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Bacterial β-glycosidases are hydrolytic enzymes that depolymerize polysaccharides such as β-cellulose, β-glucans and β-xylans from different sources, offering diverse biomedical and industrial uses. It has been shown that a conformational change of the substrate, from a relaxed 4 C 1 conformation to a distorted 1 S 3 / 1,4 B conformation of the reactive sugar, is necessary for catalysis. However, the molecular determinants that stabilize the substrate's distortion are poorly understood. Here we use quantum mechanics/molecular mechanics (QM/MM)-based molecular dynamics methods to assess the impact of the interaction between the reactive sugar, i. e. the one at subsite -1, and the catalytic nucleophile (a glutamate) on substrate conformation. We show that the hydrogen bond involving the C2 exocyclic group and the nucleophile controls substrate conformation: its presence preserves sugar distortion, whereas its absence (e.g. in an enzyme mutant) knocks it out. We also show that 2-deoxy-2-fluoro derivatives, widely used to trap the reaction intermediates by X-ray crystallography, reproduce the conformation of the hydrolysable substrate at the experimental conditions. These results highlight the importance of the 2-OH⋅⋅⋅nucleophile interaction in substrate recognition and catalysis in endo-glycosidases and can inform mutational campaigns aimed to search for more efficient enzymes., (© 2023 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)

AbnA is an extracellular GH43 α-L-arabinanase from Geobacillus stearothermophilus, a key bacterial enzyme in the degradation and utilization of arabinan. We present herein its full-length crystal structure, revealing the only ultra-multimodular architecture and the largest structure to be reported so far within the GH43 family. Additionally, the structure of AbnA appears to contain two domains belonging to new uncharacterized carbohydrate-binding module (CBM) families. Three crystallographic conformational states are determined for AbnA, and this conformational flexibility is thoroughly investigated further using the "integrative structure determination" approach, integrating molecular dynamics, metadynamics, normal mode analysis, small angle X-ray scattering, dynamic light scattering, cross-linking, and kinetic experiments to reveal large functional conformational changes for AbnA, involving up to ~100 Å movement in the relative positions of its domains. The integrative structure determination approach demonstrated here may apply also to the conformational study of other ultra-multimodular proteins of diverse functions and structures., (© 2022. The Author(s).)

Glycosynthases from more than 16 glycosidase families have been developed for the efficient synthesis of oligosaccharides and glycoconjugates. β-1,3-1,4-Glucan oligo- and polysaccharides with defined sequences can be quantitatively achieved with the glycosynthases derived from Bacillus licheniformis β-1,3-1,4-glucanase. The screening of a nucleophile saturation library of this enzyme yielded the unexpected E134D mutant which has high glycosynthase efficiency (25% higher kcat than the best glycosynthase to date, E134S) but also retains some hydrolase activity (2% relative to the wild-type enzyme). Here, we report the biochemical and structural analyses of this mutant compared to E134S and wild-type enzymes. E134D shows a pH profile of general base catalysis for the glycosynthase activity, with a kinetic pKa (on kcat/KM) assigned to Glu138 of 5.8, whereas the same residue acts as a general acid in the hydrolase activity with the same pKa value. The pKa of Glu138 in the wt enzyme was 7.0, a high value due to the presence of the catalytic nucleophile Glu134 which destabilizes the conjugate base of Glu138. Thus, the pKa of Glu138 drops 1.1 pH units in the mutant relative to the wild-type enzyme meaning that the larger distance between carboxylates in positions 138 and 134 (5.6Å for wt, 7.0Å for E134D) and/or a new hydrogen bonding interaction with a third Asp residue (Asp136) in the mutant reduces the effect of the negatively charged Asp134. In consequence, the pKa of Glu138 has a similar pKa value in the E134D mutant than in the other glycosynthase mutants having a neutral residue in position 134. The behavior of the E134D mutant shows that shortening the side chain of the nucleophile, despite maintaining a carboxylate group, confers glycosynthase activity. Therefore E134D is a transitional hydrolase to glycosynthase mutation., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Retaining glycoside hydrolases (GHs), key enzymes in the metabolism of polysaccharides and glycoconjugates and common biocatalysts used in chemoenzymatic oligosaccharide synthesis, operate via a double-displacement mechanism with the formation of a glycosyl-enzyme intermediate. However, the degree of oxocarbenium ion character of the reaction transition state and the precise conformational itinerary of the substrate during the reaction, pivotal in the design of efficient inhibitors, remain elusive for many GHs. By means of QM/MM metadynamics, we unravel the catalytic itinerary of 1,3-1,4-β-glucanase, one of the most active GHs, belonging to family 16. We show that, in the Michaelis complex, the enzyme environment restricts the conformational motion of the substrate to stabilize a (1,4)B/(1)S(3) conformation of the saccharide ring at the -1 subsite, confirming that this distortion preactivates the substrate for catalysis. The metadynamics simulation of the enzymatic reaction captures the complete conformational itinerary of the substrate during the glycosylation reaction ((1,4)B/(1)S(3) -(4)E/(4)H(3) - (4)C(1)) and shows that the transition state is not the point of maximum charge development at the anomeric carbon. The overall catalytic mechanism is of dissociative type, and proton transfer to the glycosidic oxygen is a late event, clarifying previous kinetic studies of this enzyme., (© 2011 American Chemical Society)

Using ab initio metadynamics we have computed the conformational free energy landscape of beta-D-glucopyranose as a function of the puckering coordinates. We show that the correspondence between the free energy and the Stoddard's pseudorotational itinerary for the system is rather poor. The number of free energy minima (9) is smaller than the number of ideal structures (13). Moreover, only six minima correspond to a canonical conformation. The structural features, the electronic properties, and the relative stability of the predicted conformers permit the rationalization of the occurrence of distorted sugar conformations in all the available X-ray structures of beta-glucoside hydrolase Michaelis complexes. We show that these enzymes recognize the most stable distorted conformers of the isolated substrate and at the same time the ones better prepared for catalysis in terms of bond elongation/shrinking and charge distribution. This suggests that the factors governing the distortions present in these complexes are largely dictated by the intrinsic properties of a single glucose unit.

The structure and dynamics of the enzyme-substrate complex of Bacillus 1,3-1,4-beta-glucanase, one of the most active glycoside hydrolases, is investigated by means of Car-Parrinello molecular dynamics simulations (CPMD) combined with force field molecular dynamics (QM/MM CPMD). It is found that the substrate sugar ring located at the -1 subsite adopts a distorted 1S3 skew-boat conformation upon binding to the enzyme. With respect to the undistorted 4C1 chair conformation, the 1S3 skew-boat conformation is characterized by: (a) an increase of charge at the anomeric carbon (C1), (b) an increase of the distance between C1 and the leaving group, and (c) a decrease of the intraring O5-C1 distance. Therefore, our results clearly show that the distorted conformation resembles both structurally and electronically the transition state of the reaction in which the substrate acquires oxocarbenium ion character, and the glycosidic bond is partially broken. Together with analysis of the substrate conformational dynamics, it is concluded that the main determinants of substrate distortion have a structural origin. To fit into the binding pocket, it is necessary that the aglycon leaving group is oriented toward the beta region, and the skew-boat conformation naturally fulfills this premise. Only when the aglycon is removed from the calculation the substrate recovers the all-chair conformation, in agreement with the recent determination of the enzyme product structure. The QM/MM protocol developed here is able to predict the conformational distortion of substrate binding in glycoside hydrolases because it accounts for polarization and charge reorganization at the -1 sugar ring. It thus provides a powerful tool to model E.S complexes for which experimental information is not yet available.