Temperature-Dependent Conformational Dynamics of Substrate Entrance Loops in β-Glucosidase: Insights from Molecular Dynamics Simulations

β-Glucosidase (BGL) is widely used in biofuel production, industrial value-added chemicals, and food industry applications. The substrate entrance loops of BGL play a role in substrate specificity and accessibility. To better understand the substrate entrance loops of BGL, a high-resolution crystal structure of BGL from Thermoanaerobacterium saccharolyticum (TsaBGL) was determined at 1.65 Å, and all-atom molecular dynamics (MD) simulations were performed. The crystal structure of TsaBGL exhibited both folded and straight conformations of the flexible L3 loop, along with rigid conformations of L1, L2, and L4 loops. MD simulations revealed that the folded L3 loop transitioned to a straight conformation, indicating the preference for the straight conformation. At the optimal temperature for enzyme activity, the flexibility of the L3 loop of TsaBGL decreased, whereas that of the L1 loop increased. Moreover, the positions of L1 and L2 loops shifted in a direction opposite to the substrate entrance, resulting in an expanded substrate-binding entrance and increased substrate accessibility to the active site. MD simulations of three homologous BGLs showed that, despite sequence variability, a conserved dynamic trend exists in which the L1 loop exhibits higher flexibility, whereas the L3–L4 loops maintain structural rigidity under optimal conditions. These results provide both an understanding of the loop dynamics involved in substrate accessibility in BGLs and insights into enzyme engineering to improve catalytic performance.