In the human adrenal cortex, three mitochondrial cytochromes P450 are involved in steroid hormone biosynthesis. CYP11A1 catalyses side-chain cleavage of cholesterol to yield pregnenolone, whereas P45011B1 is responsible for the conversion of 11-deoxycorticosterone and 11-deoxycortisol to the glucocorticoids, corticosterone and cortisol, respectively. The aldosterone synthase (CYP11B2) catalyses the 11β-hydroxylation, 18-hydroxylation and 18-oxidation of deoxycorticosterone to form aldosterone. All three proteins need a ferredoxin, adrenodoxin (Adx), and an FAD-dependent adrenodoxin reductase (AdR) as electron transport system to allow oxygen activation and substrate hydroxylation. The interaction between the proteins in the chain is a critical factor for the P450 activity. We have resolved the 3D structure of bovine Adx and identified critical regions of this protein, which are involved in the interaction with both, P450s and AdR. In addition, the role of phosphorylation of Adx on the interaction with its redox partners, AdR, CYP11A1 and CYP11B1 has been analysed. It could be clearly demonstrated that bovine Adx is a substrate of protein kinase CK2. CK2 phosphorylation takes place exclusively at residue Thr-71, which is located within the redox partner interaction domain of the protein. Substrate conversion assays catalyzed by CYP11A1 showed an increase in product formation when CK2-phosphorylated Adx was used as electron carrier instead of Adx-WT, whereas the activity towards CYP11B1 was not altered using these Adx species.
It was further investigated whether the competition for electrons or the supply of electrons to the mitochondrial P450s would change the product formation. Therefore, the effect of co-expression of CYP11B1 and CYP11A1 in COS cells on the product formation rate and on the specificity of product formation was tested. It was shown that there is competition for the reducing equivalents. When Adx species with improved electron transfer efficacies were used, changes in the product pattern have been observed. The results were indicative for a potential regulatory role of adrenodoxin in aldosterone production.
06 - 07 Nov 2006
Society for Endocrinology