CAREER: Computation-Enabled Rational Design of Cytochrome P450 for Ionic Liquid Biodegradation

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Ionic liquids, composed of molecular cation and anion pairs, can be designed to exist as liquids under ambient conditions. Negligible volatility and ability to design ionic liquids with desired physicochemical and biological properties are the drivers for the current and emerging development of ionic liquid technologies on an industrial scale. Such advances also elevate the concerns that the release of ionic liquids into the environment from wastewater discharges, accidental spillage or a chemical plant accident, is likely to lead to aquatic and groundwater contamination due to their water solubility and limited biodegradability. The proposed research aims to generate foundational knowledge and computational methodologies that can be extended to enzyme engineering for ionic liquid biodegradation. The goal of the proposed research is to unravel the thermodynamics and kinetics of the bacterial cytochrome P450-mediated hydroxylation, the first step in the biodegradation, of imidazolium-based ionic liquids. The knowledge will then be utilized to rationally design the P450 BM3 enzyme to activate or accelerate the hydroxylation step. The research is guided by the hypothesis that the molecular level understanding of the intermediate steps leading to ionic liquid hydroxylation can be leveraged to identify amino acid residues in the enzyme binding pocket and the substrate access channel that present a thermodynamic and/or kinetic barrier to the reaction. The hypothesis will be tested by (i) characterizing the thermodynamics of the catalytic cycle of ionic liquid hydroxylation; (ii) determining kinetic aspects of the catalytic cycle of ionic liquid hydroxylation, and (iii) evaluating the influence of in silico modification of the key P450 catalytic center residues on the thermodynamics and kinetics of ionic liquid hydroxylation. Free energy-based molecular dynamics simulations will be employed to determine the thermodynamic driving force for ionic liquid binding and product release from the catalytic center. A hybrid approach involving quantum mechanical treatment of the reacting species and molecular mechanics representation of the protein environment and the solvent will be adopted for the intermediate reaction steps. Integration of research and education will be achieved by training high school chemistry and biology teachers and aspiring teachers from the OSUTeach program in using molecular editing and visualization tools, incorporating quantum calculations into a graduate level course on modeling and simulation, and teaching undergraduate students how to utilize computation and visualization in their courses.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.