Human Cytochrome P450 Enzymes in Drug Metabolism and Chemical Activation

Abstract

The cytochrome P450 superfamily are heme-containing monooxygenases found ubiquitously in living organisms. In humans, P450 enzymes are essential in the metabolism of drugs, pollutants, and foods, as well as in endogenous biosynthetic pathways for steroids, fatty acids, and vitamins. Thus, these enzymes can be sources of deleterious effects in humans, with issues relating to drug safety and efficacy, metabolism of compounds into carcinogens or toxins, and disruptions of signaling pathways. Prediction of P450 metabolism and design of selective P450 inhibitors are of great importance for human health. Furthermore, leveraging the diversity in P450 catalysis holds promise as biocatalysts for production of synthetically difficult chemicals and green chemistry. In both health and as biocatalysts, limited understanding of P450 structure and conformational flexibility hinders the rational pursuit of these goals. Understanding P450 structure and flexibility is complicated by the diverse compounds P450 enzymes bind and metabolize, and by structural changes that may occur when P450 enzymes interact with modulatory protein partners. This dissertation explores structural features allowing human P450 enzymes to interact with a protein partner, bind and metabolize diverse substrates, and structural interactions which contribute to a thermostable P450 biocatalyst. To understand how the catalytic modulator protein cytochrome b5 exerts variable effects on P450 catalysis, b5 complexes with different human drug-metabolizing P450 enzymes were characterized using NMR. These experiments revealed that P450 enzymes bind shared and distinct surfaces on b5 and have varied complex affinity. Mutating key residues on b5 confirmed the importance of individual b5 amino acids and functional experiments helped link complex formation to b5 modulation of P450 function. The aspect of P450 flexibility in response to diverse ligands they interact with was probed through X-ray structures of the human P450 1A1, which has prominent roles in carcinogen and toxin activation. This work demonstrated that CYP1A1 undergoes conformational changes in the active site roof when bound to structurally diverse ligands, and also revealed channels allowing CYP1A1 to bind larger and nonplanar ligands. Increased understanding of CYP1A1 structural conformations provides a valuable resource for computational prediction of the binding, metabolism, and activation of drugs and chemicals. For utilization of mammalian P450 enzymes as efficient biocatalysts, obstacles such as poor thermostability and activity need to be resolved. The discovery that reconstructed ancestors of mammalian P450 enzymes have significantly improved thermostability provides a promising route for using these enzymes as biocatalysts, but structural contributors to this enhanced thermostability are unknown. Both structure and ligand binding of the mammalian ancestor of human CYP1B1 were characterized to examine sources for increased thermostability. The structures of the ancestor CYP1B1 display an open active site conformation deviating significantly in the roof elements in comparison to the closed structure of human CYP1B1. Substrate binding and activity reveal similar substrate profiles but altered regioselectivity for the metabolites. Comparison of the ancestral and human CYP1B1 structure suggest that gains in thermostability may occur through increased electrostatic and aromatic/cation stacking interactions between distinct secondary structure elements. The combined results of this dissertation provide valuable insight into P450 structure and conformational flexibility for improvements in drug design, predictions of metabolic outcomes, and development of thermostable P450 enzymes in biotechnology.