(271f) Crystallograhic Investigation of Imidazolium Ionic Liquid Effects on Enzyme Structure and Inactivation

Authors: 
Nordwald, E., University of Colorado
Plaks, J., University of Colorado
Snell, J., University of Colorado Boulder
Sousa, M., University of Colorado Boulder
Kaar, J. L., University of Colorado Boulder

Owing to their unique and diverse molecular features, ionic liquids (ILs), which are liquid salts, have been the subject of immense focus as solvents for a broad spectrum of biocatalytic reactions. Despite this immense focus, there remains a critical need to develop rational approaches to engineer enzymes for improved activity and stability in the presence of ILs. We have recently engineered a quadruple mutant of lipase A (lipA) from Bacillus subtilis that has enhanced stability towards the IL 1-butyl-3-methylimidazolium chloride ([BMIM][Cl]) via mutation of residues that alter the surface charge of the enzyme. To understand the molecular basis for the improved stability, as well as, more generally, how ILs interact with enzymes on the structural level, we have for the first time determined the x-ray crystal structure of an enzyme in the presence of ILs. Specifically, we have solved the x-ray structure of wild-type and mutant lipA crystals soaked in aqueous solutions with different concentrations (0, 5, 10, 20 % v/v) of [BMIM][Cl] at resolutions between 1.2-1.9 Å. Notably, the concentration of [BMIM][Cl] in the soaking solution had little effect on the tertiary structure of either wild-type or mutant lipA. However, significant differences were observed in the regions of Y49 and G158, which were mutated in the IL-tolerant lipA, including differences in the occupancy of [BMIM] ions. Additionally, the binding modes of [BMIM] suggested the interaction of [BMIM] with the enzyme was driven by hydrophobic interactions with the alky tail of the cation. Interestingly, binding of the cation via cation-pi stacking, which may be synergistic with interactions with the hydrophobic alkyl tail, were also observed. While painting a detailed picture of the interaction of ILs with enzymes, these results ultimately provide unprecedented insight into the mechanism of IL-induced denaturation of enzymes.