Background: The steroidogenic enzyme aromatase (CYP19A1) is a protein located in endoplasmic reticulum (ER) that catalyzes the conversion of androgens to estrogens. Both deficiency and excess of aromatase activity lead to disease states implicating its role in human biology. Cytochrome P450 (CYP) enzymes in ER use reduced nicotinamide adenine dinucleotide phosphate through cytochrome P450 oxidoreductase (POR) for their metabolic activities. Mutations in POR cause disorders of sexual development due to the deficiencies in several steroid metabolizing enzymes like CYP17A1, CYP21A2 and CYP19A1. The effect of POR mutations on different P450 activities depends on individual partner proteins. So each P450-POR mutant combination should be studied individually.
Objective: Study the impact of mutations in the flavin binding domain of POR (A115V, T142A, P284L, P284T and Q153R) on CYP19A1 activity, which can potentially influence the estrogen metabolism.
Method: The WT and mutant human POR proteins were expressed in bacteria and membranes were isolated. Human CYP19A1 was produced as His-tag recombinant protein and purified by Ni2+ metal chelate chromatography. POR variants were characterized by standard cytochrome c reduction assay and flavin content of proteins was analyzed. Bacterial membranes containing WT or mutant POR along with CYP19A1 were reconstituted into liposomes and the aromatase activity was determined by tritiated water release assay using radiolabeled androstenedione as substrate. Kinetic parameters (Km, Vmax) were calculated for each mutant and compared with WT POR.
Results: Mutations in the flavin binding domain of POR alter the cytochrome c reduction rate. We found severe effect of POR mutation on CYP19A1 enzyme activity. The POR mutants P284L, P284T, A115V and T142A showed less than 20% activity in supporting CYP19A1 reactions. Interestingly, the POR variant Q153R showed 50% higher activity than WT.
Conclusion: Our study suggests that alteration in aromatase activity may have an impact on estrogen metabolism. Lower aromatase activities due to POR mutation might affect the fetal androgen metabolism, especially in pregnant women with a male child.
10 - 12 Sep 2016
European Society for Paediatric Endocrinology