The terminal steps of cortisol and aldosterone biosynthesis

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Excess aldosterone and cortisol production from the human adrenal cortex commonly contributes to hypertension, heart failure, obesity, glucose intolerance, and low bone mass. Cytochromes P450 11B2 and P450 11B1 catalyze the final steps in the biosynthesis of aldosterone and cortisol, respectively. The zone- specific regulation of these enzymes maintains salt and water balance or carbohydrate metabolism and response to physiologic stress. The enzymes share 95% sequence similarity, and both catalyze the 11β- hydroxylation of 11-deoxycorticosterone and 11-deoxycortisol. In contrast, the 18-hydroxylase of P450 11B1 is poor compared to P450 11B2, and only P450 11B2 has 18-oxidase activity, which converts 18-hydroxy- corticosterone to aldosterone. P450 11B2 has high processivity, in that the intermediates predominantly do not dissociate before additional turnovers to aldosterone, but the reasons for this processivity and whether this property is required for aldosterone production are not known. A comparison of its microscopic steps with those of P450 11B1 offers an opportunity to understand the unique biochemistry of aldosterone production. Our long-term goal is to elucidate the biochemical and physical properties that confers high 18- hydroxylase and moderate 18-oxidase activities to P450 11B2 but not P450 11B1. Our central hypotheses are that: (1) the positioning of nascent corticosterone generated from 11-deoxycorticosterone in the active site and the conformation of P450 11B2 is different that the nature of the complex when corticosterone binds as initial substrate; and (2) that the processivity of P450 11B2 reflects a combination of slow dissociation rates and high coupling efficiency with the intermediates. Consequently, the objectives of this application are to characterize the binding constants, binding and dissociation rates, pre-steady state (single turnover) kinetics, and coupling efficiencies for P450 11B1 and P450 11B2 with several substrates and to deduce which parameters correlate best with processivity. As tools for these studies, we will employ mutations that alter the activities for P450 11B1 and P450 11B2 and alternate substrates that have specific chemical properties. In Aim 1, we will dissect the 18-hydroxylase activity of P450 11B1 and P450 11B2, wild-type and mutations. In Aim 2, we will focus on the 18-oxidase activity with a parallel series of studies as in Aim 2. In Aim 3, we will focus on extrinsic factors, specifically the lipid composition of the membrane environment, in regulating the processivity and latter two activities of P450 11B2. We will use nanodiscs to generate artificial enzyme-membrane systems and confirm key results in transfected HEK-293 or V79 cells. The work will utilize the specialized instrumentation that the Auchus and Waskell laboratories have employed to study cytochrome P450 enzymes for many years and state-of-the art tandem mass spectrometry at the University of Michigan. In this manner, we will systematically define the differences in the reaction mechanisms and properties for P450 11B1 and P450 11B2. These studies will inform strategies to screen for better drugs to safely inhibit key steps of aldosterone and cortisol production for the treatment of human diseases.