Computational Modeling and Design of Cytochrome P450 Reactivity and Substrate Specificity

0967062 Maranas酶是由自然调节的多功能结构，以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认可。然而，这些酶中的许多在期望的反应条件下具有差的稳定性，或具有不足的催化活性，或缺乏对非天然底物分子的特异性。酶的合理设计，以改善或新的催化活性仍然是一个开放的挑战，因为催化效率取决于待反应的分子的性质，并反映了活性位点的访问和结合与改善的过渡态稳定的平衡。由于筛选的难度和成本以及组合设计空间的复杂性，高通量实验在这里将不起作用。宾夕法尼亚州立大学的三位PI，Costas Maranas，帕特里克Cirino和Michael Janik，寻求以系统的方式解决这些缺点，通过首先开发一个多尺度的计算工作-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，该方法将用于设计突变体P450 BM-3单加氧酶，其在E.大肠杆菌，并用于羟基化小烷烃的醇类生物燃料。他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，它可以利用甲烷生产甲醇。甚至能够快速和选择性氧化C1-C3烷烃的酶的开发也可以有助于有效利用偏远的气体资源或低价值的炼油厂气体流来产生燃料和化学品。潜在的是一个新的综合范例酶重新设计的假设所产生的计算工作流程被用来指导实验和实验结果服务于纠正和完成计算bases.The教育和推广工作将耦合REU的方法与尝试使用现有的大学计划名为SROP，WISER和MURI，所有这些都是研究？基于计划，鼓励妇女和少数民族在职业生涯早期体验工程研究。PI计划将这项工作纳入宾夕法尼亚州立大学？该项目名为国际遗传工程机器（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.