Computational Modeling and Design of Cytochrome P450 Reactivity and Substrate Specificity

马拉纳斯酶是一种多功能的结构，可以通过自然调节来选择性地进行大量的催化功能。它们为生物质处理、生物燃料生产、生物传感、废水和环境污染物处理方面的挑战提供解决方案的潜力早已得到承认。然而，这些酶中的许多在所需的反应条件下稳定性较差，或者催化活性不足，或者缺乏对非天然底物分子的特异性。酶的合理设计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，并反映了活性部位访问和结合与改善的过渡态稳定性之间的平衡。由于筛选的难度和成本以及组合设计空间的巨大，高通量实验在这里不起作用。宾夕法尼亚州立大学的三位PI，Costas Maranas，Patrick Cirino和Michael Janik，试图以一种系统的方式解决这些缺点，首先开发一个多尺度计算工作流来设计酶，以提高活性和特异性，然后通过实验验证酶的计算重新设计。最终，该方法将被用于设计突变体P450 BM-3单加氧酶，这些酶在大肠杆菌中高水平功能表达，并用于小分子烷烃的羟基化生产酒精生物燃料。他们的目标底物是乙烷和丙烷，甲烷是最终目标。这个计划最深远的结果将是设计出一种能利用甲烷生产甲醇的酶。即使开发能够快速和选择性地氧化C1-C3烷烃的酶，也有助于有效利用偏远的天然气资源或用于燃料和化学品生产的低价值炼油厂气流。潜在的是一种新的酶重新设计的综合范式，其中由计算工作流程产生的假设被用来指导实验，实验结果用于纠正和完善计算基础。教育和推广努力将把REU方法与尝试使用现有的名为SROP、WISER和MURI的大学项目相结合，所有这些都是基于研究的项目，以激励女性和少数族裔在职业生涯早期体验工程研究。PI计划将这项工作包括在宾夕法尼亚州立大学的S项目中，该项目名为国际基因工程机器，简称IGEM。这个项目的目标是接触合成生物学的高中生和本科生。

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.