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

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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.**