Thermostabilizing mechanisms of canonical single amino acid substitutions at a GH1 β‐glucosidase probed by multiple MD and computational approaches

Abstract

β‐glucosidases play a pivotal role in second‐generation biofuel (2G‐biofuel) production. For this application, thermostable enzymes are essential due to the denaturing conditions on the bioreactors. Random amino acid substitutions have originated new thermostable β‐glucosidases, but without a clear understanding of their molecular mechanisms. Here, we probe by different molecular dynamics simulation approaches with distinct force fields and submitting the results to various computational analyses, the molecular bases of the thermostabilization of the Paenibacillus polymyxa GH1 β‐glucosidase by two‐point mutations E96K (TR1) and M416I (TR2). Equilibrium molecular dynamic simulations (eMD) at different temperatures, principal component analysis (PCA), virtual docking, metadynamics (MetaDy), accelerated molecular dynamics (aMD), Poisson‐Boltzmann surface analysis, grid inhomogeneous solvation theory and colony method estimation of conformational entropy allow to converge to the idea that the stabilization carried by both substitutions depend on different contributions of three classic mechanisms: (i) electrostatic surface stabilization; (ii) efficient isolation of the hydrophobic core from the solvent, with energetic advantages at the solvation cap; (iii) higher distribution of the protein dynamics at the mobile active site loops than at the protein core, with functional and entropic advantages. Mechanisms i and ii predominate for TR1, while in TR2, mechanism iii is dominant. Loop A integrity and loops A, C, D, and E dynamics play critical roles in such mechanisms. Comparison of the dynamic and topological changes observed between the thermostable mutants and the wildtype protein with amino acid co‐evolutive networks and thermostabilizing hotspots from the literature allow inferring that the mechanisms here recovered can be related to the thermostability obtained by different substitutions along the whole family GH1. We hope the results and insights discussed here can be helpful for future rational approaches to the engineering of optimized β‐glucosidases for 2G‐biofuel production for industry, biotechnology, and science.

Recent Advances in β-Glucosidase Sequence and Structure Engineering: A Brief Review

“…BGL plays a key role in consolidated bioprocessing; however, high temperature causes BGL denaturation [ 11 , 99 ]. A comparative analysis on the enzymatic properties and amino acid composition of mesophilic, thermophilic, and hyperthermophilic BGLs revealed a number of factors that contribute to the thermal stability of proteins, such as hydrophobic effects [ 100 , 101 ], hydrogen-bonding and electrostatic interactions [ 100 , 102 ], aromatic interactions [ 100 , 103 ], protein structural densification [ 100 ], reduction in unfolding entropy [ 99 , 101 ], etc.…”

“…Moreover, BGLs are most active only at 40–70 °C and pH 4.5–5, but industrial applications frequently go beyond these boundaries. For instance, the pre-treatment steps for removing lignin and other secondary wall components during biofuel production typically work at temperatures above 80 °C [ 11 ]. In flavor enhancement of fruit juice, enzymes with optimal activity at acidic pH 2.8–3.8 would be better adapted to release the glycosidically bound volatiles [ 7 ].…”

Liquid–Vapor Coexistence and Spontaneous Evaporation at Atmospheric Pressure of Common Rigid Three-Point Water Models in Molecular Simulations

“…However, frequently, realistic simulations at higher temperatures are also desired. This concerns, for example, temperature-dependent processes such as protein denaturation or conformational transitions of thermoresponsive polymers. − The choice of water model may influence such temperature-dependent processes in molecular simulations tremendously. − In simulations at elevated temperatures, it is important to be sure that the water model of choice is still in the liquid phase. Furthermore, exploring the correlation between the temperature-dependent properties of in silico water and the parameters of different existing models (such as geometry and charge distribution) is of interest.…”

“…This concerns, for example, temperature-dependent processes such as protein denaturation or conformational transitions of thermoresponsive polymers. 35 − 39 The choice of water model may influence such temperature-dependent processes in molecular simulations tremendously. 40 − 42 In simulations at elevated temperatures, it is important to be sure that the water model of choice is still in the liquid phase.…”

Recent Advances in β-glucosidase Engineering: A Brief Review

“…BGLs are most active at 40-70 °C and pH 4.5-5, but industrial applications frequently go beyond these boundaries. For instance, the pre-treatment steps for removing lignin and other secondary wall components at the biofuel production typically work at temperatures above 80°C [11]. In flavor enhancement of fruit juice, enzymes with optimal activity at acidic pH 2.8-3.8 would be better adapted to release the glycosidically bound volatiles [7].…”