Biophysical Characterization of Aptenodytes forsteri Cytochrome P450 19 (Aromatase)

Abstract

Cytochrome P450 19 (aromatase) catalyzes the conversion of androgens to estrogens in a sequence of three reactions that each depend on NADPH and O2. As the terminal enzyme in estrogen biosynthesis, it is critical for the maintenance of myriad tissues and has emerged as a successful target for the treatment of estrogen‐dependent breast cancer. 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 of neural circuits controlling song. Aromatase also plays a role in sex determination of fish, reptiles, and amphibians. Biophysical characterization of the human enzyme has been limited by low yields from recombinant expression systems. Accordingly, we sought to identify an aromatase homolog with enhanced expression properties in the absence of stabilizing ligands. This work describes the expression, purification, and biophysical characterization of Aptenodytes forsteri (Emperor penguin) aromatase (afCYP19A1). Using human cytochrome P450 reductase as a redox partner, the enzyme kinetic parameters for turnover of androstenedione were like the human enzyme and mass spectrometry analysis conformed that afCYP19A1 likewise catalyzes the transformation through 19‐hydroxy‐ and 19‐oxoandrostenedione intermediates. Equilibrium binding studies revealed that androstenedione and anastrozole had the highest affinity for the enzyme and were closely followed by 19‐hydroxy‐androstenedione and testosterone. The affinity of 19‐oxoandrostenedione was an order‐of‐magnitude lower. Ligand‐binding kinetics were determined by stopped‐flow UV‐visible spectroscopy. Both androstenedione and testosterone where determined to bind to the enzyme in a step‐wise fashion with a spectroscopically‐detectable intermediate. In summary, these studies describe the first biophysical characterization of a non‐human aromatase that displays strikingly similar enzyme kinetics and ligand binding properties to the human enzyme despite bearing 75% sequence identity. In addition, they provide access to an alternative model to study the catalytic mechanism of the enzyme using methods requiring substantial amounts of ligand‐free enzyme.Support or Funding InformationNational Institutes of Health National Institute of General Medical Sciences [Grant R01GM114168]This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.