Optimizing Cytochrome P450 Reductase for Efficient Electron Transfer to Cytochrome P450 Monooxygenases

该研究计划的长期目标是设计酶，使其成为生产精细化学品和生物活性天然产品的高效生物催化剂。酶是一种大的细胞分子，在非常温和的反应条件下进行化学转化，并有精细的控制（即通常只产生一种产物）。在许多情况下，这使得它们成为传统化学催化剂的成本效益和可持续替代品，传统化学催化剂通常使用高温和有毒金属。该研究计划的短期目标是提高一类称为细胞色素P450单加氧酶（P450）的生物催化可行性。p450作用于各种各样的化合物，并进行传统的“台式”化学合成无法复制的复杂化学反应。然而，P450化学通常依赖于第二种酶（细胞色素P450还原酶（CPR））的电子转移，这在大规模工业过程中是一个非常低效的催化步骤。****幸运的是，我们的实验室发现，与哺乳动物相比，青蒿中的植物细胞色素P450还原酶（aaCPR）将电子转移到人工电子受体的速度要快得多。这表明aaCPR可能改善p450催化的具有治疗价值的复杂天然产物的生产。为了验证这一假设，我们将确定aaCPR是否能够支持来自不同生物来源的p450更快的周转。我们还将确定aaCPR的独特特征，使其具有更高的催化效率。研究计划的第三个目标是设计一种aaCPR-P450融合酶，以提高电子传递过程的效率。动力学分析结合结构分析将用于实现这些目标。******P450生物催化目前被用于植物源性药物的合成，包括抗疟疾药物青蒿素和化疗药物紫杉醇。然而，低效的电子转移限制了这些药物的大规模生产。鉴于本研究旨在改善这一催化步骤，它有能力大大提高药物产量，降低公司和消费者的成本。此外，增强的P450生物催化还可以设计新的药物或精细化学品。最后，除了促进生物催化领域的快速发展外，本研究还将通过推进我们对p450和CPR的结构/功能关系的了解，对酶学领域做出重大贡献

The long-term goal of the research program is to engineer enzymes to be efficient biocatalysts for the production of fine chemicals and bioactive natural products. Enzymes, which are large cellular molecules, perform chemical transformations under very mild reaction conditions with exquisite control (i.e. one product typically is produced). In many cases, this makes them cost-effective and sustainable alternatives to traditional chemical catalysts, which often employ high temperatures and toxic metals. The short-term objective of the research program is to improve the biocatalytic feasability of a class of enzymes called cytochrome P450 monooxygenases (P450s). P450s act on a wide array of compounds and perform difficult chemical reactions that cannot be replicated through traditional "bench-top" chemical synthesis. However, P450 chemistry often relies on the transfer of electrons from a second enzyme, cytochrome P450 reductases (CPR), which is a very inefficient catalytic step in a scaled-up industrial process. ****Fortunately, our lab has discovered that a plant cytochrome P450 reductase from Artemisia annua (aaCPR) is considerably faster at electron transfer to artificial electron acceptors compared to its mammalian counterpart. This suggests that aaCPR may improve the P450-catalyzed production of complex natural products that have therapeutic value. To test this hypothesis, we will determine if aaCPR is able to support faster turnover of P450s from different biological sources. We will also determine unique features of aaCPR that make it more catalytically efficient. A third goal of the research program is to engineer an aaCPR-P450 fusion enzyme, in an effort to improve the efficiency of the electron transport process. Kinetic assays combined with structural analysis will be used to accomplish these aims. ******P450 biocatalysis is currently exploited for the synthesis of plant-derived pharmaceuticals, including the anti-malaria drug, artemisinin, and the chemotherapeutic drug, paclitaxel. However, inefficient electron transfer limits large-scale production of these drugs. Given that this research aims to ameliorate this step of catalysis, it is has the capacity to greatly increase drug production, lowering costs for the company and the consumer. Moreover, enhanced P450 biocatalysis also enables the design of new pharmaceuticals or fine chemicals. Finally, in addition to contributing to the rapidly expanding area of biocatalysis, this research will make a significant contribution to the field of enzymology by advancing our knowledge of the structural/functional relationships of P450s and CPR.**