• 2018-07
  • 2018-10
  • 2018-11
  • 2019-04
  • 2019-05
  • 2019-06
  • 2019-07
  • 2019-08
  • 2019-09
  • 2019-10
  • 2019-11
  • 2019-12
  • 2020-01
  • 2020-02
  • 2020-03
  • 2020-04
  • 2020-05
  • 2020-06
  • 2020-07
  • 2020-08
  • 2020-09
  • 2020-10
  • 2020-11
  • 2020-12
  • 2021-01
  • 2021-02
  • 2021-03
  • 2021-04
  • 2021-05
  • 2021-06
  • 2021-07
  • 2021-08
  • 2021-09
  • 2021-10
  • 2021-11
  • 2021-12
  • 2022-01
  • 2022-02
  • 2022-03
  • 2022-04
  • 2022-05
  • 2022-06
  • 2022-07
  • 2022-08
  • 2022-09
  • 2022-10
  • 2022-11
  • 2022-12
  • 2023-01
  • Introduction Cytochrome P CYP aromatase catalyzes the conver


    Introduction Cytochrome P450 19 (CYP19; aromatase) catalyzes the conversion of androgens to estrogens in a three-reaction sequence, where each step depends on NADPH and O2 [[1], [2], [3]]. The first two steps are accepted to be hydroxylations of the steroid C19 methyl group, whereas the final step relies on a debated mechanism that deformylates the C19 aldehyde and aromatizes the steroid A-ring. (Scheme 1A) [[4], [5], [6], [7]] The human enzyme converts androstenedione (ASD), testosterone (TST) and 16α-hydroxytestosterone to estrone (), 17β-estradiol () and 16α-hydroxy-17β-estradiol, respectively. E1 from adipose tissue and skin fibroblasts represents the dominant circulating estrogen [8]. Aromatase-derived estrogens mediate control of ovulation, cyclical preparation of the reproductive tract for fertilization and implantation of the blastocyst, and exert major actions on mineral, carbohydrate, protein, and lipid metabolism [9]. In the brain, gonadotropin secretion is modulated by locally-produced E2 and elsewhere, neural aromatase produces estrogens that serve to maintain brain plasticity [10]. Extremely high levels of aromatase are present in endometriosis and breast cancer tissues that greatly enhance local estrogen concentrations. Consequently, aromatase inhibitors have proven to be valuable therapies for these pathologies [11,12]. Aromatase and estrogen biosynthesis are ubiquitous among the vertebrates, extending to phylogenetically-ancient jawless fishes (hagfish and lampreys) [13]. While the physiological importance and the pharmacological value of aromatase for the treatment of a myriad of diseases are established, its physiological roles in other species are comparatively understudied. Nevertheless, it is clear from available studies that the physiological functions extend beyond those observed in mammals. Estrogens are increasingly being recognized to have pronounced effects on cognitive function [14]. In songbirds, estrogens are of critical importance in both learning and discriminating song as well as in spatial memory functions [15]. In fish, reptiles, and po1 that rely on temperature-dependent sex determination, thermosensitive mechanisms alter aromatase expression and in turn, ovarian differentiation [16]. Gonadal tissues of the invertebrate species Branchiostoma have demonstrated the ability to aromatize androgens [17] and bioinformatics analyses have identified a putative Cyp19 gene [18,19]. This discovery challenges the dogma that aromatase originated with the evolution of vertebrates [20]. When more aromatases are identified in phylogenetically-distant organisms from vertebrates, their distinct physiology will illuminate new aromatase functions and possibly provide insight into the evolutionary origins of estrogen signaling. Herein, the recombinant production and biophysical characterization of an engineered Aptenodytes forsteri aromatase (afCYP19 hereafter) are described. Enzyme kinetic analysis and characterization of products confirmed that afCYP19 and its human counterpart catalyze the transformation of ASD to E1 through a common set of intermediates and mechanistic features. In addition, yields of ligand-free afCYP19 permitted characterization of steroid and anastrozole (ATZ) (Scheme 1B) complexes by resonance Raman (RR) spectroscopy. Finally, pre-steady state kinetic analyses by stopped-flow UV–vis spectroscopy support multi-step binding mechanisms for both the steroidal ligands as well as ATZ.
    Materials and methods
    Results and discussion
    Conclusions Herein we describe the first expression and biophysical characterization of an avian cytochrome P450 aromatase from Aptenodytes forsteri. Hundreds of nanomoles of a pure, N-terminally truncated form of the enzyme can be purified L−1 of E. coli culture in the absence of a stabilizing ligand. The means to produce ligand-free afCYP19 makes it a convenient model for spectroscopic studies of ligand-aromatase interactions that typically require considerable amounts of material. afCYP19 catalysis is supported by hCPR with comparable kinetics to those previously reported for hCYP19A1. In addition, the apparently selective removal of the 1β-hydrogen atom of ASD, common oxidized intermediates in the transformation of ASD to E1, and similar heme-ligand interactions indicate that afCYP19 and hCYP19A1 share mechanistic features. Like the human enzyme, ASD appears to be the preferred substrate for afCYP19 and the affinity of the intermediates progressively decrease with increased oxidation [3,34]. In addition, afCYP19 binds the aromatase inhibitor ATZ with near identical affinity to that measured for the human enzyme. Like hCYP1A1, ASD, TST, 19-OH-ASD, and 19-oxo-ASD appear to bind to the enzyme using at least a two-step mechanism with a spectroscopically-silent intermediate. In addition, the kinetic data also support a two-step mechanism for ATZ. In summary, the similarities between the human and Aptenodytes forsteri enzymes demonstrate that the latter could serve as a convenient model system for studies of the enigmatic transformation of androgens to estrogens.