(704g) Discovery and Engineering of Cytochromes P450 from Plant Secondary Metabolism

Plant natural products are an excellent source of anti-cancer drugs and other therapeutics. For example, the defense compound indole-3-carbinol from broccoli (Brassica oleracea) arrests reproductive cancer cell lines¹, while brassinin from Chinese cabbage (Brassica rapa) increases immune response to cancerous cells². To produce these complex, small molecules on an industrial scale, we must first identify and engineer the proteins which synthesize them. Crucifers like B. rapa and B. oleraceause specific oxidizing and tailoring enzymes to create these tryptophan-derived therapeutic natural products: cytrochromes P450 (P450s). P450s possess a unique capacity for multi-step catalysis, performing not just site specific oxidation, but further tailoring of oxidized intermediates. This “catalyst within a catalyst” characteristic make P450s powerful frameworks for engineering. If structural elements causing tailoring activity are known, they may be altered, or swapped into another P450, towards the creation of new molecules. Although work has been done to understand P450 oxidation, little is known about how P450s, especially those from plants, do this specific tailoring. Because plant P450s are difficult to isolate in large numbers, the best approach for analyzing and engineering these enzymes is rational design of a small mutant library based on structural models and simulations.

Such models exist for the P450s CYP71A12 (A12) and CYP71A13 (A13) from Arabidopsis thaliana. These enzymes share 89% sequence identity but tailor molecules differently, making them an ideal case study. Both enzymes dehydrate and hydroxylate the same tryptophan-derived substrate, while only A13 promotes a unique sulfur addition³. In this study, we analyzed protein model simulations to identify structural elements correlated with substrate processing of A13. These regions were swapped from A13 into A12 in chimeric proteins which were heterologously expressed and assayed in vitro. We found distinct regions and residues responsible for specificity towards both products and the overall stability of the protein. In this work we also describe efforts to diversify the library of tryptophan-derived therapeutics by the discovery of P450s from non-model plants including B. rapaand other crucifers.  By analyzing the structure function relationships of these enzymes and by identifying new classes of P450s, we are expanding the library of novel therapeutics derived from plant natural products.

1. Hsu, J.C., et al. Biochemical pharmacology 72, 1714-1723 (2006).

2. Banerjee, T. et al. Oncogene 27,2851–2857 (2008).

3. Klein, A.P., et al. Angewandte Chemie International Edition 52, 13625-13628 (2013).