Adrenodoxin Modulates Substrate Binding in the Vitamin‐D Metabolizing Human Cytochrome P450 24A1

Abstract

Vitamin‐D insufficiency is associated with rickets, osteomalacia, cardiovascular disease, and chronic kidney disease and affects nearly 50% of the population. In humans, vitamin D is metabolized by a series of cytochrome P450 enzymes, including inactivation by the mitochondrial cytochrome P450 enzyme CYP24A1. CYP24A1 in humans hydroxylates either the carbon‐23 or carbon‐24 position of 1,25‐(OH)2D3 followed by a series of reactions to produce additional metabolites. The human isoform of CYP24A1 is not easily expressed and purified recombinantly, likely due to the labile nature of the enzyme. 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 fusion construct of human CYP24A1 with its reduction‐oxidation protein partner adrenodoxin (Adx). The fusion product contains a short linker sequence connected to a cleavable tobacco etch virus protease recognition site, thus allowing for production and then separation of human CYP24A1 from Adx. This construct provides a platform for interrogating the structural effects of an Adx‐CYP24A1 complex on substrate, analog, and inhibitor binding in CYP24A1. Notably, we’ve observed that when Adx is present, titration with 1,25(OH)2D3 primarily populates a low‐spin state. This effect is removed upon cleavage and separation of Adx. Our interpretation is that the presence of adrenodoxin stabilizes a closed form of CYP24A1 that prevents water from departing the active site as the sixth ligand. This finding points toward close structural coupling between substrate binding and redox partner recognition in mitochondrial P450 enzymes.Support or Funding InformationR00GM112862