Structure and dynamics of ionic liquid tolerant hyperthermophilic endoglucanase Cel12A from Rhodothermus marinus

Economic deconstruction of lignocellulose remains a challenge due to the complex architecture of cellulose, hemicellulose, and lignin. Advancements in pretreatment processes have introduced ionic liquids (ILs) as promising non-derivatizing solvents for reducing biomass recalcitrance and for promoting enzymatic hydrolysis. However, available commercial cellulases are destabilized or inactivated even in low concentration of residual ILs. Thus, a molecular understanding of IL-enzyme interactions is crucial for developing IL-tolerant enzymes with high catalytic activity. In this study, molecular insight behind the IL tolerance of hyperthermophilic endoglucanase Cel12A from Rhodothermus marinus (RmCel12A) has been investigated in 20%, 40%, and 60% 1-ethyl-3-methylimidazolium acetate (EmimAc) through molecular dynamic simulations at 368 K. Though the enzyme retained its stability in all EmimAc concentrations, the activity was affected due to the loss of essential dynamic motions. A protein structure network was constructed using the snapshots of protein structures from the simulation trajectories and the hub properties of residues R20, Y59, W68, W197, E203, and F220 were found to be lost in 60% EmimAc. Emim cations were observed to intrude the active site tunnel and interact with more number of catalytic residues with higher cumulative fractional occupancy in 60% EmimAc than in 20% or 40% EmimAc. Some non-catalytic residues have also been identified at the active site, which can be probable mutation targets for improving the IL tolerance. Our findings reveal the molecular understanding behind the origin of activity loss of RmCel12A and proposed insights for the further improvement of IL sensitivity.