Conformational flexibility of human aromatase.

Aromatase (CYP19A1) is one of the cytochrome P450s acting in the last steps of the steroidogenesis by converting androgens to estrogens. This enzyme has been the object of intense studies aimed at developing aromatase inhibitors to contrast some estrogen-dependent tumors, where the enzyme results pathologically overexpressed. However, only recently structural and functional characterization of the enzyme has been reported. Here, the properties of a recombinant protein purified in absence of any substrate or inhibitor are compared to those of the androstenedione bound form. In particular, circular dicroism spectroscopy and dynamic fluorescence measurements enable to detect conformational changes in the tertiary structure of the protein upon substrate binding. Furthermore, the rate of binding of carbon monoxide, used as a probe of the active site accessibility, resulted 5 fold higher (0.51±0.12 x 10-2 sec-1) than the one measured for the androstenedione-bound one (0.11±0.01 x 10-2 sec-1). Thermal and chemical unfolding studies were performed to follow the stability both of the protein (far UV circular dichroism) and the active site (visible spectroscopy). The enzyme resulted thermally stabilized by 3°C when the substrate was present both at the level of the entire structure and the active site. Furthermore, the presence of the substrate resulted in a higher chemical stability as monitored by far UV circular dichroism, whereas heme depletion from the active site of the protein was not significantly affected. These results are consistent with an increase in flexibility of the human aromatase when the substrate is not present.