3-Methylene-substituted androgens as novel aromatization inhibitors. Evidence of a requirement for C-3 oxygen in C-19 hydroxylations

Substitution of a methylene group for the C-3 oxygen in androstenedione, testosterone, and the corresponding 19-hydroxy and 19-oxo derivatives results in a new category of inhibitors of estrogen biosynthesis by human placental microsomes. The inhibition is of the competitive type with the most effective inhibitors being the 17-ketonic compounds, 3-methyleneandrost-4-en-17-one, 19-hydroxy-3-methyleneandrost-4-en-17-one, and 3-methylene-19-oxoandrost-4-en-17-one with apparent Ki values of 4.7, 13, and 24 nM, respectively. The 3-methylene derivatives of androstenedione and 19-hydroxyandrostenedione were effective substrates for the placental microsomal 17 beta-hydroxy-steroid oxidoreductase but were only marginally hydroxylated at the C-19 position to the respective 19-hydroxy and 19-oxo derivatives. The 3-methylene analogs are thus competitive inhibitors of aromatization but are not substrates for this enzyme complex. Time-dependent inhibition of aromatization by 10 beta-difluoromethylestr-4-ene-3,17-dione and 10 beta-(2-propynyl)estr-4-ene,3,17-dione was abolished by substitution of a methylene function for the C-3 oxygen, suggesting that the presence of an oxygen at C-3 is required for an oxidative transformation at C-19, an initial step in aromatization. The essential role of the C-19 hydroxylation in aromatization is supported by the observation that the 3-methylene derivatives of 19-hydroxy- and 19-oxoandrostenedione showed time-dependent inhibition, but the corresponding 19-methyl compound did not. The 3-methylene androgens are potent inhibitors of placental aromatization but are themselves only marginal substrates for the enzyme. Their high affinity for and inertness to the placental aromatase complex makes them valuable probes of the aromatization process.     

Microbial 16β-Hydroxylation of Steroids with Aspergillus niger

Microbial 16β-hydroxylation of some steroids with Wojnowicia graminis, Corticium centrifugum and Bacillus megaterium has been reported, but not 16β-hydroxylation of normal 17-oxo steroids with Aspergillus niger. This time, we tried microbial transformation of dehydroepiandrosterone with this fungus, and obtained 4-androstene-3,17-dione, 17β-hydroxy-4-androstene-3,16-dione, 16β,17β-dihydroxy-4-androsten-3-one and a new compound, 16β-hydroxy-4-androstene-3,17-dione. This new compound was also obtained by the fermentation of 4-androstene-3,17-dione and testosterone.     

Metabolism of progesterone and testosterone by a Bacillus sp.

Microbial transformations by a Bacillus sp. were employed as a means of preparing potentially important derivatives of progesterone and testosterone. Each microbial metabolite was subjected to structure elucidation employing ¹H and ¹³C nmr, mass spectral and cd analysis. Hplc was used for the determination of the percentages of the metabolites formed. The progesterone metabolites were characterised as 14-hydroxy-4-pregnene-3,20-dione (II), 14-hydroxy-5 α -pregnane-3,6,20-trione (III)., 11 α — hydroxy-5 α — pregnane-3, 6,20-trione (IV) and 11 α, 14-dihydroxy-4-pregnene-3,20-dione (V). The testosterone analogs were identified as 4-androstene-3,17-dione (VII), 17 β-hydroxy-5 α -androstene-3,6-dione (VIII), 14-hydroxy-4-androstene-3,17-dione (IX) and 14, 17 β-dihydroxy-4-androsten -3-one (X)₁. The availability of the metabolites enabled complete elucidation of their ¹³C nmr spectra.     

Inhibitor specificity of the placental microsomal oxidase system responsible for the aromatization of epitestosterone (17 alpha-hydroxy-4-androsten-3-one)

Human placental microsomes converted epitestosterone to estradiol-17 alpha at rates of 23-48 pmol/min X mg protein with a Km of 113 microM. Activity was inhibited 70-90% by concentrations of CO, metyrapone, n-octylamine, 7,8-benzoflavone and 7-ethoxycoumarin which had no effect on the aromatization of 4-androstene-3, 17-dione. Conversely, cyanide and azide were more effective inhibitors of the conversion of the latter androgen. A variety of neutral steroids inhibited the aromatization of epitestosterone with 19-norsteroids being particularly effective, but competitive effects could not be demonstrated. Both 17 beta-hydroxy-4-estren-3-one and 16 alpha-hydroxy-4-androstene-3,17-dione caused a mixed inhibition. A number of phenolic steroids were also inhibitory with 16-oxo compounds being particularly effective. Inhibition by estrone was non-competitive (Ki = 16 microM). The aromatization of epitestosterone resembles placental microsomal oxidase activities against estrone and benzo [a]pyrene in its inhibitor specificity and epitestosterone may be the native substrate for an oxidase also active in the metabolism of aromatic xenobiotic chemicals.     

Identification by gas chromatography-mass spectrometry of intermediates in the aromatization of modified C19 steroids by human placental microsomes

Analogs of 4-androstene-3,17-dione and testosterone were tested as substrates for the aromatizing enzyme complex of human placenta. Compounds modified in rings B, C and D were found to be aromatized via a pathway similar to that postulated for 4-androstene-3,17-dione and testosterone, in which oxidation to the 19-hydroxy and 19-oxo (or corresponding gem-diol) intermediates occurs. No evidence of additional intermediates was obtained.     

The stereospecific removal of a C-19 hydrogen atom in oestrogen biosynthesis

1. The synthesis of a number of 19-substituted androgens is described. 2. A method for the partially stereospecific introduction of a tritium label at C-19 in 19-hydroxyandrost-5-ene-3beta,17beta-diol was developed. The 19-(3)H-labelled triol produced by reduction of 19-oxoandrost-5-ene-3beta,17beta-diol with tritiated sodium borohydride is tentatively formulated as 19-hydroxy[(19-R)-19-(3)H]androst-5-ene-3beta,17beta-diol and the 19-(3)H-labelled triol produced by reduction of 19-oxo[19-(3)H]-androst-5-ene-3beta,17beta-diol with sodium borohydride as 19-hydroxy[(19-S)-19-(3)H]-androst-5-ene-3beta,17beta-diol. 3. In the conversion of the (19-R)-19-(3)H-labelled compound into oestrogen by a microsomal preparation from human term placenta more radioactivity was liberated in formic acid (61.6%) than in water (38.4%). In a parallel experiment with the (19-S)-19-(3)H-labelled compound the order of radioactivity was reversed: formic acid (23.4%), water (76.2%). 4. These observations are interpreted in terms of the removal of the 19-S-hydrogen atom in the conversion of a 19-hydroxy androgen into a 19-oxo androgen during oestrogen biosynthesis. 5. It is suggested that the removal of C-19 in oestrogen biosynthesis occurs compulsorily at the oxidation state of a 19-aldehyde with the liberation of formic acid.     

Role of reduced glutathione in the delta(5)-3-kitosteroid isomerase reaction of liver.

Partially purified rat liver Δ⁵-3-ketosteroid isomerase (EC 5.3.3.1) is profoundly and specifically activated by reduced glutathione (GSH). This stimulating effect shows normal saturating kinetics, and both K_m and V_max are pH-dependent. The binding of GSH is independent of the concentration of Δ⁵-androstene-3,17-dione, whereas the K_m for Δ⁵-androstene-3,17-dione is markedly reduced by saturating levels of GSH. The same catalytic site appears to isomerize both Δ⁵-androstene-3,17-dione and Δ⁵-pregnene-3,20-dione. Several steroidal inhibitors compete with Δ⁵-androstene-3,17-dione, whereas $\text{S}$-methyl-glutathione competes with GSH. This activation of Δ⁵-3-ketosteroid isomerase is also observed in the livers of other species (calf, guinea pig, human), and represents a hitherto unrecognized function of reduced glutathione.     

Effects of 4-hydroxy-4-androstene-3,17-dione and 10-propargylestr-4-ene-3,17-dione on the metabolism of androstenedione in human breast carcinoma and breast adipose tissues

The effects of 4-hydroxy-4-androstene-3,17-dione (4-OH-A) and 10-propargylestr-4-ene-3,17-dione (PED) on the aromatization of androstenedione (A) and the conversion of A to testosterone (T) were studied in incubations with breast carcinoma and breast adipose tissues. Parallel studies were carried out to determine the effects of 4-OH-A and PED on A metabolism in tissue from 5 patients with breast carcinoma. At 11 μM, both compounds fully inhibited aromatization, whereas the conversion of A to T was decreased in only 2 incubations.Studies with varying concentrations of 4-OH-A and PED demonstrated that both compounds inhibited estrone (E₁) formation by 80% at a concentration of 0.085 μM, with maximum effect at 0.34 μM. 90% inhibition of estradiol (E₂) formation was observed at inhibitor concentrations of 0.17 μM or greater. T formation was slightly affected at 0.67 μM, but was progressively inhibited with increasing 4-OH-A or PED concentrations, reaching 70% at 11 μM.Similar experiments with 4-OH-A in breast adipose tissue homogenates showed that a concentration of 0.1 μM was sufficient to inhibit aromatization while T inhibition required 11 μM.4-OH-A and PED are selective inhibitors of aromatization in human breast tissues and may provide a mechanism for controlling estrogen responsive processes.     

Concerning the pathway from 19-oxoandrost-4-ene-3,17-dione to estrone

The conversion of a molecule of 19-oxoandrost-4-ene-3,17-dione [1a] to estrone [2a] by human placental aromatase requires a molecule of oxygen and of NADPH. An atom of this molecule of oxygen is incorporated into the extruded formic acid derived from C-19 of [1a]. It was proposed that the 0₂ is utilized for the enzymatic 2β-hydroxylation of [1a] and the released intermediate 2β-hydroxy-19-oxoandrost-4-ene-3, 17-dione [5a]aromatizes nonenzymatically. Should [5a] be an obligatory intermediate of estrogen biosynthesis, then all the oxygen of its 2β-hydroxyl must be incorporated into the extruded formic acid. We have previously synthesized [2β-¹⁸0;19-³H][5c] and proved that none of its 2β-¹⁸0 was incorporated in the formic acid extruded in the aromatization. On this basis we concluded that [5a] can not be an obligatory precursor of estrogen biosynthesis.

The trapping of radioactive androst-4-ene-2β,3β,17β,19-tetrol in a reductively terminated incubation of a mixture of radioactive androst-4-ene-3, 17-dione and [5a] with crude placental aromatase was interpreted as evidence in support of the intermediacy of [5a]. We confirmed that the tetrol can indeed be trapped in the reductively terminated incubations. However, considering that the crude placental enzyme preparation very likely contains numerous activated oxygen species capable of a variety of oxidation reactions, most of which may not be related to estrogen elaboration, and in view of our results quoted above, the origin and the eventual biosynthetic role of the parent compound of the tetrol remains to be determined.     

Aromatase reaction of 3-deoxyandrogens: steric mode of the C-19 oxygenation and cleavage of the C10-C19 bond by human placental aromatase

Aromatase is a cytochrome P-450 enzyme complex that catalyzes the conversion of androst-4-ene-3,17-dione (AD) to estrone and formic acid through three sequential oxygenations of the 19-methyl group. To gain insight into the catalytic function of aromatase as well as the mechanism of the hitherto uncertain third oxygenation step, we focused on the aromatase-catalyzed 19-oxygenation of 3-deoxyandrogens: 3-deoxy-AD (1), which is a very powerful competitive inhibitor but poor substrate of aromatase, and its 5-ene isomer 4, which is a good competitive inhibitor and effective substrate of the enzyme. In incubations of their 19S-(3)H-labeled 19-hydroxy derivatives 2 and 5 and the corresponding 19R-(3)H isomers with human placental microsomes in the presence of NADPH under air, the radioactivity was liberated in both water and formic acid. The productions of (3)H(2)O and (3)HCOOH were blocked by the substrate AD or the inhibitor 4-hydroxy-AD, indicating that these productions are due to a catalytic function of aromatase. A comparison of the (3)H(2)O production from S-(3)H substrates 2 and 5 with that from the corresponding R-(3)H isomers revealed that the 19-pro-R hydrogen atom was stereospecifically (pro-R:pro-S = 100:0) removed in the conversion of 5-ene substrate 5 into the 19-oxo product 6, whereas 75:25 stereoselectivity for the loss of the pro-R and pro-S hydrogen atoms was observed in the oxygenation of the other substrate, 2. The present results reveal that human placental aromatase catalyzes three sequential oxygenations at C-19 of 3-deoxyandrogens 1 and 4 to cause the cleavage of the C(10)-C(19) bond through their 19-hydroxy (2 and 5) and 19-oxo (3 and 6) intermediates, respectively, where there is a difference in the stereochemistry between the two androgens in the second 19-hydroxylation. It is implied that the aromatase-catalyzed 19-oxygenation of 5-ene steroid 4 but not the 4-ene isomer 1 would proceed in the same steric mechanism as that involved in the AD aromatization.     

Microbial degradation of the phytosterol side-chain to 24-oxo products

A mutant of the potent sterol degrader Mycobacteriun fortuitum (ATCC6842) has been isolated which is defective in its ability to degrade both the steroid nucleus and sterol side-chains that are branched at the 24-position. Bioconversions of phytosterol mixtures by this mutant resulted in the accumulation of the novel 24-oxo intermediates 9-hydroxy-27-nor-4-cholestene-3,24-dione (II) and 9-hydroxy-26,27-dinor-4-cholestene-3,24-dione (III). Under the same conditions, cholesterol is degraded mainly to 9-hydroxy-4-androstene-3,17-dione (I) by this organism.     

Spectrophotometric estimation of 4-androstene-3,17-dione and 1,4-androstadiene-3,17-dione during C-1(2)-dehydrogenation by Mycobacterium fortuitum NRRL B-8153

A spectrophotometric method for simultaneously estimating 4-androstene-3,17-dione (AD) and 1,4-androstadiene-3,17-dione (ADD) in a binary mixture has been developed using sulphuric acid chromogens. The method has been used to estimate both AD and ADD during C-1(2)-dehydrogenation by Mycobacterium fortuitum NRRL B-8153.The authors are with the School of Life Sciences, Devi Ahilya Vishwavidyalaya. Vigyan Bhawan, Khandwa Road, Indore-452 001, India.     

Hepatic microsomal metabolism of androst-4-ene-3,17-dione: Relative importance of ring hydroxylation and aromatization in control and induced rat liver

The purpose of these studies was to determine whether oestrogen production is a quantitatively important pathway in the hepatic microsomal metabolism of androst-4-ene-3,17-dione. The effects of the enzyme inducing agents phenobarbitone and β-naphthoflavone on microsomal cytochrome P-450-mediated androst-4-ene-3,17-dione hydroxylation and aromatization was investigated in the rat in vitro. In microsomal fractions from untreated rats the ratio of hydroxylated products to aromatized (oestrogenic) metabolites was 33:1. Phenobarbitone pretreatment of rats increased total hydroxylation by about 20% but did not change the ratio of hydroxylated to aromatized products (27:1). In contrast, β-naphthoflavone induction decreased total hydroxylation to about 35% of control but did not affect total aromatization. Thus the ratio of hydroxylation to aromatization was significantly lower than in control microsomes (17:1).The principal aromatized products were oestriol and 2-hydroxyoestradiol-17β, with oestradiol-17β and its 4-hydroxy metabolite as minor products; no oestrone was observed. In further studies of the microsomal metabolism of oestrone, the major product was oestradiol-17β whereas hydroxylated metabolites were only minor products. Oestradiol-17β, in contrast, was hydroxylated to a considerable extent. These findings suggest that oestrone is a better substrate for the microsomal 17β-oxidoreductase than it is for cytochrome P-450. It therefore appears likely that any oestrone formed from the aromatization of androst-4-ene-3,17-dione would be readily converted to oestradiol-17β which, in turn, is subject to cytochrome P-450-mediated hydroxylation. Although the liver is a site of C₁₉-steroid aromatization, it appears unlikely that this organ could contribute significantly to serum oestrogen levels since microsomal hydroxylases are readily able to convert aromatized products to biologically inactive metabolites.     

Synthesis of 3 beta,16 beta,19-trihydroxyandrost-5-en-17-one and 16 beta,19-dihydroxyandrost-4-ene-3,17-dione and their 19-oxo derivatives

3 beta,16 beta,19-Trihydroxyandrost-5-en-17-one (12) was synthesized from 5 alpha-bromo-3 beta-acetoxy-6 beta,19-epoxyandrostan-17-one (2) through acetoxylation at C-16 beta of the enol acetate 4 with lead tetraacetate and reductive cleavage of the epoxide ring with zinc dust yielding the 3 beta,16 beta-diacetoxy-19-hydroxy steroid 11, followed by hydrolysis of the acetoxy groups with sulfuric acid. Jones oxidation of compound 11 followed by the acid hydrolysis gave the 19-oxo steroid 15. 5 alpha-Bromo-3 beta-hydroxy-16 beta-acetoxy-6 beta,19-epoxyandrostan-17-one (8), obtained by selective hydrolysis of the 3-formate 5 with ammonium hydroxide, was oxidized with Jones reagent to afford the 3-oxo steroid 16, which was converted into the 19-hydroxy derivative 17 by treatment with zinc dust. 16 beta,19-Dihydroxyandrost-4-ene-3,17-dione (18) and its 19-oxo derivative 21 were obtained from compound 17 through a similar reaction sequence.     

Differential inhibition of androst-4-en-3,17-dione aromatization by carbon monoxide in the presence of estr-4-en-3,17-dione.

The aromatization of androst-4-en-3,17-dione or 17beta hydroxyandrost-4-en-3-one (testosterone) is not inhibited by carbon monoxide under normal incubation conditions, whereas the aromatization of corresponding 19-nor steroids (estr-4-en-3,17-dione and 17beta-hydroxyestr-4-en-3-one) is readily inhibited under the same conditions. A possible explanation was found when it was shown that androst-4-en-3,17-dione and testosterone could displace bound carbon monoxide from human placental microsomal cytochrome P-450. The 19-nor steroids did not displace carbon monoxide, even at very high concentrations. These C-18 compounds appeared to facilitate complex formation and reversed the effects of the C-19 steroids. A mutual antagonism was observed with regard to effects on the formation of the ce titrated. These observations suggested that the aromatization of androst-4-en-3,17-dione should be inhibited by carbon monoxide if sufficient concentrations of the 19-nor steroids were present in reaction flasks. This hypotheses was tested and positive results were obtained, providing strong evidence for the involvement of cytochrome P-450 in normal estrogen biosynthesis.     

Synthesis of (4-C)-labeled and non-labeled 3β,15α-dihydroxy-5-pregnen-20-one and 3β,15α-dihydroxy-5-androsten-17-one

The synthesis of labeled and non-labeled 3β,15α-dihydroxy-5-pregnen-20-one (V) and 3β, 15α-dihydroxy-5-androsten-17-one (XI) is described. Treatment of 15α-hydroxy-4-pregnene-3,20-dione (I) with acetic anhydride and acetyl chloride gave 3,15α-diacetoxy-3,5-pregnadien-20-one (II). The enol acetate (II) was ketalized by a modification of the general procedure to yield 3,15α-diacetoxy-3,5-pregnadien-20-one cyclic ethylene ketal (III) which was then reduced with NaBH₄ and LiAlH₄ to give 3β, 15α-dihydroxy-5-pregnen-20-one cyclic ethylene ketal (IV). Cleavage of the ketal group of IV gave V. Similarly, XI was prepared by starting with 15α-hydroxy-4-androstene-3,17-dione (VII). The (4-¹⁴C)-3β,15α-dihydroxy-5-pregnen-20-one was prepared by a modification of the above procedure in that the enol acetate (II)was directly reduced with NaBH₄ and LiAlH₄ to yield 5-pregnene-3β,15α,20β-triol (XIII) which was then oxidized enzymatically with 20β-hydroxysteroid dehydrogenase to V.     

Catalysis of the isomerization of Δ5-3-ketosteroids to Δ4-3-ketosteroids by primary amines: Evidence for an imine intermediate

The isomerization of 5-androstene-3,17-dione and 17β-hydroxy-5-androstene-3-one to 4-androstene-3,17-dione and 17β-hydroxy-4-androstene-3-one, respectively, is catalyzed by primary amines. In the case of the isomerization catalyzed by glycylglycine the reaction proceeds through an intermediate which absorbs maximally at 275 nm. Based on spectral similarities to appropriate model compounds and structural analysis of the intermediate after its reduction by sodium borohydride, the intermediate has been tentatively identified as the Δ⁴-3-imine.     

Identification of genes involved in inversion of stereochemistry of a C-12 hydroxyl group in the catabolism of cholic acid by Comamonas testosteroni TA441

Comamonas testosteroni TA441 degrades steroids such as testosterone via aromatization of the A ring, followed by meta-cleavage of the ring. In the DNA region upstream of the meta-cleavage enzyme gene tesB, two genes required during cholic acid degradation for the inversion of an α-oriented hydroxyl group on C-12 were identified. A dehydrogenase, SteA, converts 7α,12α-dihydroxyandrosta-1,4-diene-3,17-dione to 7α-hydroxyandrosta-1,4-diene-3,12,17-trione, and a hydrogenase, SteB, converts the latter to 7α,12β-dihydroxyandrosta-1,4-diene-3,17-dione. Both enzymes are members of the short-chain dehydrogenase/reductase superfamily. The transformation of 7α,12α-dihydroxyandrosta-1,4-diene-3,17-dione to 7α,12β-dihydroxyandrosta-1,4-diene-3,17-dione is carried out far more effectively when both SteA and SteB are involved together. These two enzymes are encoded by two adjacent genes and are presumed to be expressed together. Inversion of the hydroxyl group at C-12 is indispensable for the subsequent effective B-ring cleavage of the androstane compound. In addition to the compounds already mentioned, 12α-hydroxyandrosta-1,4,6-triene-3,17-dione and 12β-hydroxyandrosta-1,4,6-triene-3,17-dione were identified as minor intermediate compounds in cholic acid degradation by C. testosteroni TA441.     

Studies on the Microbiological Transformation of Steroids

An attempt was made to clarify how Pellicularia filamentosa f. sp. microsclerotia IFO 6298 capable of hydroxylating C₂₁-steroids at the C-19 position converts C₁₉-steroids, especially monohydroxyderivatives of androst-4-ene-3, 17-dione. Such substrates as 11β-hydroxyandrost-4-ene-3,17-dione (I), androst-4-ene-3, 11, 17-trione (II), androsta-1,4-diene-3, 17-dione (III), 11β-hydroxyandrosta-1,4-diene-3,17-dione (IV), 14α-hydroxyandrost-4-ene-3, 17-dione (V), 15α-hydroxyandrost-4-ene-3, 17-dione (VI) and 9α-hydroxyandrost-4-ene-3, 17-dione (VII) were converted by the organism. All the main and several minor products were then isolated and identified. As a result it is concluded that this organism converts I and II into 14α-hydroxyandrost-4-ene-3,11,17-trione, III and IV into 14α-hydroxyandrosta-1,4-diene-3,1l,17-trione, V into 11α 14α dihydroxyandrost-4-ene-3, 17-dione (main) and 11β, 14α-dihydroxyandrost-4-ene-3, 17-dione (minor, a tentative structure), VI into 11β, 15α-dihydroxyandrost-4-ene-3,17-dione (main) and 15α-hydroxyandrost-4-ene-3,11,17-trione (minor, a tentative structure) and VII into 9α, 14α-dihydroxyandrost-4-ene-3, 17-dione (main) and 6β, 9α-dihydroxyandrost-4-ene-3,17-dione (minor).

In addition, the structural requirement of substrate for the 19-hydroxylation catalyzed by the organism and the influence of a hydroxyl group on steroid nucleus upon the 11β- and 14α-hydroxylations and the 11β-OH-dehydrogenation was discussed.     

Role of Neutral Metabolites in Microbial Conversion of 3β-Acetoxy-19-Hydroxycholest-5-Ene into Estrone

Biotransformation of 3β-acetoxy-19-hydroxycholest-5-ene (19-HCA, 6 g) by Moraxella sp. was studied. Estrone (712 mg) was the major metabolite formed. Minor metabolites identified were 5α-androst-1-en-19-ol-3,17-dione (33 mg), androst-4-en-19-ol-3,17-dione (58 mg), androst-4-en-9α,19-diol-3,17-dione (12 mg), and androstan-19-ol-3,17-dione (1 mg). Acidic metabolites were not formed. Time course experiments on the fermentation of 19-HCA indicated that androst-4-en-19-ol-3,17-dione was the major metabolite formed during the early stages of incubation. However, with continuing fermentation its level dropped, with a concomitant increase in estrone. Fermentation of 19-HCA in the presence of specific inhibitors or performing the fermentation for a shorter period (48 h) did not result in the formation of acidic metabolites. Resting-cell experiments carried out with 19-HCA (200 mg) in the presence of α,α′-bipyridyl led to the isolation of three additional metabolites, viz., cholestan-19-ol-3-one (2 mg), cholest-4-en-19-ol-3-one (10 mg), and cholest-5-en-3β,19-diol (12 mg). Similar results were also obtained when n-propanol was used instead of α,α′-bipyridyl. Resting cells grown on 19-HCA readily converted both 5α-androst-1-en-19-ol-3,17-dione and androst-4-en-19-ol-3,17-dione into estrone. Partially purified 1,2-dehydrogenase from steroid-induced Moraxella cells transformed androst-4-en-19-ol-3,17-dione into estrone and formaldehyde in the presence of phenazine methosulfate, an artificial electron acceptor. These results suggest that the degradation of the hydrocarbon side chain of 19-HCA does not proceed via C₂₂ phenolic acid intermediates and complete removal of the C₁₇ side chain takes place prior to the aromatization of the A ring in estrone. The mode of degradation of the sterol side chain appears to be through the fission of the C₁₇-C₂₀ bond. On the basis of these observations, a new pathway for the formation of estrone from 19-HCA in Moraxella sp. has been proposed.