Aqueous ionic liquids redistribute local enzyme stability via long-range perturbation pathways

Area of Science:

• Biocatalysis
• Protein Engineering
• Computational Chemistry

Background:

• Ionic liquids (IL) and aqueous ionic liquids (aIL) are promising solvents for biocatalysis.
• Enzyme activity is often reduced in ILs/aILs due to direct and indirect effects.
• The molecular mechanisms behind indirect effects, such as structural perturbations, remain unclear.

Purpose of the Study:

• To elucidate the molecular origin of aIL-induced indirect effects on enzyme structure and activity.
• To identify specific enzyme residues and interactions responsible for altered stability in aILs.
• To develop a predictive strategy for engineering enzymes with enhanced aIL tolerance.

Main Methods:

• Multi-microsecond molecular dynamics simulations.
• Free energy computations and protein rigidity analyses.
• Validation using a comprehensive site saturation mutagenesis library of *Bacillus subtilis* Lipase A (BsLipA).

Main Results:

• Aqueous ionic liquids favorably interact with specific BsLipA residues, inducing long-range perturbations in noncovalent interactions.
• These perturbations affect local structural stability, propagating to the catalytic site and protein core.
• Residues within identified perturbation pathways are critical for aIL tolerance, with substitutions significantly improving residual activity.

Conclusions:

• The study reveals the molecular basis of indirect aIL effects on enzyme structure via perturbation pathways.
• Identifying specific IL-residue interactions and perturbation pathways enables prediction of enzyme variants with improved aIL tolerance.
• This work provides a rational approach for designing robust biocatalysts for use in aqueous ionic liquid media.