Human Endobiotic Cytochrome P450 Enzymes: Structure, Dynamics, and Small Molecule Interactions

Abstract

Cytochrome P450 enzymes are a highly diverse superfamily of heme-containing monooxygenases that catalyze challenging biochemical reactions in all domains of life. In humans, these membrane-bound enzymes metabolize xenobiotic (e.g. therapeutics, toxins) and endobiotic compounds (e.g. steroid hormones, fatty acids, vitamins, nutrients) and are thus essential to human health. The structure and function of P450 enzymes that maintain the homeostasis of steroids and fatty acids are the focus of this work. Detailed methods to express and purify five recombinant human steroidogenic P450 enzymes (CYP11A1, CYP11B1, CYP11B2, CYP17A1, CYP21A2) for biophysical investigation are presented in Chapter 2. Chapters 3 and 4 focus on the study of steroidogenic cytochrome P450 17A1 (CYP17A1) by solution NMR spectroscopy. This enzyme catalyzes early steps in the production of cortisol or sex steroids, and is implicated in the pathogenesis of prostate and breast cancers, as well as many other diseases. Chapter 3 introduces a novel active site ligand designed to facilitate NMR data collection of the ferrous state of CYP17A1. This ligand—a steroid-derived isonitrile—binds CYP17A1 in both ferric and ferrous states, with the isonitrile group positioned linearly above the heme iron. Collecting NMR data of CYP17A1 bound to this molecule should facilitate the completion of sequential backbone assignments. Chapter 4 investigates the changes in the conformational dynamics of CYP17A1 when interacting with b5, an interaction that promotes the androgenic activity of CYP17A1. This chapter proposes that the B, E, and H helices along with the β3-1/3-2 strands may communicate b5 binding to the active site through a series of structural adjustments to favor sex steroid synthesis. xviii Chapter 5 focuses on the early-stage study of a different human endobiotic P450 enzyme—the eicosanoid-metabolizing cytochrome P450 5A1 (CYP5A1), which promotes the innate immune response, including blood clotting and vasoconstriction. This enzyme is investigated for its role in cardiovascular diseases including atherosclerosis and ischemic stroke. Enzyme expression, purification, binding of small molecules, and crystallization trials are discussed. I conclude this work with the proposal of future directions for the study of both CYP17A1 and CYP5A1 in Chapter 6, with recommendations for future researchers and lingering questions.