Crystallographic Investigation of Imidazolium Ionic Liquid Effects on Enzyme Structure

Abstract

We present the first crystallographic insight into the interactions of an ionic liquid (IL) with an enzyme, which has widespread implications for stabilizing enzymes in IL media for biocatalysis. Structures of Bacillus subtilis lipase A (lipA) and an IL stable varaint (QM-lipA) were obtained in the presence of increasing concentrations of 1-butyl-3-methylimidazolium chloride ([BMIM][Cl]). These studies revealed that the [BMIM] cation interacts to surface residues via hydrophobic and cation-π interactions. Of specific interest was the disruption of internal stacking interactions of aromatic side chains by [BMIM], which provide structural evidence for the mechanism of enzyme denaturation by ILs. The interaction with [BMIM] and Cl ions to lipA was reduced by the stabilizing mutations Y49E and G158E in QM-lipA. Ultimately, these findings present the molecular basis for stabilizing enzymes from IL-induced inactivation as well as the selection of ILs that are less denaturing.

Enantioselectivity ofd-amino acid oxidase in the presence of ionic liquids

“…In buffer, L-Ala is tightly bound to the active site of the enzyme by noncovalent interaction, forming a quite stable transient intermediate, in which a-NH 3 group faces away from the carbonyl moiety. However, [MIM] cation can interact with surface residue (Tyr) through hydrophobic p-p interactions due to a permanent positive charge and a N-methyl group of the imidazolium ring, which agrees with the previous report that most of the cations interact with hydrophobic regions on the surface of the enzyme 41. Thus, in presence of [MIM][COO], the interaction between [MIM] cation and active site places L-Ala in a better attacking position to transfer of a-proton to FAD or the nucleophilic attack to the amino group by water.…”

Protein Crystallization in Ionic-Liquid Hydrogel Composite Membranes

“…For some P2 1 2 1 2 structures present in the PDB, a strong pseudo-I222 symmetry was found, where deviation from the true I222 was only hinted [38]. A similar effect of inducing a space group transition was observed for lipase A crystals soaked with a solution containing imidazolium IL as additive [39]. The symmetry transition, occurred at higher IL concentrations, was there attributed to stronger IL-protein interactions.…”

Genetic and chemical approaches for surface charge engineering of enzymes and their applicability in biocatalysis: A review

“…In comparison, nonspecific charge modification via acetylation and succinylation resulted in only a 1.2-and 1.9-fold improvement in stability, respectively, over wild-type (WT) BsLA (Nordwald, Armstrong et al, 2014). A crystallography study also proved that these stabilizing mutations in this mutant indeed reduced the extent of enzyme binding to [BMIM] and Cl ions (Nordwald, Plaks, Snell, Sousa, & Kaar, 2015).…”

“…Meanwhile, the engineered halotolerant enzymes were also more resistant to the destabilizing effects of selected ILs and denaturants such as SDS sodium dodecyl sulphate that cause unfolding of proteins through the disruption of secondary structural elements (α-helices and β-sheets). The increased frequency of surface negative charges that electrostatically bind cations has the dual effect of stabilization of the surface structure and increased repulsion of anions, which has been reported to render enzymes resistant against ILs (i.e., [BMIM][Cl]; Nordwald & Kaar, 2013a;Nordwald, Armstrong et al, 2014;Nordwald et al, 2015).…”

“…The increased frequency of surface negative charges that electrostatically bind cations has the dual effect of stabilization of the surface structure and increased repulsion of anions, which has been reported to render enzymes resistant against ILs (i.e., [BMIM][Cl]; Nordwald & Kaar, 2013a;Nordwald, Armstrong et al, 2014;Nordwald et al, 2015). Meanwhile, the engineered halotolerant enzymes were also more resistant to the destabilizing effects of selected ILs and denaturants such as SDS sodium dodecyl sulphate that cause unfolding of proteins through the disruption of secondary structural elements (α-helices and β-sheets).…”