Purification and reconstitution properties of human placental aromatase. A cytochrome P-450-type monooxygenase

Abstract

The hemoprotein component of human placental aromatase (estrogen synthetase) has been purified to a high degree of homogeneity by a combination of affinity and adsorption chromatography on aminohexyl-Sepharose, concanavalin-A-Sepharose, and hydroxyapatite. The monomeric form of the enzyme has an M , of 55000 +_ 1000 as estimated by sodium dodecyl sulfate gel electrophoresis. Its absolute spectrum shows a high-spin Soret band at 394 nm while its reduced, CO-difference spectrum has a maximum at 447 1 nm. Full reconstitution of aromatase activity was obtained when it was recombined with a homogeneous preparation of the higher-M, form of either human placental, or bovine hepatic NADPH -cytochrome P-450 reductase. Critical factors for purification of the very unstable, membrane-bound hemoprotein with good retention of activity were, besides the chromatographic sequence, the use of the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (Chaps) during the solubilization, and the stabilizing effect of the aromatase substrate, 4-androstene-3,17-dione, throughout the procedure. In the presence of NADPH, the reconstituted enzyme system smoothly aromatizes 19-oxoandrostenedione, 19-hydroxyandrostenedione and androstenedione in this order of reactivity. The same reconstituted system also aromatized testosterone, but it was inactive towards 19-norandrostenedione. Known cytochrome P-450 inhibitors decreased its activity. We conclude: (a) the terminal oxidase of human placental aromatase is indeed a cytochrome P-450-type monooxygenase; (b) the multistep aromatization reaction of CI9 androstenes is catalyzed by a single enzyme; (c) aromatization of 19-norsteroids reported by other authors must be due to a different aromatase. Experimental data obtained with the reconstituted enzyme are fully compatible with the concept of a reaction mechanism for the aromatization sequence involving an all-trans, antiparallel elimination of the 19-methyl group, the 2p proton and the l a proton, rather than the 1p proton, as generally assumed.The conversion of male to female steroid hormones that takes place in the microsomes is catalyzed by a mixed-function oxidase, i.e. an enzyme that concurrently reduces molecular oxygen, but oxidizes NADPH. In this respect, the aromatase enzyme is no different from the other well-known steroid hydroxylases. However, whereas these hydroxylases catalyze a single C -H to C -OH substitution reaction, in contrast, the aromatase (estrogen synthetase) is implicated in a sequence of four reactions. Initial hydroxylation at the angular 19-methyl group is followed by a second hydroxylation at the same site. According to Fishman and co-workers [l -31, a third hydroxylation then takes place stereospecifically at the 28 position and the 2p-hydroxy-19,19-di-hydroxy(or 19-oxo)-4-androstene-3-one thus formed is the true end product of this three-step enzymic reaction. Because this compound is thermodynamically labile, upon release into the medium by

Dimethandrolone (7α,11β-dimethyl-19-nortestosterone) and 11β-methyl-19-nortestosterone are not converted to aromatic A-ring products in the presence of recombinant human aromatase

“…Thus, published findings have claimed that aromatization of 19-norandrogens is both possible [11][12][13][14] or not possible [7][8][9][10] using various in vitro systems including placental microsomes and granulosa or Leydig cells. Previous publications describing a variety of aromatase preparations and detection methods suggested that both MENT and 19-NT could be aromatized.…”

“…Aromatization of C19 androgens such as T and androstenedione (AD) to their C18 aromatic A-ring products, estradiol and estrone (E 1 ), respectively, takes place in three sequential steps [4][5][6]: (1) hydroxylation of the C19-methyl group; (2) a second hydroxylation of the C19-methyl group to form a 19-oxo-compound; (3) cleavage of the bond between C10 and C19 to yield formic acid and the aromatized A-ring product with loss of the hydrogen atoms at the 1β and 2β positions. Although the mechanism of action of human aromatase involves the C19-methyl group, published findings concerning the potential aromatization of 19-norandrogens in vitro and in vivo have been contradictory [7][8][9][10][11][12][13][14]. Most of these studies have employed human placental microsomes or Leydig or granulosa cells which contain other enzymatic activities, and detection methods involving thin layer or gas chromatography, HPLC, or radioimmunoassays.…”

Aromatase inhibitors and enzyme stability.

“…Stabilization and increased content caused by substrates or inhibitors has been found for many enzymes: for example, the cellular concentrations of arginase (Schimke 1964) and tryptophan pyrrolase (Schimke et al 1965) in rat liver are known to become increased as a result of decreased protein degradation dependent on the substrates, arginine and tryptophan. Furthermore, steroidogenic cytochrome P450 11β and aromatase were successfully purified in the presence of deoxycorticosterone (Takemori et al 1975) and testosterone (Harada 1988) or androstenedione (Tan & Muto 1986) used as substrate stabilizers to prevent inactivation of the enzyme. More recently, immunoreactive aromatase in the quail brain was found to be increased by the specific inhibitors, fadrozole and vorozole (Foidart et al 1994).…”

Constitutional genetic variation at the human aromatase gene (Cyp19) and breast cancer risk

“…Cyp19 encodes for a cytochrome P450 aromatase, an enzyme catalysing the conversion of androgens into oestrogens (Tan and Muto, 1986;Mendelson et al, 1990). Aromatase activity has been demonstrated in multiple tissues including normal and transformed breast tissues (James et al, 1987;Bulun and Simpson, 1994;Santen et al, 1994;Sasano et al, 1994).…”