A Novel Testosterone Catabolic Pathway in Bacteria�‡Yann-Lii Leu,1† Po-Hsiang Wang,2† Ming-Shi Shiao,3 Wael Ismail,4 and Yin-Ru Chiang2*
Natural Products Laboratory, Graduate Institute of Natural Products, College of Medicine, Chang-Gung University, Taoyuan, Taiwan1;Microbiology Laboratory, Graduate Institute of Natural Products, College of Medicine, Chang-Gung University, Taoyuan,Taiwan2; Department of Biomedical Sciences, Chang-Gung University, Taoyuan, Taiwan3; and Biotechnology Program,
Arabian Gulf University, Al-Manamah, Kingdom of Bahrain4
Received 8 March 2011/Accepted 22 June 2011
Forty years ago, Coulter and Talalay (A. W. Coulter and P. Talalay, J. Biol. Chem. 243:3238–3247, 1968)established the oxygenase-dependent pathway for the degradation of testosterone by aerobes. The oxic testos-terone catabolic pathway involves several oxygen-dependent reactions and is not available for anaerobes. Sincethen, a variety of anaerobic bacteria have been described for the ability to degrade testosterone in the absenceof oxygen. Here, a novel, oxygenase-independent testosterone catabolic pathway in such organisms is described.Steroidobacter denitrificans DSMZ18526 was shown to be capable of degrading testosterone in the absence ofoxygen and was selected as the model organism in this study. In a previous investigation, we identified theinitial intermediates involved in an anoxic testosterone catabolic pathway, most of which are identical to thoseof the oxic pathway demonstrated in Comamonas testosteroni. In this study, five additional intermediates of theanoxic pathway were identified. We demonstrated that subsequent steps of the anoxic pathway greatly differfrom those of the established oxic pathway, which suggests that a novel pathway for testosterone catabolism ispresent. In the proposed anoxic pathway, a reduction reaction occurs at C-4 and C-5 of androsta-1,4-diene-3,17-dione, the last common intermediate of both the oxic and anoxic pathways. After that, a novel hydrationreaction occurs and a hydroxyl group is thus introduced to the C-1� position of C19 steroid substrates. To ourknowledge, an enzymatic hydration reaction occurring at the A ring of steroid compounds has not beenreported before.
Isoprenoids are built up from one or more five-carbon unitsand constitute a large group of natural compounds. Amongthem cholesterol, a triterpenoid, is present in the membranesof eukaryotes, and its major role is to modulate the toughnessand permeability of membranes. In addition, cholesterol alsoserves as a precursor to all steroid hormones, vitamin D, andbile acids (3). One of its derivatives, testosterone, belongs tothe C19 androgen group and is primarily secreted by the testesof males and ovaries of females. Compared to cholesterol, thealiphatic side chain present on C-17 is absent from testoster-one (for its structure, see Fig. 1), which makes it more watersoluble than its biosynthetic precursor. Mammals are able tosynthesize, but cannot degrade, testosterone and other steroidhormones. After a slight modification to enhance the solubility,these steroid compounds are excreted into the environmentthrough the urinary tract (3).
A variety of androgens and estrogens have been detected ineffluents of American, Brazilian, Canadian, and Germanwastewater treatment plants and in surface water in Americanand Dutch rivers at concentrations in the ng liter�1 range (2,18, 20, 32). Most steroid hormones present in the environmentare produced by humans and livestock (25). One of the majorconcerns about these natural steroid hormones is their ability
to alter the sexual behavior and endocrine systems of wildlife(22, 31). Because of the negative environmental impacts ofsteroid hormones, the removal of these compounds from theenvironment has attracted increasing interest (1). Recent stud-ies indicated that anoxic riverbed sediments and soil have thepotential to be a reservoir for steroid compounds (13, 33). Thus,in order to improve the removal of steroids from the environ-ment, it is necessary to understand the biochemical processesinvolved in anoxic mineralization of steroid hormones.
In addition, because of their diverse physiological functionsin the human body, steroid compounds may be ranked amongthe most widely marketed chemicals by the pharmaceuticalindustry (11). This interest is due to the biotechnological ap-plications of steroid-transforming enzymes with high regio-and stereospecificity in the industrial synthesis of steroids (19).The complex structure of steroid compounds and the highregio- and stereoselectivity of the enzymatic reactions renderthe utility of biocatalysts from microbial sources particularlyfascinating.
The complete oxic mineralization of testosterone by variousspecies such as Comamonas testosteroni was studied in somedetail (7, 16, 17) (Fig. 1A). The oxic catabolism of testosteroneis initiated by dehydrogenation of the 17�-hydroxyl group toproduce androst-4-en-3,17-dione, which then undergoes an-other dehydrogenation to form androsta-1,4-diene-3,17-dione.The subsequent cleavage of the core ring system is catalyzed byseveral oxygenases that utilize oxygen as a cosubstrate (12, 16,17, 26) (see also Fig. 1A).
On the contrary, little is known about the anoxic degradationof steroids (4). It is obvious that anaerobic microorganismswould have to utilize a novel oxygen-independent catabolic
pathway to degrade testosterone in the absence of molecularoxygen. Studies on anoxic testosterone metabolism should re-veal many unprecedented reactions and novel enzymes withmany potential applications. In the last decade, a few bacteriathat can mineralize steroids under denitrifying conditions wereisolated and characterized (9, 10, 15, 30). All of them areproteobacteria and have a relatively narrow substrate spec-trum. One of them, Steroidobacter denitrificans DSMZ18526,can anaerobically utilize certain C18 estrogen or C19 androgensteroids as the sole carbon and energy source via an unknowncatabolic pathway (10). Recently, we reported on the initialsteps in the anoxic catabolism of testosterone by this denitri-
fying bacterium (5). We demonstrated that under anoxic con-ditions, S. denitrificans initially oxidizes testosterone to 1-de-hydrotestosterone, which is then transformed to androsta-1,4-diene-3,17-dione. Moreover, it seems that testosterone canalso be transformed to androst-4-en-3,17-dione by Steroidobac-ter cells. In general, the initial steps of anoxic testosteronedegradation by S. denitrificans are very similar to those of theoxic pathway demonstrated in Comamonas testosteroni (7, 16,17). In the present study, we report on subsequent intermedi-ates of the anoxic testosterone catabolic pathway in S. denitri-ficans. From our current data, a novel pathway for testosteronecatabolism is proposed (Fig. 1B).
FIG. 1. Initial steps involved in the catabolism of testosterone by bacteria. (A) Established oxic pathway (7, 17). (B) Proposed initial steps inthe anoxic pathway, as studied in S. denitrificans DSMZ18526. The marked WPS3 and WPS4 are the presumed intermediates that were not foundin the present study. The ring identification system (A to D) and carbon numbering system of steroids is as shown for testosterone.
Materials and bacterial strain. [4C-14C]testosterone and 18O-labeledwater(99 atom%) were respectively obtained from Perkin-Elmer and Sigma. Thechemicals used were of analytical grade and were purchased from MallinckrodtBaker, Merck, or Sigma-Aldrich. Steroidobacter denitrificans DSMZ18526 (10)was obtained from the Deutsche Sammlung fur Mikroorganismen und Zellkul-turen (Braunschweig, Germany).
The denitrifying growth of bacteria. S. denitrificans was grown anaerobically at28°C under a nitrogen atmosphere. Large-scale fed-batch cultures were carriedout in 5-liter glass bottles sealed with rubber stoppers. The medium and methodused for the cultivation of S. denitrificans under denitrifying conditions aredescribed elsewhere (5). Cells were harvested by centrifugation in the exponen-tial growth phase at an optical density at 600 nm (OD600) of 0.8 to 1.0 (opticalpath, 1 cm), and the cell pellet was then stored at �80°C.
Bacterial culture grown anaerobically on [4C-14C]testosterone. In a small-scale fed-batch culture (250 ml), S. denitrificans cells were incubated with 2.5 mMtestosterone under denitrifying conditions, to which [4C-14C]testosterone (108
dpm) was added as a tracer. The fed-batch culture was carried out in a 300-mlglass bottle sealed with a rubber stopper. The headspace (ca. 50 ml) of theculture was connected to a 120-ml glass tube containing 100 ml of 3 M NaOH,which trapped 14CO2 produced by S. denitrificans cells during the mineralizationof [4C-14C]testosterone. A Tygon tube (2 mm in inner diameter, 25 cm long) wasused for the connection, one end of which was connected to a ceramic cylinder(15 by 8 mm) to minimize the size of the CO2 bubbles. The ceramic cylinder waslocated near the bottom of the 120-ml glass tube containing a 3 M NaOHsolution. After different time intervals of incubation (0, 8, 16, 24, 32, 40, and48 h), samples (1.5 ml) were withdrawn from the 3 M NaOH solution (100 ml).One hour before each sampling, nitrogen gas (ca. 300 ml) was used as the carriergas to expel the residual 14CO2 from the 300-ml glass bottle with a flow rate ofca. 5 ml min�1. The amount of the trapped 14CO2 was determined as describedbelow. At the same time intervals, samples (5 ml) were withdrawn from thebacterial culture to measure the growth of bacterial cells (measured as totalproteins), the residual amount of nitrate and testosterone in the medium, theamount of 14C remaining in the growth medium, and the amount of 14C assim-ilated in the biomass. Separation of S. denitrificans cells from the residual tes-tosterone by centrifugation (10,000 � g for 15 min) was not successful. There-fore, the culture samples (0.5 ml) were extracted with the same volume of ethylacetate three times to isolate the residual [4C-14C]testosterone from the waterfraction. After centrifugation (10,000 � g for 10 min) the biomass, including celldebris and lipids, remained in the water phase and interface, whereas [4C-14C]testosterone remained in the ethyl acetate phase. The ethyl acetate fractionswere combined, evaporated, and the residue was redissolved in 0.5 ml of ethanol.The amount of 14C remaining in the 0.5-ml water phase (mainly the assimilatedbiomass) and 14C extracted by ethyl acetate (mostly the remaining [4C-14C]tes-tosterone) were determined as described below.
Measurement of protein content. Culture samples (0.2 ml) were centrifuged at10,000 � g for 10 min. After centrifugation, the pellet was resuspended in 1 mlof reaction reagent (Pierce BCA protein assay kit; Thermo Scientific). Theprotein content in the culture samples and in cell extracts were determined usinga BCA protein assay according to manufacturer’s instructions with bovine serumalbumin as the standard.
Measurement of testosterone and nitrate concentrations. Testosterone wasquantified by using high-performance liquid chromatography (HPLC) as de-scribed below. Culture samples of 0.5 ml were extracted three times with anequal volume of ethyl acetate. After evaporation of ethyl acetate under a vac-uum, the residue was dissolved in 0.5 ml of 2-propanol for the HPLC analysis.Nitrate was determined by using the 2,6-dimethylphenol photometric method asdescribed previously (6).
Measurement of the amount of 14C. The amount of 14C in water-solublesamples (0.5 ml) was determined by liquid scintillation counting (liquid scintil-lation analyzer, Tri-Carb 2900 TR [Perkin-Elmer]) using 1.5 ml of an UltimaGold high flash-point LSC scintillation cocktail (Perkin-Elmer). In contrast, theamount of 14C in ethanol-soluble samples (0.5 ml) was determined by liquidscintillation counting using the Ecoscint O scintillation cocktail (National Diag-nostics). The counting efficiency was determined via the channel ratio method.
In vivo transformation of 1-testosterone. S. denitrificans cells grown anaerobi-cally with testosterone in a 2-liter glass bottle were harvested by centrifugation(10,000 � g for 10 min, 4°C) when the culture was incubated until an OD600 of0.9 (optical path, 1 cm) was obtained. The pellet with a wet weight of 2.1 g wasresuspended in 23 ml of ice-cold 50 mM potassium phosphate buffer (pH 7)containing 15 mM sodium nitrate and 1 mM 1-testosterone. The assay (25 ml)was performed under anoxic conditions at 28°C for 1 h under a nitrogen gas
phase. After that, the cell suspension was kept on ice, and steroid compounds inthe 25-ml cell suspension were immediately extracted three times with equalvolumes of ethyl acetate. The extracts were combined, and the solvent wasevaporated to dryness. The residue was redissolved in 2 ml of methanol. Theextremely nonpolar contaminants were removed by passing the methanol-solublesamples (2 ml) through a solid-phase extraction cartridge (Bakerbond [C18] SPE,40-�m particles, 1 ml [J. T. Baker]). The presence of 1-testosterone-derivedsteroid intermediates in the samples was detected by ultraperformance liquidchromatography-atmospheric pressure chemical ionization-mass spectrometry(UPLC-APCI-MS) as described below.
Preparation of cell extracts. All steps used for preparation of cell extracts wereperformed at 4°C under anoxic conditions. Frozen cells were suspended in twicethe volume of 150 mM Tris-HCl buffer (pH 9) containing 0.1 mg of DNase Iml�1. Cells were broken by passing the cell suspension through a French pres-sure cell (Thermo Fisher Scientific) twice at 137 MPa. The cell lysate wasfractionated by two steps of centrifugation: the first step involved centrifugationfor 30 min at 20,000 � g to get rid of the cell debris, unbroken cells, and residualtestosterone. The supernatant (crude cell extract) was then centrifuged at100,000 � g for 1.5 h to separate soluble proteins from membrane-bound pro-teins.
In vitro biotransformation assays. In vitro assays were routinely performedunder anoxic conditions at 30°C for 16 h under a nitrogen gas phase. The assaymixtures (1.5 ml) for exclusively producing the steroid products, WPS1 andWPS2, contained 100 mM Tris-HCl buffer (pH 9), soluble proteins (7.5 mg)extracted from S. denitrificans cells, 4.5 mM NADH, and 100 �l of a 67.5 mMandrosta-1,4-diene-3,17-dione solution (in 2-propanol). The final concentrationof androsta-1,4-diene-3,17-dione in the reaction mixture was 4.5 mM. The final2-propanol content was 6.67%. To produce the steroid products WPS5�,WPS5�, and WPS6, the reaction mixture (1.5 ml) contained 100 mM Tris-HClbuffer (pH 9), soluble proteins (7.5 mg) extracted from S. denitrificans cells, 75 �lof a 90 mM androsta-1,4-diene-3,17-dione solution (in 2-propanol), and 25 �l ofa 90 mM testosterone solution (in 2-propanol). The final concentrations ofandrosta-1,4-diene-3,17-dione and testosterone in the reaction mixture were 4.5and 1.5 mM, respectively. The final 2-propanol content was also 6.67%. In thebiotransformation assays to produce WPS5�, WPS5�, and WPS6, no artificialelectron acceptor was added. To produce the steroid products on a large scale,the reaction mixture was enlarged to 100 ml.
18O-incorporation experiments. To elucidate the origin of the oxygen atom atthe C-1 position of three hydroxylated steroid products (WPS5�, WPS5�, andWPS6), two in vitro assays were performed. The two reaction mixtures (3 ml foreach assay) were prepared anaerobically and incubated at 30°C for 16 h withshaking. Three hydroxylated steroid products were purified from the assays andthen analyzed by UPLC-APCI-MS as described below.
(i) Control assay. The 3-ml reaction mixture contained 100 mM Tris-HClbuffer (pH 9), soluble proteins (15 mg) of S. denitrificans, 4.5 mM androsta-1,4-diene-3,17-dione, and 1.5 mM testosterone. The stock solution (in 2-propanol) oftestosterone and androsta-1,4-diene-3,17-dione was prepared as described in thebiotransformation assays for producing the intermediates, WPS5�, WPS5�, andWPS6. The 2-propanol content of the control assay was 6.67%.
(ii) 18O-labeled water-treated assay. 18O-labeled water (1.5 ml) was added to1.5 ml of 200 mM Tris-HCl buffer (pH 9) containing soluble proteins of S.denitrificans (15 mg). The final 18O-labeled water content was ca. 50%. Thereaction was begun by adding 1.5 mM testosterone and 4.5 mM androsta-1,4-diene-3,17-dione to the anoxic assay. The 2-propanol content of the 18O-labeledwater-treated assay was also 6.67%.
TLC. Steroid products were first extracted three times with an equal volume ofethyl acetate, and the ethyl acetate-soluble fraction was concentrated under avacuum. The steroid standards and extracted products were separated on silicagel aluminum thin-layer chromatography (TLC) plates (Silica gel 60 F254; thick-ness, 0.2 mm; 20 by 20 cm [Merck]). The following developing solvent system wasused: dichloromethane-ethyl acetate-ethanol (14:4:1 [vol/vol]). The steroid com-pounds were visualized under UV light at 254 nm or visualized by spraying theTLC plates with 30% (vol/vol) H2SO4.
HPLC. A reversed-phase Hitachi HPLC system was used for the separation,isolation, and identification of steroid standards and steroid products trans-formed from androsta-1,4-diene-3,17-dione.
(i) System 1. For the separation and isolation of WPS1 and WPS2, an analyt-ical RP-C18 column [Luna C18(2), 5 �m, 150 by 4.6 mm (Phenomenex)] was usedwith flow rate of 0.4 ml min�1 at room temperature. The mobile phase was 70%(vol/vol) methanol. Steroid standards and products were detected with a UVdetector (L-2400; Hitachi) at 225 nm.
(ii) System 2. The column used for the separation and isolation of WPS5�,WPS5�, and WPS6 was the same as that described for system 1. Separation was
performed isocratically with 50% (vol/vol) methanol as an eluent and a flow rateof 0.4 ml min�1. Steroid products were detected using two detectors: a UVdetector (Hitachi) monitored at 205 nm and a refractive-index detector(Bischoff) in series.
UV-VIS spectroscopy. Standards and HPLC-purified steroid products weredissolved in acetonitrile in the range of 5 to 10 �g ml�1. UV absorption spectraof these compounds were obtained using a U-1900 UV/VIS spectrometer (Hi-tachi).
UPLC-APCI-MS. The ethyl acetate-extractable samples or HPLC-purifiedsteroid intermediates were analyzed by UPLC-MS with UPLC coupled to anAPCI mass spectrometer. Mass spectral data were obtained using a MicromassZQ quadrupole mass spectrometer (Waters) equipped with a standard APCIsource operating in the positive-ion mode. Separation was achieved on a re-versed-phase C18 column (Acquity UPLC BEH C18, 1.7 �m, 100 by 1.0 mm[Waters]) with a flow rate of 0.1 ml min�1 at 35°C (column oven temperature).The mobile phase comprised a mixture of two solvents: solvent A (1% [vol/vol]acetonitrile containing 0.1% formic acid to enable good ionization in the APCI)and solvent B (methanol containing 0.1% formic acid). Separation was achievedwith a linear gradient of solvent B from 90 to 95% in 17 min. In the APCI-MSanalysis, the temperature of the ion source was maintained at 100°C. Nitrogendesolvation gas was set at a flow rate of 500 liters h�1, and the probe was heatedto 400°C. Nitrogen was used as an APCI carrier gas. The corona current wasmaintained at 20 �A, and the electron multiplier voltage was set to 650 eV. Theparent scan was in the range of 250 to 350 (m/z).
ESI-MS. Electrospray ionization (ESI)-MS data were recorded on a BrukerAPEX II mass spectrometer running in the positive-ion mode.
Nuclear magnetic resonance (NMR) spectroscopy. 1H- and 13C-NMR spectrawere recorded at 27°C with a Bruker Avance-400 FT-NMR spectrometer. Chem-ical shifts (�) were recorded and are shown as parts per million (ppm) values withdeuterated chloroform (99.5%;1H, � � 7.26 ppm; 13C, � � 77.0 ppm) as thesolvent and internal reference.
RESULTS
Anoxic mineralization of testosterone by S. denitrificans. Inour previous study, we showed that S. denitrificans should beable to anaerobically degrade testosterone using a stoichiomet-ric method. However, the possibility of partial oxidation oftestosterone by our model organism could not be excluded (5).We presented here clear and direct evidence to show thattestosterone was mineralized to CO2 during the denitrifyinggrowth of S. denitrificans (Fig. 2).
As shown in Fig. 2A, bacterial growth (measured as theincrease in the protein concentration) was accompanied by adecrease in the concentration of total testosterone and theconsumption of nitrate. The results were very consistent withthe theoretical stoichiometry for anoxic testosterone mineral-ization under denitrifying conditions, which follows the equa-tion (see the explanation in the legend to Fig. 2): C19H28O2 �20 NO3� � 20 H� 3 19 CO2 � 10 N2 � 24 H2O.
Furthermore, bacterial growth was accompanied by a de-crease in the residual [4C-14C]testosterone in the medium, theaccumulation of radioactive 14C in the bacterial cells, and anincrease in the amount of trapped 14CO2 (Fig. 2B). However,it seemed that 14CO2 was not efficiently captured using 3 MNaOH as the trap. Thus, after 48 h of incubation, only 20% ofthe 14C (in the form of CO2) had been trapped (Fig. 2B). After48 h of anaerobic growth of S. denitrificans, the total 14Crecovered was 74% (10% of the 14C remained mainly as tes-tosterone in the medium, 44% of the 14C was assimilated incells, and 20% the 14C was trapped by the 3 M NaOH solution;Fig. 2B). Compared to the stoichiometric results (Fig. 2A), itseems that most of the lost 14C had escaped in the form ofCO2. Our results clearly indicated that S. denitrificans is able to
completely degrade testosterone to CO2 under anaerobic con-ditions.
In vitro transformation of steroid substrates by cell extractsof S. denitrificans. In our previous study (5), androsta-1,4-diene-3,17-dione was the last identified intermediate of theanoxic testosterone catabolic pathway. To identify subsequentintermediates, in vitro transformation of androsta-1,4-diene-3,17-dione under anoxic conditions was carried out. In the firstexperiment, the crude cell extract of S. denitrificans was frac-tionated by ultracentrifugation into soluble and membrane-
FIG. 2. (A) Growth of S. denitrificans DSMZ18526 with 2.5 mMtotal testosterone under denitrifying conditions. Symbols: F, bacterialgrowth (measured as the total protein concentration in the culture); f,residual total testosterone; Œ, total nitrate consumption. (B) Assimi-lation and mineralization of [4C-14C]testosterone (original amountwas 400,000 dpm ml�1) with time in the same bacterial culture (250ml). Symbols: �, residual 14C-labeled carbon in the medium; E, as-similated 14C-labeled carbon in the biomass; ƒ, trapped 14CO2. Thedata are the average of three experimental measurements. After 48 hof incubation, testosterone (2.5 mM) was consumed to a residualconcentration of 0.2 mM (8%), which was accompanied by the con-sumption of 25.5 mM nitrate. Hence, based on the nitrate consumption(see the equation in Results), 1.3 mM testosterone (52%) should havebeen completely degraded to CO2, and 1.0 mM testosterone (40%)should have been assimilated in cells. The measured 14C distribution isin good agreement with this inference: 10% of the 14C remained in themedium (mainly as [4C-14C]testosterone), whereas 44% of the 14C wasassimilated in the biomass.
bound proteins. These different protein fractions were incu-bated with androsta-1,4-diene-3,17-dione and testosteronewithout the addition of any artificial electron acceptor. Whenthe total proteins of Steroidobacter cells were incubated withandrosta-1,4-diene-3,17-dione and testosterone, five interme-diates, named WPS1, WPS2, WPS5�, WPS5�, and WPS6,were observed (Fig. 3, lane D). The same intermediates werealso produced when soluble proteins extracted from Steroido-bacter cells were used (Fig. 3, lane F). It is noteworthy that thesoluble proteins or membrane proteins alone (without the ad-dition of steroid substrates) did not cause the accumulation ofany apparent steroid product after the overnight incubation(Fig. 3, lanes E and G). The soluble proteins of S. denitrificanswere then incubated with the steroid substrates (androsta-1,4-diene-3,17-dione and testosterone) and different artificial elec-tron acceptors (nitrate, ferric ion, NAD�, NADP�, 2,6-dichlo-rophenolineophenol [DCPIP], or methylene blue). However,no additional intermediates were observed (data not shown).Intermediate WPS2 highly overlapped with the wax contami-nants on the TLC plates (Fig. 3, lanes D and F). Fortunately,because of their apparent UV absorption at 225 nm, WPS1and WPS2 could be separated by applying the HPLC systemwith a UV detector (see the supplemental material). The in-termediates, WPS1 and WPS2, were exclusively produced by
incubating soluble proteins of Steroidobacter cells with andro-sta-1,4-diene-3,17-dione and NADH. The transformation ofandrosta-1,4-diene-3,17-dione to WPS1 and WPS2 occurred,since soluble proteins of S. denitrificans were present (see thesupplemental material). The addition of NADH to the reac-tion mixture apparently improved the accumulation of WPS1and WPS2. In an in vitro biotransformation assay, differentconcentrations of NADH were added, and the production ofthe intermediates, WPS1 and WPS2, from androsta-1,4-diene-3,17-dione occurred in an NADH-dependent manner (data notshown).
Identification and structural elucidation of products de-rived from androsta-1,4-diene-3,17-dione. The Rf values, re-tention times, UV absorption maxima, and molecular weightsof HPLC-purified steroid products are summarized in Table 1.The 13C- and 1H-NMR spectral data for these steroid productsare shown in Table 2 and Fig. 4, respectively. The structures of
FIG. 3. Thin-layer chromatograms showing the production of in-termediates from androsta-1,4-diene-3,17-dione and testosterone bythe cell extract, soluble proteins, and membrane proteins of S. denitri-ficans in 100 mM Tris-HCl (pH 9). Lanes: A, four steroid standards; B,negative control 1 (steroid substrates only, without the addition of theproteins of S. denitrificans); C, negative control 2 (cell extract only,without the addition of steroid substrates); D, cell extract with steroidsubstrates; E, negative control 3 (soluble proteins only, without theaddition of steroid substrates); F, soluble proteins with steroid sub-strates; G, negative control 4 (membrane proteins only, without theaddition of steroid substrates); H, membrane proteins with steroidsubstrates. The total protein concentrations of the cell extract, solubleprotein, and membrane protein fractions in different assays were alldiluted to 5 mg ml�1. The assays (1.5 ml) containing different proteinfractions, 4.5 mM androsta-1,4-diene-3,17-dione, and 1.5 mM testos-terone were incubated at 30°C for 16 h under anoxic conditions. Prod-ucts were extracted with ethyl acetate, separated by TLC, and visual-ized by spraying the TLC plates with 30% (vol/vol) H2SO4. WPS2*,WPS2 highly overlapped with the wax contaminants in the TLC sys-tem. Abbreviations: AD, androst-4-en-3,17-dione; ADD, androsta-1,4-diene-3,17-dione; DT, 1-dehydrotestosterone; T, testosterone.
TABLE 1. TLC, HPLC, UV absorption, and mass assignments ofHPLC-purified products and the authentic compound
a TLC separation was performed using the developing solvent: dichlorometh-ane-ethyl acetate-ethanol (14:4:1 vol/vol�). Steroid standard and products wereanalyzed by HPLC in triplicate under two different separation conditions asdescribed in Materials and Methods.
b ADD, androsta-1,4-diene-3,17-dione.
TABLE 2. 13C-NMR chemical shifts for androsta-1,4-diene-3,17-dione compared to those of HPLC-purified steroid
WPS1 and WPS2 were identified by comparing their spectraldata to assignments reported in the literature (34).
In the 1H-NMR spectrum of WPS6, two oxygenated methineprotons were present at �H 4.04 (1H, m, H-3) and 3.84 (1H,br.s, H-1) (Fig. 4). In the 13C-NMR spectrum of WPS6, onecarbonyl signal revealed at �C 220.5 (C-17) and two hetero-atom-substituted carbons were observed at �C 73.3 (C-1) and67.0 (C-3) (Table 2). Final structural elucidation of WPS6 wascarried out using two-dimensional (2D)-NMR (COSY,NOESY, HSQC, and HMBC). The location of a carbonylgroup at C-17 was confirmed by the 3J correlation signal be-tween H-18 (�H 0.83) and a carbonyl carbon (�C 220.5). Hy-droxylated C-1 was proven by the 2J and 3J correlation signalsbetween H-1 (�H 3.84)/C-10 (�C 51.9) and H-19 (�H 0.87)/C-1(�C 73.3), respectively. In the NOESY spectrum of WPS6, thepresence of an NOE signal between H-1 and 19-CH3 indicatedthat H-1 should be in a �-conformation (data not shown). Allof the data described above suggested that WPS6 was 1�,3�-dihydroxy-5�-androstan-17-one.
In the case of WPS5�, an additional oxygenated methineproton and a corresponding oxygenated carbon were present at�H 3.64 (1H, t, J � 8.4 Hz, H-17; Fig. 4) and �C 82.3 (Table 2),respectively. The final structural elucidation of WPS5� wasperformed using 2D-NMR. HMBC correlations between H-18(�H 0.76)/C-17 (�C 82.3) and H-1 (�H 3.84)/C-19 (�C 13.4)suggested that WPS5� was androstan-1,3,17-triol. The NOEsignal between H-1 and 19-CH3 was present, indicating the�-conformation of H-1 of this compound. These data sug-gested that WPS5� was androstan-1�,3�,17�-triol.
The 1H and 13C-NMR spectra of WPS5� and WPS5� werevery similar (Fig. 4, Table 2). An H-3 signal of WPS5� waspresent at �H 4.14 (1H, br.s), and the signal of C-3 of WPS5�was upfield shifted to 63.3 ppm (Table 2) compared to that ofWPS5�. From the results of the splitting pattern and chemicalshifts in their NMR spectra, H-3 of products WPS5� andWPS5� was determined to be in the �- and �-conformations,
respectively. Our data suggested that WPS5� was androstan-1�,3�,17�-triol.
Evidence for the hydration reaction at the A ring of testos-terone. To look for the origin of the hydroxyl groups at C-1 ofWPS5�, WPS5�, and WPS6, two in vitro transformation assayswere performed: (i) an 18O-labeled water-treated assay con-tained ca. 50% 18O-labeled water (mol/mol) in the anoxicreaction mixture (3 ml), and (ii) a control assay (3 ml) wasincubated under anoxic conditions without the addition of 18O-labeled water. After overnight incubation, the steroid productsWPS5�, WPS5�, and WPS6 were purified from these assays,and their molecular weights were determined by APCI-MS(Fig. 5).
The WPS5� samples purified from the control assay had amolecular weight of 308, and a protonated molecular ion([M�H]�, m/z at 309) and a significant dehydrated fragmention ([M-H2O�H]�, m/z at 291) derived from WPS5� wereobserved (Fig. 5A). In contrast, around one-half of the productWPS5� purified from the 18O-labeled water-treated assayshowed an increase in the molecular weight from 308 to 310(Fig. 5B). An increase in the molecular weight from 306 to 308was also observed in product WPS6 purified from the 18O-labeled water-treated assay (Fig. 5F). The proportion of prod-ucts WPS5� and WPS6 with increased molecular weight (ca.50%) exactly matched the proportion of 18O-labeled water(ca. 50%) present in the in vitro biotransformation assay(18O-labeled water-treated assay). For WPS5� purified fromthe control assay, [M�H]� at m/z 309 was omitted, and asignificant [M-H2O�H]� at m/z 291 was present (Fig. 5C).Around one-third of the product WPS5� purified from the18O-labeled water-treated assay showed an increase in the de-hydrated fragment ion ([M-H2O�H]� from m/z at 291 to 293(Fig. 5D). According to the ESI-MS analysis, the molecularweights of WPS5� and WPS5� were both 308 (Table 1). It isworth mentioning that for product WPS5�, the proportion (ca.33%) of the dehydrated fragment ion [M-H2O�H]� (Fig. 5D)with an increased m/z ratio exactly matched that of WPS5�(Fig. 5B). These results clearly demonstrate that the oxygenatom of the incorporated hydroxyl groups at C-1 of productsWPS5�, WPS5�, and WPS6 originated from water and notfrom molecular oxygen.
DISCUSSION
Proposed initial reactions of the anoxic testosterone cata-bolic pathway. According to data from our previous work (5)and the present study, an outline for the initial reactions of anovel testosterone catabolic pathway is proposed using S. deni-trificans as the model organism (Fig. 1B). In our previousreport (5), we showed that testosterone is oxidized to androsta-1,4-diene-3,17-dione via two dehydrogenation reactions at C-1/C-2 and the hydroxyl group at C-17 of testosterone. In thepresent study, we used androsta-1,4-diene-3,17-dione as thesubstrate and NADH as the electron donor in the in vitrotransformation assays, which resulted in the production andaccumulation of WPS1 and WPS2. Compared to androsta-1,4-diene-3,17-dione, the double bond at C-4/C-5 of WPS1 andWPS2 was saturated by a reduction reaction. The fact that S.denitrificans is able to grow anaerobically on these steroidcompounds (testosterone, 1-dehydrotestosterone, androst-4-en-
FIG. 5. APCI-MS spectra (positive-ion mode) of WPS5�, WPS5�, and WPS6. (A) WPS5� purified from the anoxic control assay. (B) WPS5�purified from the 18O-labeled H2O-treated assay. (C) WPS5� purified from the anoxic control assay. (D) WPS5� purified from the 18O-labeledH2O-treated assay. (E) WPS6 purified from the anoxic control assay. (F) WPS6 purified from the 18O-labeled H2O-treated assay.
3,17-dione, androsta-1,4-diene-3,17-dione, WPS1, or WPS2) asthe sole carbon source corroborates the conclusion that thesesteroids should be true intermediates of an anoxic testosteronecatabolic pathway. WPS1 and WPS2 were produced from andro-sta-1,4-diene-3,17-dione in an NADH-dependent manner, sug-gesting that the biocatalyst responsible for the reduction reactionat C4/C5 of steroids may be an NADH-dependent enzyme. OurNMR data showed that the hydrogen atom at C-5 of WPS1 andWPS2 is of the �-form. Thus, the transformation of androsta-1,4-diene-3,17-dione to WPS2 may be catalyzed by an enzyme fromthe steroid-5� reductase subfamily. Major research efforts weredevoted to the study of steroid-5�-reductase (SRD5�) and ste-roid-5�-reductase (SRD5�) in mammalian model organisms be-cause of their implications in numerous human diseases such asprostate carcinoma and hepatic dysfunction, and it seems that thetwo enzymes have different ancestors (21). In contrast, very littleis known about nonmammalian species.
Missed steroid intermediates between WPS1/2 and WPS5/6.In the proposed anoxic testosterone catabolic pathway (Fig.1B), two presumed intermediates, WPS3 and WPS4, were stillnot found in the present study. According to current data, wesupposed that WPS3 (for its structure, see Fig. 1B) is the directprecursor of WPS5�, which might be produced from WPS3 byan unidentified 3�/17�-hydroxysteroid dehydrogenase (3�/17�-HSD) of S. denitrificans. The same enzyme may also cat-alyze the transformation of WPS4 to WPS6 (for its structure,see Fig. 1B). Such an enzyme was purified and characterizedfrom Comamonas testosteroni, which has the ability to degradetestosterone under oxic conditions (28, 29). On the other hand,WPS3 may be transformed to WPS5� by another unidentifiedenzyme of S. denitrificans, 3�-hydroxysteroid dehydrogenase.This enzyme was also purified and characterized from the sameGram-negative bacterium, C. testosteroni (24). Nevertheless,the biocatalysts of S. denitrificans responsible for these func-tions remain to be identified.
Unprecedented hydration reaction of the steroid A ring. Inthe present work, we identified three hydroxylated steroidproducts (WPS5�, WPS5�, and WPS6) produced from andro-sta-1,4-diene-3,17-dione by soluble proteins of S. denitrificansin the absence of molecular oxygen (for their structures, seeFig. 1B). All of these products have a hydroxyl group at theC-1� position. It is known that almost all of the hydroxylgroups of steroid compounds result from hydroxylation reac-tions catalyzed by cytochrome P450 monooxygenases (3). Inaddition, we previously reported that oxygen-independent hy-droxylation catalyzed by a molybdenum-containing hydroxy-lase results in the addition of a hydroxyl group at C-25 of C27steroid compounds, with water as the oxygen donor (6). Wepresent here the first report concerning the unprecedentedhydration reaction of the A ring of steroid compounds. Thefollowing lines of evidence suggest that the hydroxyl group atC-1 of WPS5�/� and WPS6 was introduced via a hydrationreaction: (i) WPS1 (with a double bond at C-1/C-2) was trans-formed to WPS5�/� and WPS6 by a soluble protein fraction ofS. denitrificans. (ii) The addition of electron acceptors (e.g.,ferric ion) or electron donors (NADH or NADPH) did notimprove the production of WPS5�/� or WPS6 in the in vitrobiotransformation, indicating that the introduction of a hy-droxyl group at C-1 of WPS5�/� and WPS6 was catalyzed byneither a cytochrome P450 monooxygenase (which requires
NADPH) nor an Mo-containing hydroxylase (which requiresferric ion, 8). (iii) The oxygen atom of the hydroxyl group atC-1 of steroid products WPS5�/� and WPS6 originated fromwater. Considering that a variety of anaerobes utilize enoylcoenzyme A hydratase and fumarase, respectively, involved inthe �-oxidation pathway and citric acid cycle to oxidize differ-ent organic compounds, it is not surprising to see that anaer-obes apply hydration reactions to oxidize and activate steroidsubstrates in the absence of oxygen. It is known that thiocya-nate is a noncompetitive inhibitor of fumarase (8, 14, 23, 27).However, the addition of 500 mM thiocyanate to the in vitrobiotransformation assay did not inhibit the production of threehydroxylated steroid intermediates (data not shown). The hy-dration reaction implies a novel type of hydratase acting onC-1 and C-2 of steroid compounds. It is noteworthy that thehydration reaction at C-1/C-2 of steroids also occurred in thepresence of molecular oxygen (data not shown), suggestingthat the corresponding enzyme is not oxygen-labile.
In order to validate the roles of these hydroxylated steroidsin the anoxic testosterone catabolism, we also conducted an invivo assay with whole cells of S. denitrificans to transform1-testosterone (WPS1). The ethyl acetate-extractable samplewas then analyzed by UPLC-APCI-MS, and WPS2, WPS5�,and WPS6 were present in the extract (data not shown). Theresult corroborates the conclusion that these steroids should betrue intermediates of the anoxic pathway.
ACKNOWLEDGMENTS
This research was funded by Chang-Gung Memorial Hospital(CMRPD180312) and the National Science Council (NSC 98-2312-B-182-003-MY3) of Taiwan.
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