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.

Conformational flexibility of human aromatase. / Giovanna Di Nardo; Maximilian Breitner; Sheila J. Sadeghi; Eleonora Nicolai; Giampiero Mei; Gianfranco Gilardi. - In: THE FEBS JOURNAL. - ISSN 1742-464X. - STAMPA. - 278(2011), pp. 102-103. ((Intervento presentato al convegno FEBS 2011 tenutosi a Torino nel 25-30 Giugno.

Conformational flexibility of human aromatase.

DI NARDO, Giovanna;SADEGHI, JILA;GILARDI, Gianfranco
2011

Abstract

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.
FEBS 2011
Torino
25-30 Giugno
278
102
103
Cytochrome P450; Aromatase; Protein Structure
Giovanna Di Nardo; Maximilian Breitner; Sheila J. Sadeghi; Eleonora Nicolai; Giampiero Mei; Gianfranco Gilardi
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/2318/130408
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