Abstract
Cytochrome P450 19 (CYP19, aromatase) catalyzes the conversion of androgens to estrogens in a sequence of three reactions that each depend on NADPH and O2. Aromatase is a phylogenetically-ancient enzyme and its breadth of expression in other species has highlighted distinct physiological functions. In songbirds, estrogen production is required for programming the neural circuits controlling song and in the determination of sex in fish and reptiles. This work describes the expression, purification, and biophysical characterization of Aptenodytes forsteri (Emperor penguin, af) aromatase. Using human cytochrome P450 reductase as a redox partner, afCYP19 displayed similar substrate turnover and LC/MS/MS confirmed that afCYP19 catalyzes the transformations through the intermediates 19-hydroxy- and 19-oxo-androstenedione. Androstenedione and anastrozole had the highest affinity for the enzyme and were followed closely by 19-hydroxyandrostenedione and testosterone. The affinity of 19-oxo-androstenedione for afCYP19 was ten-fold lower. The time-dependent changes in the Soret bands observed in stopped-flow mixing experiments of the steroidal ligands and the inhibitor anastrozole with afCYP19 were best described by a two-step binding mechanism. In summary, these studies describe the first biophysical characterization of an avian aromatase that displays strikingly similar enzyme kinetics and ligand binding properties to the human enzyme and could serve as a convenient model system for studies of the enigmatic transformation of androgens to estrogens.