Anaerobic and aerobic cleavage of the steroid core ring structure by Steroidobacter denitrificans

The aerobic degradation of steroids by bacteria has been studied in some detail. In contrast, only little is known about the anaerobic steroid catabolism. Steroidobacter denitrificans can utilize testosterone under both oxic and anoxic conditions. By conducting metabolomic investigations, we demonstrated that S. denitrificans adopts the 9,10-seco-pathway to degrade testosterone under oxic conditions. This pathway depends on the use of oxygenases for oxygenolytic ring fission. Conversely, the detected degradation intermediates under anoxic conditions suggest a novel, oxygenase-independent testosterone catabolic pathway, the 2,3-seco-pathway, which differs significantly from the aerobic route. In this anaerobic pathway, testosterone is first transformed to 1-dehydrotestosterone, which is then reduced to produce 1-testosterone followed by water addition to the C-1/C-2 double bond of 1-testosterone. Subsequently, the C-1 hydroxyl group is oxidized to produce 17-hydroxy-androstan-1,3-dione. The A-ring of this compound is cleaved by hydrolysis as evidenced by H218O-incorporation experiments. Regardless of the growth conditions, testosterone is initially transformed to 1-dehydrotestosterone. This intermediate is a divergence point at which the downstream degradation pathway is governed by oxygen availability. Our results shed light into the previously unknown cleavage of the sterane ring structure without oxygen. We show that, under anoxic conditions, the microbial cleavage of steroidal core ring system begins at the A-ring. The aerobic degradation of steroids by bacteria has been studied in some detail. In contrast, only little is known about the anaerobic steroid catabolism. Steroidobacter denitrificans can utilize testosterone under both oxic and anoxic conditions. By conducting metabolomic investigations, we demonstrated that S. denitrificans adopts the 9,10-seco-pathway to degrade testosterone under oxic conditions. This pathway depends on the use of oxygenases for oxygenolytic ring fission. Conversely, the detected degradation intermediates under anoxic conditions suggest a novel, oxygenase-independent testosterone catabolic pathway, the 2,3-seco-pathway, which differs significantly from the aerobic route. In this anaerobic pathway, testosterone is first transformed to 1-dehydrotestosterone, which is then reduced to produce 1-testosterone followed by water addition to the C-1/C-2 double bond of 1-testosterone. Subsequently, the C-1 hydroxyl group is oxidized to produce 17-hydroxy-androstan-1,3-dione. The A-ring of this compound is cleaved by hydrolysis as evidenced by H218O-incorporation experiments. Regardless of the growth conditions, testosterone is initially transformed to 1-dehydrotestosterone. This intermediate is a divergence point at which the downstream degradation pathway is governed by oxygen availability. Our results shed light into the previously unknown cleavage of the sterane ring structure without oxygen. We show that, under anoxic conditions, the microbial cleavage of steroidal core ring system begins at the A-ring. Steroids are ubiquitous and abundant in nature. Considering their divergent functions, the structural similarity of steroids is remarkable. Mammals are unable to degrade steroids. After a modification (e.g., glucuronide and sulfate conjugations) to enhance the solubility, steroid hormones are excreted into the environment through the urinary tract of mammals (1Shore L.S. Shemesh M. Naturally produced steroid hormones and their release into the environment.Pure Appl. Chem. 2003; 75: 1859-1871Crossref Scopus (193) Google Scholar). In addition, large amounts of steroid drugs are released from the pharmaceutical industry as environmental pollutants (2Daughton C.G. Heggenhougen K. Quah S. Pharmaceuticals as environmental pollutants: the ramifications for human exposure. In International Encyclopedia of Public Health. Vol. 5. Academic Press, Oxford2008: 66-102Google Scholar). Members of androgens and estrogens have been detected in a number of effluents of wastewater treatment plants and rivers at concentrations in the ng l−1 range (3Belfroid A.C. Van der Horst A. Vethaak A.D. Schäfer A.J. Rijs G.B.J. Wegener J. Cofino W.P. Analysis and occurrence of estrogenic hormones and their glucuronides in surface water and waste water in the Netherlands.Sci. Total Environ. 1999; 225: 101-108Crossref PubMed Scopus (614) Google Scholar, 4Huang C.H. Sedlak D.L. Analysis of estrogenic hormones in municipal wastewater effluent and surface water using enzyme-linked immunosorbent assay and gas chromatography/tandem mass spectrometry.Environ. Toxicol. Chem. 2001; 20: 133-139Crossref PubMed Scopus (329) Google Scholar, 5Kolodziej E.P. Gray J.L. Sedlak D.L. Quantification of steroid hormones with pheromonal properties in municipal wastewater effluent.Environ. Toxicol. Chem. 2003; 22: 2622-2629Crossref PubMed Scopus (146) Google Scholar, 6Ternes T.A. Stumpf M. Mueller J. Haberer K. Wilken R.D. Servos M. Behavior and occurrence of estrogens in municipal sewage treatment plants–I. Investigations in Germany, Canada, and Brazil.Sci. Total Environ. 1999; 225: 81-90Crossref PubMed Scopus (1227) Google Scholar). Because of the negative environmental effects of steroid hormones, the removal of these compounds from the environment has attracted considerable interest (7Panter G.H. Thompson R.S. Sumpter J.P. Adverse reproductive effects in male fathead minnows (Pimephales promelas) exposed to environmentally relevant concentrations of the natural oestrogens, oestradiol and oestrone.Aquat. Toxicol. 1998; 42: 243-253Crossref Scopus (273) Google Scholar, 8Larsson D.G.J. Hällman H. Förlin L. More male fish embryos near a pulp mill.Environ. Toxicol. Chem. 2000; 19: 2911-2917Crossref Scopus (160) Google Scholar, 9Teles M. Gravato C. Pacheco M. Santos M.A. Juvenile sea bass biotransformation, genotoxic and endocrine responses to β-naphthoflavone, 4-nonylphenol and 17β-estradiol individual and combined exposures.Chemosphere. 2004; 57: 147-158Crossref PubMed Scopus (70) Google Scholar). The biotransformation of steroids by microorganisms is a crucial example of the successful application of microbial technology in industrial processes (10Fernandes P. Cruz A. Angelova B. Pinheiro H.M. Cabral J.M.S. Microbial conversion of steroid compounds: recent developments.Enzyme Microb. Technol. 2003; 32: 688-705Crossref Scopus (460) Google Scholar). Several species of bacteria, such as Comamonas testosteroni, can degrade testosterone under oxic conditions. In 1968, Coulter and Talalay (11Coulter A.W. Talalay P. Studies on the microbiological degradation of steroid ring A.J. Biol. Chem. 1968; 243: 3238-3247Abstract Full Text PDF PubMed Google Scholar) established the oxygenase-dependent pathway (9,10-seco-pathway; Fig. 1) for the degradation of testosterone by aerobes. The aerobic testosterone degradation by C. testosteroni starts with the oxidation of the C-17 hydroxyl group and the introduction of a double bond at C-1/C-2. Subsequently, hydroxylation at C-9 occurs. The resulting compound, 9α-hydroxy-androsta-1,4-diene-3,17-dione, is unstable and undergoes spontaneous aromatization of the A-ring and nonenzymatic cleavage of the B-ring. The aromatized A-ring is subsequently split through the meta-cleavage. Investigations of the details of the aerobic steroid catabolic pathway are in progress. Overall, during the aerobic degradation of testosterone, three reactions are catalyzed by oxygenases. In addition to testosterone, cholesterol and phytosterols are also degraded through the common 9,10-seco-pathway, with C19 androgens as the intermediates (12Kieslich K. Microbial side-chain degradation of sterols.J. Basic Microbiol. 1985; 25: 461-474Crossref PubMed Scopus (112) Google Scholar). By contrast, information on the biochemical and molecular details of anaerobic steroid degradation is very limited. Steroids, especially sterols, may remain in anoxic sediments over hundreds of millions of years (13Mackenzie A.S. Brassell S.C. Eglinton G. Maxwell J.R. Chemical fossils: the geological fate of steroids.Science. 1982; 217: 491-504Crossref PubMed Scopus (520) Google Scholar, 14Wakeham S.G. Reduction of stenols to stanols in particulate matter at oxic-anoxic boundaries in sea water.Nature. 1989; 342: 787-790Crossref Scopus (105) Google Scholar), indicating that steroids are not degraded facilely by anaerobes. Obviously, anaerobes must use a novel, oxygenase-independent catabolic strategy to degrade steroids in the absence of oxygen. Denitrifying bacteria are facultative aerobes that can use various aromatic compounds and terpenoids as sole sources of carbon and energy; thus, they play a crucial role in carbon cycling in the environment. In the last decade, a few denitrifying bacteria that can anaerobically mineralize steroids were isolated and characterized (15Harder J. Probian C. Anaerobic mineralization of cholesterol by a novel type of denitrifying bacterium.Arch. Microbiol. 1997; 167: 269-274Crossref PubMed Scopus (43) Google Scholar, 16Tarlera S. Denner E.B.M. Sterolibacterium denitrificans gen. nov., sp. nov., a novel cholesterol-oxidizing, denitrifying member of the β-Proteobacteria.Int. J. Syst. Evol. Microbiol. 2003; 53: 1085-1091Crossref PubMed Scopus (85) Google Scholar, 17Fahrbach M. Kuever J. Meinke R. Kämpfer P. Hollender J. Denitratisoma oestradiolicum gen. nov., sp. nov., a 17β-oestradiol-degrading, denitrifying betaproteobacterium.Int. J. Syst. Evol. Microbiol. 2006; 56: 1547-1552Crossref PubMed Scopus (200) Google Scholar, 18Fahrbach M. Kuever J. Remesch M. Huber B.E. Kämpfer P. Dott W. Hollender J. Steroidobacter denitrificans gen. nov., sp. nov., a steroidal hormone-degrading gammaproteobacterium.Int. J. Syst. Evol. Microbiol. 2008; 58: 2215-2223Crossref PubMed Scopus (141) Google Scholar). Among them, S. denitrificans DSMZ18526 has an unusual ability to degrade testosterone under both oxic and anoxic conditions. The Blast results showed that S. denitrificans strains are widely distributed in diverse oxic and anoxic ecosystems, e.g., agriculture soil, bioremediated soil, anoxic sediment, activated sludge, and anoxic sludge (supplementary Fig. I). Recently, the initial reactions involved in the anaerobic metabolism of cholesterol and testosterone were reported (19Chiang Y.R. Ismail W. Müller M. Fuchs G. Study of anoxic and oxic cholesterol metabolism by Sterolibacterium denitrificans..J. Biol. Chem. 2007; 282: 13240-13249Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 20Chiang Y.R. Fang J.Y. Ismail W. Wang P.H. Initial steps in anoxic testosterone degradation by Steroidobacter denitrificans.Microbiology. 2010; 156: 2253-2259Crossref PubMed Scopus (22) Google Scholar, 21Leu Y.L. Wang P.H. Shiao M.S. Ismail W. Chiang Y.R. A novel testosterone catabolic pathway in bacteria.J. Bacteriol. 2011; 193: 4447-4455Crossref PubMed Scopus (23) Google Scholar), albeit the ring cleavage details of the anaerobic pathways are yet to be unraveled. In this study, we adopted a 13C metabolomic approach to investigate the anaerobic degradation of testosterone using S. denitrificans as a model organism. The aerobic testosterone degradation by the same model organism was also studied for comparison. The results obtained shed light into the previously unknown cleavage of the sterane ring structure without oxygen. To our knowledge, this is the first study showing that under anoxic conditions, the microbial cleavage of steroidal core ring system begins at the A-ring. The [2,3,4C-13C]testosterone was purchased from Isosciences. The chemicals were analytical grade and were purchased from Mallinckrodt Baker, Merck, or Sigma-Aldrich. Steroidobacter denitrificans DSMZ18526 was obtained from the Deutsche Sammlung für Mikroorganismen und Zellkulturen (Braunschweig, Germany). S. denitrificans was grown with 2 mM of unlabeled testosterone in a 250 ml glass bottle. After the unlabeled testosterone was completely consumed, 10 ml of the anoxic culture was transferred into a 12 ml glass bottle sealed with a rubber stopper. The S. denitrificans cells were subsequently fed with 2 mM testosterone (unlabeled testosterone and [2,3,4C-13C]testosterone were mixed in a 1:1 molar ratio) under denitrifying conditions. The samples (1 ml) were withdrawn after 10 and 12 of at After the mM of of H. a of oxidation in Biol. Chem. 1985; Full Text PDF PubMed Google was to the anoxic The culture samples were three with the same of to The were the was and the was in of The intermediates were using The S. denitrificans was grown with mM of unlabeled testosterone at in anoxic to Y.R. Fang J.Y. Ismail W. Wang P.H. Initial steps in anoxic testosterone degradation by Steroidobacter denitrificans.Microbiology. 2010; 156: 2253-2259Crossref PubMed Scopus (22) Google Scholar). The amounts of testosterone in the anoxic culture were using After the of 2 mM testosterone, mM was to the and was for an 12 The were subsequently three with the same of to testosterone and from the of was using and The of intermediates were using The S. denitrificans was grown in ml in 2 mM The were at in an After the of 2 mM testosterone, mM of of from by Environ. Microbiol. PubMed Google was to the and for an 12 The were using and intermediates in the were using of was using and A S. denitrificans culture ml) was first anaerobically grown on 2 mM After testosterone and were completely consumed, ml of the was mixed with ml of mM testosterone, 10 mM and 10 mM The resulting ml) were transferred to glass sealed with rubber and were under various concentrations of oxygen and The culture oxygen in was in The were in an anaerobic and gas was into the after through a The were at with ml) were to the growth of cells as the of and testosterone, and the of ring cleavage intermediates and was to 10 mM the initially was After the of mM testosterone, mM and mM were to the and was for an 12 The in the culture samples and in was using a assay to with as the was by using the as Y.L. Wang P.H. Shiao M.S. Ismail W. Chiang Y.R. A novel testosterone catabolic pathway in bacteria.J. Bacteriol. 2011; 193: 4447-4455Crossref PubMed Scopus (23) Google Scholar). A ml was with 2 of The in ml was to the and with the same system at a of 2 ml The was in ml and a ml was withdrawn from The was and the was in 10 of The samples after were using The that the same compounds were and to and of was to the of intermediates was using The steroid and were on The system was The steroid compounds were under light at or by the with A system was for the The was on an analytical (2Daughton C.G. Heggenhougen K. Quah S. Pharmaceuticals as environmental pollutants: the ramifications for human exposure. In International Encyclopedia of Public Health. Vol. 5. Academic Press, Oxford2008: 66-102Google Scholar), with a of ml The was at with as an The steroid were detected in the range of using a In addition, was for the of steroids in the S. denitrificans The of steroids and was from their using a of individual The for the were are of three The denitrifying growth of S. denitrificans with testosterone and the of were as previously Y.L. Wang P.H. Shiao M.S. Ismail W. Chiang Y.R. A novel testosterone catabolic pathway in bacteria.J. Bacteriol. 2011; 193: 4447-4455Crossref PubMed Scopus (23) Google Scholar). 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Chem. Full Text PDF PubMed Google Scholar, R. 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PubMed Scopus Google Scholar). The microbial oxygenolytic cleavage of and aromatic under oxic conditions is widely distributed in for aerobic of and by bacteria The metabolism of by J. PubMed Scopus Google Scholar). The of hydroxyl into the by oxygenases is a common catabolic strategy that the microbial cells to the of these compounds The Microb. PubMed Scopus Google Scholar, The pathway and the of Microbiol. PubMed Scopus Google Scholar). The metabolomic in this study show that anaerobic degradation of testosterone by S. denitrificans through a catabolic that differs from the aerobic degradation The crucial in the adopted for the cleavage of the core ring structure of testosterone In the aerobic pathway, the cleavage starts with the in the anaerobic pathway, the A-ring is the of oxygen on the ring cleavage In to the oxygenolytic ring under oxic conditions, the of the A-ring under anoxic conditions through the The the ring cleavage is established that the microbial cleavage of the of and aromatic compounds under anoxic conditions through W. A. Fuchs G. Anaerobic metabolism of by denitrifying Microbiol. PubMed Scopus Google Scholar, W. A. Fuchs G. reactions involved in anaerobic metabolism by a denitrifying Microbiol. 1989; PubMed Scopus Google Scholar, M. Fuchs G. J. Anaerobic oxidation of aromatic compounds and Chem. Biol. PubMed Scopus Google Scholar, G. Anaerobic metabolism of aromatic 2008; PubMed Scopus Google Scholar). W. A. Fuchs G. Anaerobic metabolism of by denitrifying Microbiol. PubMed Scopus Google Scholar, W. A. Fuchs G. reactions involved in anaerobic metabolism by a denitrifying Microbiol. 1989; PubMed Scopus Google Scholar) showed that the ring cleavage in anaerobic degradation is in the anaerobic testosterone the ring cleavage has a structure in A-ring Recently, the addition of water to the bond of compounds was and characterized from the denitrificans J. and of a with and Microbiol. 2011; PubMed Scopus Google Scholar). This and and to the Our showed that the of that this may be catalyzed by a the ring cleavage involved in anaerobic was only as a for this W. A. Fuchs G. reactions involved in anaerobic metabolism by a denitrifying Microbiol. 1989; PubMed Scopus Google Scholar). the addition of to to the in or in not the A-ring cleavage of steroid not A crucial of our is that testosterone is transformed to of the growth conditions. Subsequently, the through divergent pathways on the of oxygen can be a common intermediate or a divergence point for testosterone to our S. denitrificans adopts only catabolic pathway the or to degrade testosterone, on oxygen This of have an is to the degradation of a through some common with the same of the conditions. Subsequently, the last common intermediate can be into the relevant This denitrifying bacteria to their catabolic the oxic and anoxic and their This is by the of of S. denitrificans strains in oxic and anoxic not as were reported in the for a number of facultative such as K. J. M. M. H. Fuchs G. A from an in anaerobic and aerobic Bacteriol. 2003; PubMed Scopus Google Scholar) and J. A. M. H. Fuchs G. for a pathway of aerobic metabolism in Bacteriol. PubMed Scopus Google Scholar). bacteria can use and under oxic and anoxic conditions G. Anaerobic metabolism of aromatic 2008; PubMed Scopus Google Scholar, K. J. M. M. H. Fuchs G. A from an in anaerobic and aerobic Bacteriol. 2003; PubMed Scopus Google Scholar, J. A. M. H. Fuchs G. for a pathway of aerobic metabolism in Bacteriol. PubMed Scopus Google Scholar, G. M. J. Microbial degradation of aromatic compounds from strategy to Microbiol. 2011; PubMed Scopus Google Scholar). In both the is initially transformed to a common intermediate or by the same or on the of the common is into the relevant oxic conditions, the or pathway is in the absence of the anaerobic is The the core by the of and Microbial for with bond mass

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