Mechanistic Insight into Species‐specific Redox Partner Interactions in the Vitamin D Carbon‐24 Hydroxylase CYP24A1

Abstract

The mitochondrial cytochrome P450 24A1 (CYP24A1) is responsible for side chain hydroxylation of vitamin D, the initial step in deactivation of the bioactive hormone. Mutations in CYP24A1 lead to incomplete clearance of active vitamin D and correlate with idiopathic infantile hypercalcemia, or elevated calcium in blood serum, and can lead to loss of appetite, nausea, vomiting, and failure to thrive in newborns. The regiospecific hydroxylation of the vitamin D side chain at either carbon‐23 (C23) or carbon‐24 (C24) relies in part on differences between species; while human isoforms are both C23 or C24 hydroxylases, other species are either C23 or C24 pure hydroxylases, despite an overall sequence identity of 80% between isoforms. Recent work from our group using solution NMR to evaluate the interaction between CYP24A1 and the redox accessory protein adrenodoxin (from bovine) suggests that species displaying distinct regioselectivity for vitamin D also form distinct P450‐redox partner complexes. Evidence of species‐specific complexes combined with the fact that mix‐species P450‐redox complexes are widely used to examine P450 function, highlights the need to examine this redox complex using proteins from the same species. Therefore, in this work we have used a combined approach of site‐directed mutagenesis, protein NMR, chemical cross‐linking, and vitamin D hydroxylation assays to evaluate the CYP24A1‐adrenodoxin complex from rat (a C24 hydroxylase). Differences in the 2D NMR spectra between bovine and rat adrenodoxin led to the complete re‐assignment of the protein backbone for rat adrenodoxin. Nonetheless, differential broadening of the NMR signal when titrated with rat CYP24A1 bound to 1α‐hydroxyvitamin D suggests the complex is mediated by surface charges. Dependence on surface charges was also verified by site‐directed mutagenesis of both proteins as well as zero‐length chemical cross‐linking using 1‐Ethyl‐3‐(3‐dimethylaminopropyl)carbodiimide. While preliminary, this works demonstrates the combined use of biophysical and biochemical approaches to evaluate nuanced differences in a P450‐redox complex that is critical to the maintenance of proper levels of bioactive vitamin D.Support or Funding InformationNIGMSR00GM112862 (DFE)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.