Molecular basis for the interaction of four different classes of substrates and inhibitors with human aromatase

Aromatase cytochrome P450 (CYP19) converts androgen to estrogen. In this study, the interactions of four classes of compounds, 17β-estradiol (the product of aromatase), 17-methyltestosterone (a synthetic androgen), dibenzylfluorescein (a synthetic substrate of aromatase), and coumestrol (a phytoestrogen), with aromatase were investigated through spectral analysis using purified human recombinant aromatase and site-directed mutagenesis studies using CHO cells expressing wild-type human aromatase or five aromatase mutants, E302D, D309A, T310S, S478T and H480Q. Spectral analysis showed that a type I binding spectrum was produced by the binding of 17-methyltestosterone to aromatase and a novel binding spectrum of aromatase was induced by dibenzylfluorescein. Mutagenesis experiments demonstrated that residues S478 and H480 in the β-4 sheet play an important role in the binding of all four compounds. Computer-assisted docking of these compounds into the three-dimensional model of aromatase revealed that: (1) weak interaction between 17β-estradiol and the β-4 sheet of aromatase facilitates the release of 17β-estradiol from the active site of aromatase; (2) 17-methyl group of 17-methyltestosterone affects its binding to aromatase; (3) dibenzylfluorescein binds to the active site of aromatase with its O-dealkylation site near the heme iron and residue T310; and (4) coumestrol binds to aromatase in a manner such that rings A and C of coumestrol mimic rings A and B of steroid. These structure–function studies help us to evaluate the structural model of aromatase, and to accelerate the structure-based design for new aromatase inhibitors.