Computational Insights into Glucose Tolerance and Stimulation in a Family 1 β-glucosidase

Abstract

β-Glucosidases catalyze the hydrolysis of cellobiose to glucose during lignocellulosic biomass depolymerization. A significant limitation of many β-glucosidases is product inhibition by glucose, leading to reduced conversion efficiency. However, certain β-glucosidases exhibit tolerance or even stimulation by glucose. The mechanisms underlying this remarkable feature remain poorly elucidated. Here, we employ molecular dynamics simulations to investigate the molecular basis of glucose tolerance and stimulation within the family 1 β-glucosidase from Humicola insolens (HiBgl). Potential of mean force calculations reveal a substantial difference in binding free energies between cellobiose (-12.5 kcal/mol) and glucose (-4.3 kcal/mol) at the HiBgl active site, indicating that the glucose product is a considerably weaker ligand than the cellobiose substrate. These findings are consistent with our observations that HiBgl undergoes conformational changes in its substrate binding site, specifically involving the Trp349 side chain, in the presence of glucose, potentially facilitating glucose expulsion and mitigating product inhibition. Simulations of HiBgl solvated in a 200 mM aqueous glucose environment show that glucose molecules from the bulk solution are capable of penetrating and widening the substrate binding pocket, forming direct interactions with cellobiose in the active site, which may contribute to catalytic stimulation. Additionally, we identify seven distinct secondary glucose binding sites located on the HiBgl surface, spatially distant from the active site, implying a potential role in allosteric regulation. Finally, we demonstrate that glucose at subsite +1 can adopt multiple orientations relative to glucose at subsite -1, a prerequisite for transglycosylation reactions in HiBgl. Our findings elucidate the molecular mechanisms governing HiBgl's glucose tolerance and stimulation, thereby enabling the design of site-directed mutagenesis experiments to improve enzyme efficiency for industrial applications, particularly in biofuel production and oligosaccharide synthesis.

1

(A) Snapshot of HiBgl + cellobiose (system A) showing residue Trp349 as part of subsite +1. (B) Distance between the C1 atom of the glucosyl residue of cellobiose (in green) located at subsite −1 and the CH₂ atom of Trp349, represented as a dotted line in panel A. Snapshot of HiBgl + glucose (system B) with subsite +1 (C) intact and (D) disassembled. (E) Distance between the C1 atom of the glucose (in cyan) and the CH2 atom of Trp349, represented as a dotted line in panels C and D. In panels B and E, different colors represent different independent MD simulations.

2

Distance between glucose and Trp349 in 5 independent simulations of system C, where HiBgl is complexed to glucose initially at the subsite +2. The short initial distances correspond to the glucose molecule bound to subsite +1, where Trp349 is located. We notice that in less than 100 ns the glucose molecule exits the binding pocket, as indicated by the abrupt increase in the distance.

3

Scheme showing the reaction coordinates employed to compute the PMF of (A) cellobiose and (C) glucose dissociation from HiBgl. PMF of (B) cellobiose and (D) glucose dissociation. The enzyme’s affinity for its substrate is higher than for its product, which is consistent with the fact that HiBgl is tolerant to, and not inhibited by, glucose. Error bars were computed by bootstrap error analysis, and are much smaller than the thermal energy.

4

(A) Simulation box showing the aqueous solution of glucose surrounding HiBgl in systems F and G. Distance between the CD atom of Glu377 and (B) cellobiose in system F and (C) the closest glucose molecule in system G. While cellobiose remains bound to its binding site in HiBgl, glucose binds only temporarily, which is consistent with the product tolerance of the enzyme.

5

Direct interaction of glucose and cellobiose. (A) Snapshot showing a glucose molecule from the solution (in cyan) hydrogen bonding the cellobiose (in green) in the active site of HiBgl. The catalytic residues are shown. (B) Distance between cellobiose and the closest glucose molecule along the simulation. (C) Probability density of the cellobiose–glucose distance.

6

Open and closed conformations at the HiBgl binding site entrance. (A) Snapshots of HiBgl in system A showing the closed (pink) and open (cyan) states. The spheres represent the α-carbon of residues Tyr179 and Phe348. The dotted lines on the right panel indicate the distance employed to characterize open/closed conformations. Distribution of the distance between the α-carbons of Tyr179 and Phe348 for systems (B) A and F and (C) B and G.

7

Secondary glucose binding sites. Frequency that there is a glucose molecule within 3 Å of each residue of HiBgl in (A) system F and in (B) system G. Residues that interacted with glucose more than 80% of the time (dashed lines) were clustered in different secondary binding sites. (C) Spatial distribution of the secondary binding sites on the HiBgl’s surface.

8

HiBgl bound to two glucose molecules at subsites −1 and +1 (system E). (A) Distance between the C1 atom of glucose bound to subsite −1 and the O2, O3, O4, and O6 atoms of the glucose bound to subsite +1 along a 500 ns long trajectory in which no dissociation was observed. (B) Probability density of the distances shown in panel (A). The dashed vertical line indicates minimum equilibrium distance between the pairs of atoms and their probabilities. (C–F) Representative snapshots showing different orientations of the glucose bound to subsite +1. The dotted lines connect atoms for which the distance is minimum in each case. Asp237 hydrogen bonds the glucose molecule in the configurations (C), (D), and (F).