Insights into Glucose-6-phosphate Allosteric Activation of β-Glucosidase A

Abstract

Second-generation ethanol production involves the use of agricultural and forestry waste as feedstock, being an alternative to the first-generation technology as it relies on low-cost abundant residues and does not affect food agriculture. However, the success of second-generation biorefineries relies on energetically efficient processes and effective enzyme cocktails to convert cellulose into fermentable sugars. β-glucosidases catalyze the last step on the enzymatic hydrolysis of cellulose; however, they are often inhibited by glucose. Previous studies demonstrated that glucose-6-phosphate (G6P) is a positive allosteric modulator of Bacillus polymyxa β-glucosidase A, improving enzymatic efficiency, providing thermoresistance, and imparting glucose tolerance. However, the precise molecular details of G6P-β-glucosidase A interactions have not yet been described so far. We investigated the molecular details of G6P binding into B. polymyxa β-glucosidase A through in silico docking using the site identification by ligand competitive saturation technology followed by site-directed mutagenesis studies, from which an allosteric binding site for G6P was identified. In addition, a mechanistic shift toward the transglycosylation reaction as opposed to hydrolysis was observed in the presence of G6P, suggesting a new role of G6P allosteric modulation of the catalytic activity of β-glucosidase A.

In silico identification of a β 2 -adrenoceptor allosteric site that selectively augments canonical β 2 AR-Gs signaling and function

“…Notably, the SILCS-Hotspots method allows for docking a collection of fragments on the entire protein to identify putative binding sites suitable for drug-like molecules ( 30 ) in a highly computational efficient fashion with a level of accuracy comparable with other state-of-the-art computational methods such as free energy perturbation ( 44 ). The present results further represent the capability of the SILCS method to identify allosteric binding sites showing, notably, its utility against a GPCR ( 30 , 32 , 35 ). The technology takes advantage of the inclusion of protein flexibility in SILCS combined with the use of GCMC water and solute sampling to identify possible binding sites on the interior of a protein not evident in experimental crystallographic or cryo-EM structures.…”

“…Notably, the SILCS-Hotspots method allows for docking a collection of fragments on the entire protein to identify putative binding sites suitable for drug-like molecules ( 30 ). The SILCS approach has previously been used to identify allosteric sites on the proteins heme oxygenase ( 31 ) and β-Glucosidase A ( 32 ). In addition, the SILCS-Pharmacophore ( 33 ) and Monte Carlo (SILCS-MC) ( 34 ) methods are of utility for identifying drug-like molecules that bind to the targeted sites, an approach we have previously used to identify novel agonists of the β 2 AR ( 35 ).…”

SILCS-RNA: Toward a Structure-Based Drug Design Approach for Targeting RNAs with Small Molecules

“…Selection of these sites includes one or more adjacent hotspots without steric hindrance between them based on the exclusion maps that would preclude linking fragments between those sites. The success of this approach was verified by the identification of known allosteric and orthosteric binding sites in proteins and the identification of novel allosteric sites in β-glucosidase A …”

“…The success of this approach was verified by the identification of known allosteric and orthosteric binding sites in proteins 75 and the identification of novel allosteric sites in β-glucosidase A. 108 Visual analysis was thus undertaken to identify novel putative binding sites followed by the application of SILCS-MC docking on a database of 380 chemically diverse FDA approved compounds to quantitatively evaluate the suitability that could be subjected to subsequent docking calculations and experimental validation.…”

Identifying and Assessing Putative Allosteric Sites and Modulators for CXCR4 Predicted through Network Modeling and Site Identification by Ligand Competitive Saturation

“…SILCS-Hotspots was used for identifying and characterizing putative allosteric binding sites. − The approach is based on the SILCS-MC docking protocol in which translation, rotational, and dihedral degrees of freedom of ligands are subjected to MC sampling using the LGFE plus the CGenFF intramolecular energy with a 4r distance-dependent dielectric constant for the electrostatic interactions as the Metropolis criteria, as previously described . In SILCS-Hotspots SILCS-MC docking of chemical fragment molecules is conducted on the entire simulation box partition into 10 Å 3 subvolumes with each fragment docked 1000 times in each subspace.…”