Substrate Distortion in the Michaelis Complex of Bacillus 1,3–1,4-β-Glucanase

Abstract

The structure and dynamics of the enzyme-substrate complex of Bacillus 1,3-1,4-␤-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 1 S 3 skew-boat conformation upon binding to the enzyme. With respect to the undistorted 4 C 1 chair conformation, the 1 S 3 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 ␤ 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 E⅐S complexes for which experimental information is not yet available. Glycoside hydrolases (GHs)2 are the enzymes responsible for the hydrolysis of glycosidic bonds and play important biological functions such as glycan processing in glycoproteins, remodeling the cell walls, and polysaccharide modification and degradation. The reaction mechanism, a classical textbook example of enzymatic reaction, has attracted much interest because genetically inherited disorders of glycoside hydrolysis often occur and because inhibitors of these enzymes can act as new therapeutic agents for the treatment of viral infections (1, 2). Despite the large number of GHs known, classified into more than 90 families (1), the catalytic mechanism is similar. They typically operate by means of acid/base catalysis with retention or inversion of the anomeric configuration, although a different mechanism has recently been proposed for the GH family 4 (3). The acid/base reaction is assisted by two essential residues: a proton donor and a nucleophile or general base residue (4). Inverting enzymes operate by a single nucleophilic substitution, whereas retaining glycosidases follow a double displacement mechanism via formation and hydrolysis of a covalent glycosyl-enzyme intermediate. Both steps involve oxocarbenium ion-like transition states (F...

Molecular simulations of carbohydrates and protein–carbohydrate interactions: motivation, issues and prospects

“…This equilibrium is akin to an interconversion between α- and β-anomers, which is not straightforward to simulate with force fields that employ unique torsion terms for α- and β-anomers. This may also be expected to be a significant issue when simulating oligosaccharides bound to carbohydrate-degrading enzymes, in which non-standard ring conformations are a hallmark of the transition state [108]. In point of fact, if the transition state is a half chair, where the anomeric center is neither axial (α-anomer in most pyranoses) nor equatorial (β-anomer in most pyranoses), it is not immediately clear which anomeric parameter set should be employed.…”

Mechanism of Cellulose Hydrolysis by Inverting GH8 Endoglucanases: A QM/MM Metadynamics Study

“…This functional was chosen based on its reliability in describing hydrogen bonds, 44 and it is the one that we used in our previous QM/ MM work on a retaining GH. 8 A constant temperature of 300 K was reached by coupling the system to a Nosé-Hoover thermostat 45 of 3500 cm -1 frequency.…”

“…5,6 The conformational interconversions that occur in the active sites of specific GHs have been the subject of many recent studies. [7][8][9][10][11][12][13][14] Nevertheless, direct evidence of conformational itineraries is still missing for many GH families. Furthermore, detailed structural features of the enzymatic transition state (TS) in GHs are still fairly unknown.…”

β-d-Xylosidase from Selenomonas ruminantium: Role of Glutamate 186 in Catalysis Revealed by Site-Directed Mutagenesis, Alternate Substrates, and Active-Site Inhibitor

“…The strategy was first applied to glycoside hydrolases in the case of SXA acting on 4NPX and 4NPA [11]. The energy barrier for conformational inversion of pyranoses (10 kcal/mol) is considerably higher than that of furanoses (3-4 kcal/mol) [17], and distortion of pyranoside substrates from the ground-state chair conformation is well recognized as a major task for the catalyst [22][23][24][25][26][27][28][29][30]. SXA active-site residues R290 and F31, important for promoting both 4NPA and 4NPX reactions, were identified as being more important for the SXA-catalyzed hydrolysis of 4NPX than that of 4NPA, strongly suggesting involvement of the two residues in promoting conformational inversion.…”