Adrenodoxin and Substrate Binding Modulate the Vitamin‐D Metabolizing Human Cytochrome P450 24A1

Abstract

Vitamin-D3 insufficiency is associated with rickets, cardiovascular disease, and chronic kidney disease, and therefore impacts nearly 50% of the U.S. population. In humans, vitamin-D3 is metabolized by a series of cytochrome P450 enzymes, including inactivation by the mitochondrial cytochrome P450 enzyme CYP24A1. CYP24A1 in humans first hydroxylates either the carbon-23 or carbon-24 position of 1,25-(OH)2D3. The human isoform of CYP24A1 is not easily expressed and purified recombinantly. Therefore, most current structural studies are based on the rat isoform, which does not display carbon-23 regioselective hydroxylation. As a way to study the human isoform, we have initiated use of a cleavable fusion construct of human CYP24A1 with its reduction-oxidation protein partner adrenodoxin. Cleaved and isolated human CYP24A1 provides a platform for interrogating the structural effects of an Adx-CYP24A1 complex on substrate, analog, and inhibitor binding. Notably, we observed that the presence Adx weakens the interaction between human CYP24A1 1.5-fold, while also increasing the population of the high spin shift that results from displacement of a water ligand. This effect is not seen with 1-(OH)D3, a vitamin-D3 analog, suggesting that specific substrate binding relates allosterically to Adx recognition. The path of this allostery is investigated by specific point mutations. These findings provide mechanistic insight into the interrelated nature of step I (substrate binding) and step II (redox partner binding) of a multi-functional mitochondrial cytochrome P450 enzyme.