2013; Badieyan et al, 2012). They corroborated the predicted oxocarbenium-like nature of both TSs, which present the corresponding breaking bond already broken and the forming one far from established.…”