Study of substrate specificity of human aromatase by site directed mutagenesis

Abstract

Human aromatase is responsible for estrogen biosynthesis and is implicated, in particular, in reproduction and estrogen-dependent tumor proliferation. The molecular structure model is largely derived from the X-ray structure of bacterial cytochromes sharing only 15-20% identities with hP-450arom. In the present study, site directed mutagenesis experiments were performed to examine the role of K119, C124, I125, K130, E302, F320, D309, H475, D476, S470, I471 and I474 of aromatase in catalysis and for substrate binding. The catalytic properties of mutants, transfected in 293 cells, were evaluated using androstenedione, testosterone or nor-testosterone as substrates. In addition, inhibition profiles for these mutants with indane or indolizinone derivatives were obtained. Our results, together with computer modeling, show that catalytic properties of mutants vary in accordance with the substrate used, suggesting possible differences in substrates positioning within the active site. In this respect, importance of residues H475, D476 and K130 was discussed. These results allow us to hypothesize that E302 could be involved in the aromatization mechanism with nor-androgens, whereas D309 remains involved in androgen aromatization. This study highlights the flexibility of the substrate-enzyme complex conformation, and thus sheds new light on residues that may be responsible for substrate specificity between species or aromatase isoforms.Keywords: aromatase; site-directed mutagenesis; molecular modeling; androgens; inhibitors.Estrogens are known to be implicated in reproduction and estrogen-dependent tumor proliferation [1]. Moreover, an abnormal expression of aromatase, the enzyme involved in the conversion of androgens to estrogens, has been detected in breast tumors and in surrounding adipose stromal cells [2,3]. The aromatase enzyme comprises a specific cytochrome P-450 aromatase and the ubiquitous cytochrome P-450 NADPH reductase. A common treatment for estrogen-dependent cancers is the use of antiestrogens and/or aromatase inhibitors [4]. The usual way to develop new aromatase inhibitors is to screen in vitro chemical compounds [5][6][7][8][9][10] from the knowledge of the substrate structure, or by comparisons with other aromatases [11][12][13]. The design of more specific and efficient aromatase inhibitors could be improved by a better knowledge of the enzyme's active site. A precise modeling of this part of the molecule is therefore necessary. Despite success in obtaining aromatase purified to homogeneity [14], crystallization of this microsomal membrane-anchored protein has not been reported. Knowledge of the aromatase structure is largely derived from comparisons with soluble bacterial cytochromes Pseudomons putida camphor P-450 (P-450cam), Bacillus megaterium P-450 (P-450BM-3), Pseudomonas putida a-terpineol P-450 (P-450terp) and Saccharopolyspora erythreae erythromycin F P-450 (P-450eryF), these proteins being well characterized and crystallized [15][16][17][18][19]. However, human aromatase shares only 15-20% ...

Association of CYP17, CYP19, CYP1B1 , and COMT Polymorphisms with Serum and Urinary Sex Hormone Concentrations in Postmenopausal Women

“…Our results, in combination with those of Haiman et al (17), suggest that this polymorphism or one in linkage disequilibrium with it may alter the binding affinity of the CYP19 protein (aromatase) to testosterone and androstenedione versus 16␣-hydroxyandrostenedione. Data suggest that 16␣-hydroxyandrostenedione has a separate binding site from that of testosterone and androstenedione but that each hormone inhibits the aromatization of the others (40,41). Thus, individuals with the 7r(Ϫ3) allele may produce proteins with a higher binding affinity for 16␣-hydroxyandrostenedione, which would inhibit the binding of androstenedione and testosterone.…”

Active site acidic residues and structural analysis of modelled human aromatase: A potential drug target for breast cancer

“…However, the D309A mutant which induced activity loss with androstenedione as well as testosterone, exhibited activity with nor-testosterone. In contrast, the mutant E302A was found to be inactive with all the three substrates [10]. This suggests that E302 has an important part to play in the enzymatic mechanism involving both androgens as well as nor-androgens.…”

“…Earlier site directed mutagenesis studies on D309 of human aromatase implied the probable role of this aspartate in decarboxylation of androstenedione or testosterone and attracting a proton at C2 of the steroids during aromatization [7][8][9]. This process is assumed to be assisted by basic residues H475 and K473 present in proximity to C3 [10]. Previous site directed mutagenesis studies suggested that the repulsive force between D476 and E309 may be important for efficient aromatization of C19-androgens.…”

Aromatase and breast cancer: W39R, an inactive protein

“…Aromatase activity 'in cell' was evaluated 48 h post-transfection. Transfection efficiency was evaluated by ELISA of the wild-type and several mutated proteins without showing differences in recombinant aromatase expressions (16).…”