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

离子液体由分子阳离子和阴离子对组成，可以设计成在环境条件下以液体形式存在。可忽略的挥发性和设计具有所需物理化学和生物学性质的离子液体的能力是工业规模上离子液体技术的当前和新兴发展的驱动力。这些进展也引起了人们的关注，即由于离子液体的水溶性和有限的生物降解性，离子液体从废水排放、意外泄漏或化工厂事故释放到环境中可能导致水生物和地下水污染。拟议的研究旨在产生基础知识和计算方法，可以扩展到酶工程的离子液体生物降解。该研究的目的是揭示细菌细胞色素P450介导的羟基化的热力学和动力学，在生物降解的第一步，咪唑类离子液体。然后将利用这些知识来合理设计P450 BM 3酶以激活或加速羟基化步骤。该研究是由以下假设指导的，即可以利用对导致离子液体羟基化的中间步骤的分子水平理解来识别酶结合口袋和底物进入通道中的氨基酸残基，所述氨基酸残基对反应呈现热力学和/或动力学屏障。该假设将通过（i）表征离子液体羟基化的催化循环的热力学;（ii）确定离子液体羟基化的催化循环的动力学方面，和（iii）评估关键P450催化中心残基的计算机修饰对离子液体羟基化的热力学和动力学的影响来测试。基于自由能的分子动力学模拟将用于确定离子液体结合和产物从催化中心释放的热力学驱动力。一个混合的方法，涉及量子力学处理的反应物种和分子力学表示的蛋白质环境和溶剂将被采用的中间反应步骤。研究和教育的整合将通过培训高中化学和生物学教师以及来自OSUTeach计划的有抱负的教师使用分子编辑和可视化工具来实现，将量子计算纳入建模和模拟的研究生课程，以及教授本科生如何在他们的课程中利用计算和可视化。这个奖项反映了NSF的法定使命，并被认为是值得的通过使用基金会的知识价值和更广泛的影响审查标准进行评估来提供支持。

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.