Computational Modeling and Design of Cytochrome P450 Reactivity and Substrate Specificity

酶是一种多用途的结构，由大自然调节，可以选择性地执行大量的催化功能。它们在解决生物质处理、生物燃料生产、生物传感、废水和环境污染物处理等方面的挑战方面的潜力早已得到认可。然而，许多这些酶在期望的反应条件下稳定性差，或者催化活性不足，或者对非天然底物分子缺乏特异性。合理设计酶以提高或新颖的催化活性仍然是一个开放的挑战，因为催化效率取决于要反应的分子的性质，并反映了活性位点进入和结合与改善过渡态稳定性的平衡。由于筛选的难度和成本以及组合设计空间的巨大，高通量实验将无法在这里工作。宾夕法尼亚州立大学的科斯塔斯·马拉纳斯、帕特里克·奇里诺和迈克尔·雅尼克三位pi试图以一种系统的方式解决这些缺点，他们首先开发了一个多尺度的计算工作流程来设计酶，以提高酶的活性和特异性，然后通过实验验证酶的计算重新设计。最终，该方法将用于设计突变体P450 BM-3单加氧酶，该酶在大肠杆菌中具有高水平的功能表达，并用于酒精生物燃料的小烷烃羟基化。它们的目标底物是乙烷和丙烷，最终目标是甲烷。这个项目最深远的成果将是设计出一种酶，它可以利用甲烷产生甲醇。甚至能够快速和选择性氧化C1-C3烷烃的酶的开发也有助于有效利用远程天然气资源或低价值的炼油气流用于燃料和化学生产。这有可能为酶的重新设计提供一种新的综合范式，其中由计算工作流产生的假设用于指导实验，实验结果用于纠正和完善计算基础。教育和推广工作将结合REU的方法，并尝试使用现有的大学项目，如SROP、WISER和MURI，这些都是研究项目。鼓励女性和少数族裔在职业生涯早期体验工程研究的项目。pi计划将这项工作纳入宾夕法尼亚州立大学？这一项目名为“国际基因工程机器”（IGEM）。该计划的目标是高中和本科生动手接触合成生物学。

0967062Maranas Enzymes are versatile structures tuned by nature to selectively carry on a vast array of catalytic functions. Their potential to provide solutions to challenges in biomass treatment, biofuels production, biosensing, wastewater and environmental pollutants treatment has long been recognized. However, many of these enzymes suffer from poor stability under the desired reaction conditions, or have inadequate catalytic activity, or a lack of specificity for non-native substrate molecules. The rational design of enzymes for improved or novel catalytic activity remains an open challenge because catalytic efficiency depends on the nature of the molecules to be reacted, and reflects a balance of active site access and binding with improved transition state stabilization. High throughput experimentation will not work here due to the difficulty and cost of screening and the enormity of the combinatorial design space.The trio of PIs, Costas Maranas, Patrick Cirino and Michael Janik, all at Pennsylvania State University, seek to address these shortcomings in a systematic fashion, by first developing a multi-scale computational work-flow to design enzymes for improved activity and specificity and then experimentally validating the computational redesign of the enzymes. Ultimately the methodology will be used to design mutant P450 BM-3 monooxygenase enzymes which are functionally expressed at high levels in E. coli and are used for hydroxylation of small alkanes for alcoholic biofuels. Their target substrates are ethane and propane, with methane the ultimate goal.The most far-reaching results for this program would be to devise an enzyme that works on methane to produce methanol. Even the development of enzymes capable of rapid and selective oxidation of C1-C3 alkanes could aid in the efficient utilization of remote gas resources or low value refinery gas streams for fuel and chemical generation. The potential is for a new integrated paradigm for enzyme redesign where hypotheses generated by the computational workflow are used to guide the experiment and experimental results serve to correct and complete the computational base.The educational and outreach efforts will couple the REU approach with attempts to use existing University programs titled SROP, WISER and MURI, all of which are research ?based programs to inspire women and minorities to experience engineering research early in their careers. The PIs plan to include this work in Penn State?s program titled International Genetically Engineered Machines, or IGEM. This program targets high school and undergraduates with hands-on exposure to synthetic biology.