Anoxic Androgen Degradation by the Denitrifying Bacterium Sterolibacterium denitrificans via the 2,3-seco Pathway

The biodegradation of steroids is a crucial biochemical process mediated exclusively by bacteria. So far, information concerning the anoxic catabolic pathways of androgens is largely unknown, which has prevented many environmental investigations. In this work, we show that Sterolibacterium denitrificans DSMZ 13999 can anaerobically mineralize testosterone and some C₁₉ androgens. By using a ¹³C-metabolomics approach and monitoring the sequential appearance of the intermediates, we demonstrated that S. denitrificans uses the 2,3-seco pathway to degrade testosterone under anoxic conditions. Furthermore, based on the identification of a C₁₇ intermediate, we propose that the A-ring cleavage may be followed by the removal of a C₂ side chain at C-5 of 17-hydroxy-1-oxo-2,3-seco-androstan-3-oic acid (the A-ring cleavage product) via retro-aldol reaction. The androgenic activities of the bacterial culture and the identified intermediates were assessed using the lacZ-based yeast androgen assay. The androgenic activity in the testosterone-grown S. denitrificans culture decreased significantly over time, indicating its ability to eliminate androgens. The A-ring cleavage intermediate (≤500 μM) did not exhibit androgenic activity, whereas the sterane-containing intermediates did. So far, only two androgen-degrading anaerobes (Sterolibacterium denitrificans DSMZ 13999 [a betaproteobacterium] and Steroidobacter denitrificans DSMZ 18526 [a gammaproteobacterium]) have been isolated and characterized, and both of them use the 2,3-seco pathway to anaerobically degrade androgens. The key intermediate 2,3-seco-androstan-3-oic acid can be used as a signature intermediate for culture-independent environmental investigations of anaerobic degradation of C₁₉ androgens.     

Omega oxidation of fatty acids and the pathway of 3-hydroxybutyric acid formation

Pathways followed by the carbons of long chain fatty acids in their conversion to 3-hydroxybutyric acid were traced and the contribution of ω-oxidation to fatty acid oxidation was determined in the cellular environment where ketone body formation occurs. 1-¹⁴C-, 2-¹⁴C-, and ω-¹⁴C-labeled fatty acids were injected into alloxan-induced diabetic rats in ketosis. 3-Hydroxybutyric acid was isolated from their urines and degraded. About 1.2 to 1.4 times as much ¹⁴C was found in carbon 1 as carbon 3 of 3-hydroxybutyric acid when the 1-¹⁴C-labeled fatty acids were injected and in carbon 2 as carbon 4 when the 2-¹⁴C-labeled fatty acids were injected. There was about 4 times as much incorporation into carbon 4 as carbon 2 of 3-hydroxybutyric acid formed from the ω-¹⁴C-labeled fatty acids. This means that 50% or more of the fatty acids were oxidized, so that the terminal two carbons of the fatty acids were converted to acetoacetyl-CoA without acetyl-CoA as an intermediate. Incorporation of ¹⁴C into carbons 1 and 2 of the hydroxybutyric acid reflects the distribution of ¹⁴C in acetyl-CoA. Incorporation into carbon 1 was very small when the ω-¹⁴C-labeled fatty acids were substrate. This means that ω-oxidation of fatty acids makes, at most, a small contribution to the formation of the acetyl-CoA pool from which acetoacetate is derived.     

Microbial Hydroxylation of (−)-α-Santonin by Aspergillus niger

The microbiological transformation of a sesquiterpene lactone, (−)-α-santonin was carried out by using Aspergillus niger MIL 5024 and MIL 5025. Strain MIL 5024 brings about the transformation of (−)-α-santonin (400 mg) to 11-hydroxy-(−)-α-santonin (P1) (50.4 mg), 3,6,9-trihydroxy-9,10-seco-selina-1,3,5(10)-trien-12-oic acid-12,6-lactone (P2) (22.4 mg), and 3,6-dihydroxy-9,10-seco-selina-1,3,5(10)-trien-9,12-dioic acid-12,6-lactone (P3) (27.1 mg), which were isolated and characterized by UV, IR, mass and ¹H-NMR spectroanalyses. All of these products are described for the first time. Results similar to those with MIL 5024 were also obtained from the transformation of (−)-α-santonin by the other isolate, strain MIL 5025.     

Biosynthesis of ecdysterone from cholesterol in Taxus baccata

Biosynthesis of ecdysterone from [4-¹⁴C, 3-³H]cholesterol in Taxus baccata does not involve obligatory oxidation at C-3 during the formation of the A/B-cis ring junction.     

Stable Isotopic Studies of n-Alkane Metabolism by a Sulfate-Reducing Bacterial Enrichment Culture

Gas chromatography-mass spectrometry and nuclear magnetic resonance spectroscopy were used to study the metabolism of deuterated n-alkanes (C₆ to C₁₂) and 1-¹³C-labeled n-hexane by a highly enriched sulfate-reducing bacterial culture. All substrates were activated via fumarate addition to form the corresponding alkylsuccinic acid derivatives as transient metabolites. Formation of d₁₄-hexylsuccinic acid in cell extracts from exogenously added, fully deuterated n-hexane confirmed that this reaction was the initial step in anaerobic alkane metabolism. Analysis of resting cell suspensions amended with 1-¹³C-labeled n-hexane confirmed that addition of the fumarate occurred at the C-2 carbon of the parent substrate. Subsequent metabolism of hexylsuccinic acid resulted in the formation of 4-methyloctanoic acid, and 3-hydroxy-4-methyloctanoic acid was tentatively identified. We also found that ¹³C nuclei from 1-¹³C-labeled n-hexane became incorporated into the succinyl portion of the initial metabolite in a manner that indicated that ¹³C-labeled fumarate was formed and recycled during alkane metabolism. Collectively, the findings obtained with a sulfate-reducing culture using isotopically labeled alkanes augment and support the previously proposed pathway (H. Wilkes, R. Rabus, T. Fischer, A. Armstroff, A. Behrends, and F. Widdel, Arch. Microbiol. 177:235-243, 2002) for metabolism of deuterated n-hexane by a denitrifying bacterium.     

Biosynthesis of Bisorbicillinoid in Trichoderma sp. USF-2690; Evidence for the Biosynthetic Pathway,via Sorbicillinol,of Sorbicillin,Bisorbicillinol, Bisorbibutenolide,and…

An incorporation study of [1-¹³C] and [1,2-¹³C₂] labeled sodium acetates into sorbicillinol 1 established a ring closure system between C-1 and C-6 and the positions that were oxidized and/or methylated on a hexaketide chain. Subsequent investigations, using ¹³C-labeled 1 prepared from [1-¹³C] labeled sodium acetate, clearly demonstrated that both bisorbicillinol 2 and sorbicillin 6 incorporated ¹³C-labeled 1 into their carbon skeletons. ¹³C-labeled bisorbicillinols 2 derived from [1-¹³C]- and [2-¹³C]-labeled sodium acetates clearly indicate that these were on the biosynthetic route from 1 to bisorbibutenolide (bislongiquinolide) 3 and bisorbicillinolide 4 via 2 as a branching point in the fungus.     

C-nuclear magnetic resonance studies of the biosynthesis of 5-aminolevulinic acid destined for chlorophyll formation in dark-grown Scenedesmus obliquus

The ¹³C-nuclear magnetic resonance (NMR) spectra of chlorophyll a formed in dark-grown Scenedesmus obliquus (Turp.) Kützing in the presence of [1-¹³C]glutamate, [2-¹³C]- and [1-¹³C]glycineshowed that the ¹³C of glutamate was specifically incorporated into the eight-carbon atoms in the tetrapyrrole macrocycles derived from C-5 of 5-aminolevulinic acid (ALA), while the C-2 of glycine was only incorporated into the methyl carbon of the methoxycarbonyl group attached to the isocyclic ring of chlorophyll a. No specific enrichment of these nine carbon atoms was observed in the spectrum of chlorophyll a formed in the presence of [1-¹³C]-glycine. These labeling patterns provide evidence for the operation of the C₅-pathway and against the operation of the ALA synthase pathway for chlorophyll formation in darkness.     

C nuclear magnetic resonance study of acetate incorporation into malate during ca-uptake by isolated leaf tissues

¹³C Nuclear magnetic resonance spectroscopy of leaflets of Gleditsia triacanthos and Albizia julibrisin was used to determine the fate of acetate taken up during the absorption of calcium from ¹³C-labeled Ca-acetate solution. Small amounts of acetate accumulated temporarily in the leaf tissues, but the bulk of acetate was incorporated into malate. The initial rate of malate synthesis was very low, but increased rapidly during acetate treatment and reached its maximum after 8 hours; the enzymes involved in malate synthesis thus appear to be substrate induced. Use of acetate-2-¹³C yielded malate labeled in C-3, indicating that vacuolar malate accumulating during Ca-uptake might be synthesized via malate synthase from acetate and glyoxalate. However, a source of glyoxalate condensing with acetate during malate synthesis could not be identified. Glycolate produced in photorespiration is an unlikely source, because glycolate-2-¹³C was absorbed and metabolized by the leaf tissues into products of the glycolate pathway, but was not a major precursor in malate synthesis. Malate synthesis via the glyoxalate cycle is also unlikely, because no evidence for the recycling of a ¹³C-labeled 4-carbon organic acid was found. Malate synthesis in the leaflets of Gleditsia and Albizia thus appears to involve the inducible condensation of acetate with a 2-carbon compound of unidentified nature and origin.     

Mode of incorporation of precursors into alizarin (1,2-dihydroxy-9,10-anthraquinone)

The biosynthesis of alizarin (1,2-dihydroxy-9,10-anthraquinone) in Rubia tinctorum L. has been studied by tracer techniques. Specific incorporation of label from carboxyl-¹⁴C-d-shikimic acid, 2-¹⁴C-dl-glutamic acid and 5-¹⁴C-dl-Mevalonic acid suggests that these compounds provide the carbon skeleton of alizarin. Nonsymmetrical incorporation of label from carboxyl-¹⁴C-d-shikimic acid and 2-¹⁴C-dl-glutamic acid into alizarin indicates that the symmetrical 1,4-naphthoquinone is probably not an intermediate. Activity from o-(succinyl-2,3¹⁴C)-benzoic acid was found in the substituted benzene ring of alizarin. These data indicate that α-ketoglutaric acid or a derivative thereof combines with shikimic acid, chorismic acid or phrephenic acid to give o-succinylbenzoic acid which is then transformed to a nonsymmetrical intermediate γ,γ-Dimethylallylpyrophosphate is then attached, ring closure and further modification leading to alizarin.     

A Flavin-dependent Monooxygenase from Mycobacterium tuberculosis Involved in Cholesterol Catabolism

Mycobacterium tuberculosis (Mtb) and Rhodococcus jostii RHA1 have similar cholesterol catabolic pathways. This pathway contributes to the pathogenicity of Mtb. The hsaAB cholesterol catabolic genes have been predicted to encode the oxygenase and reductase, respectively, of a flavin-dependent mono-oxygenase that hydroxylates 3-hydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione (3-HSA) to a catechol. An hsaA deletion mutant of RHA1 did not grow on cholesterol but transformed the latter to 3-HSA and related metabolites in which each of the two keto groups was reduced: 3,9-dihydroxy-9,10-seconandrost-1,3,5(10)-triene-17-one (3,9-DHSA) and 3,17-dihydroxy-9,10-seconandrost-1,3,5(10)-triene-9-one (3,17-DHSA). Purified 3-hydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione 4-hydroxylase (HsaAB) from Mtb had higher specificity for 3-HSA than for 3,17-DHSA (apparent k_cat/K_m = 1000 ± 100 m⁻¹ s⁻¹ versus 700 ± 100 m⁻¹ s⁻¹). However, 3,9-DHSA was a poorer substrate than 3-hydroxybiphenyl (apparent k_cat/K_m = 80 ± 40 m⁻¹ s⁻¹). In the presence of 3-HSA the K_mapp for O₂ was 100 ± 10 μm. The crystal structure of HsaA to 2.5-Å resolution revealed that the enzyme has the same fold, flavin-binding site, and catalytic residues as p-hydroxyphenyl acetate hydroxylase. However, HsaA has a much larger phenol-binding site, consistent with the enzyme''s substrate specificity. In addition, a second crystal form of HsaA revealed that a C-terminal flap (Val³⁶⁷–Val³⁹⁴) could adopt two conformations differing by a rigid body rotation of 25° around Arg³⁶⁶. This rotation appears to gate the likely flavin entrance to the active site. In docking studies with 3-HSA and flavin, the closed conformation provided a rationale for the enzyme''s substrate specificity. Overall, the structural and functional data establish the physiological role of HsaAB and provide a basis to further investigate an important class of monooxygenases as well as the bacterial catabolism of steroids.     

Biosynthesis of a heterocycle formed by iron-deficient Azotobacter vinelandii Strain 0

Biosynthesis of the heterocycle, 1H-2,3,5,6-tetrahydro-5-oxo-9,10-dihydroxyimidazo[3,4,5-de]pyrimido[1,2-a]quinolinium-1- carboxylate, produced by iron-deficient Azotobacter vinelandii, Strain 0 was investigated using ¹⁴C-labeled aminto acids and formate. The incorporation of ¹⁴C is consistent with this unusual heterocycle beinig derived from l-3(3,4-dihydroxyphenyl)alanine, l-asparagine, formate and glycine.     

Biosynthesis of hygrine from [5-14c]ornithine via a symmetrical intermediate in Nicandra physaloides1

Radioactive hygrine (2.2% incorporation) was isolated from Nicandra physaloides plants which had been fed Dl-[5-¹⁴C]ornithine. A systematic degradation of the hygrine yielded products whose activity was consistent with the pyrrolidine ring of this alkaloid being labeled equally at the C-2 and C-5 positions. The result does not agree with the previous work of O′Donovan and Keogh, whose publication is critically examined.     

The mechanism of the non-oxidative segment of the pentose cycle in the liver

[1-¹⁴C] Xylitol was used as substrate for isolated liver parenchymal cells from fasted rats or hamsters, and the glucose formed was isolated and degraded. Over 90% of the total radioactivity was in carbons 1 and 3, and the ratio of specific activity of C-1 to that of C-3 was only slightly under two, the expected ratio for the classical mechanism of the non-oxidative pentose phosphate pathway.     

Pathway of Propionate Oxidation by a Syntrophic Culture of Smithella propionica and Methanospirillum hungatei

The pathway of propionate conversion in a syntrophic coculture of Smithella propionica and Methanospirillum hungatei JF1 was investigated by ¹³C-NMR spectroscopy. Cocultures produced acetate and butyrate from propionate. [3-¹³C]propionate was converted to [2-¹³C]acetate, with no [1-¹³C]acetate formed. Butyrate from [3-¹³C]propionate was labeled at the C2 and C4 positions in a ratio of about 1:1.5. Double-labeled propionate (2,3-¹³C) yielded not only double-labeled acetate but also single-labeled acetate at the C1 or C2 position. Most butyrate formed from [2,3-¹³C]propionate was also double labeled in either the C1 and C2 atoms or the C3 and C4 atoms in a ratio of about 1:1.5. Smaller amounts of single-labeled butyrate and other combinations were also produced. 1-¹³C-labeled propionate yielded both [1-¹³C]acetate and [2-¹³C]acetate. When ¹³C-labeled bicarbonate was present, label was not incorporated into acetate, propionate, or butyrate. In each of the incubations described above, ¹³C was never recovered in bicarbonate or methane. These results indicate that S. propionica does not degrade propionate via the methyl-malonyl-coenzyme A (CoA) pathway or any other of the known pathways, such as the acryloyl-CoA pathway or the reductive carboxylation pathway. Our results strongly suggest that propionate is dismutated to acetate and butyrate via a six-carbon intermediate.     

Kreb's TCA cycle in Halobacterium salinarum investigated by 13C nuclear magnetic resonance spectroscopy

Kreb's tricarboxylic (TCA) cycle was studied in Halobacterium salinarum cells grown in the presence of glucose or alanine. The cells were incubated with ¹³C-labeled substrate and the labeling pattern of various carbon positions in glutamate was monitored by ¹³C-NMR spectroscopy. [2-¹³C]pyruvate, when used as a substrate, led mainly to signals for C-1 and C-5 glutamate, with some C-3 glutamate. [3-¹³C]pyruvate as a substrate produced signals, mainly C-2, C-3, and C-4 glutamate, with some C-1 and C-5 glutamate. The multiplicity of the signals and observation of a C-1 signal in this case indicates extensive cycling of the label in the TCA cycle. Isotopomer analysis of glutamate labeling suggested that of the total pyruvate entering the TCA cycle, the flux through pyruvate:ferredoxin oxidoreductase was 90% while that through pyruvate caboxylase was 10%. Only 53% of the total acetyl-CoA was produced from the added labeled pyruvate, the rest being generated endogenously. In the presence of nitrogen, mainly transamination reaction products were formed in the case of both these substrates. Received: November 26, 1997 / Accepted: May 11, 1998     

Fungal hydroxylation of (−)-santonin and its analogues

Santonin (1) was incubated with separate growing cultures of Aspergillus niger ATCC 9142, Mucor plumbeus ATCC 4740, Whetzelinia sclerotiorum ATCC 18687, Cunninghamella echinulata var. elegans ATCC 8688a and Phanerochaete chrysosporium ATCC 24725. Three novel metabolites were isolated: 11β,13-dihydroxysantonin (3), 6,7-dehydosantonin (5) and 3,6-dihydroxy-9-keto-9,10-seco-selina-1,3,5(10)-trien-12-oic acid-12,6-lactone (7). 11β-Hydroxysantonin (2), 14-hydroxysantonin (4) and 3,6,9-trihydroxy-9,10-seco-selina-1,3,5(10)-trien-12-oic acid-12,6-lactone (6) were also isolated. Hydroxylation at C-9 followed by a retro-aldol reaction was postulated to have produced 6 and 7. Through the synthesis and fermentation of the santonin analogues: tetrahydrosantonin (8) and α-desmotroposantonin (12), several new compounds were obtained; the most significant being 9-keto-desmotroposantonin (14), which was indicative of C-9 monohydroxylation.     

A novel testosterone catabolic pathway in bacteria

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 testosterone catabolic pathway involves several oxygen-dependent reactions and is not available for anaerobes. Since then, a variety of anaerobic bacteria have been described for the ability to degrade testosterone in the absence of 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 of oxygen and was selected as the model organism in this study. In a previous investigation, we identified the initial intermediates involved in an anoxic testosterone catabolic pathway, most of which are identical to those of the oxic pathway demonstrated in Comamonas testosteroni. In this study, five additional intermediates of the anoxic pathway were identified. We demonstrated that subsequent steps of the anoxic pathway greatly differ from those of the established oxic pathway, which suggests that a novel pathway for testosterone catabolism is present. 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 hydration reaction occurs and a hydroxyl group is thus introduced to the C-1α position of C(19)steroid substrates. To our knowledge, an enzymatic hydration reaction occurring at the A ring of steroid compounds has not been reported before.     

Biosynthesis of the 2-arylbenzofuran phytoalexin vignafuran in Vigna unguiculata

Feeding experiments using l-phenylalanine-[U-¹⁴C], dl-phenylalanine-[1-¹⁴C] and -[2-¹⁴C] together with degradative studies have been used to investigate the biosynthesis of the 2-arylbenzofuran phytoalexin vignafuran in UV-treated seedlings of cowpea (Vigna unguiculata). During the biosynthetic process, C-3 of phenylalanine appears to be lost, and the resulting labelling pattern is consistent with vignafuran being derived from an isoflavonoid precursor, but the phenylalanine-derived aromatic ring becomes the 2-aryl substituent and not part of the benzofuran system. A previously proposed pathway to 2-arylbenzofurans by loss of C-6 from a coumestan is thus excluded. Alternative routes are suggested.     

Incorporation of shikimate and 4-(2′-carboxyphenyl)-4-oxobutyrate into phylloquinone

The patterns of incorporation of d-[G-¹⁴C]shikimate and variously labelled ¹⁴C-4-(2′-carboxy-phenyl)-4-oxobutyrate into the naphthoquinone nucleus of phylloquinone by maize shoots have been investigated. The results show that (a) the alicyclic ring and C-7 of shikimate give rise to Ring A and either C-1 or C-4, and (b) the phenyl ring, 2′-carboxy and C-4, and C-2 and -3 of 4-(2′-carboxyphenyl)-4-oxobutyrate give rise to Ring A, C-1 and -4 and C-2 and -3. Radioactivity from α-[1-¹⁴C]naphthol, 1,4-[1,4-¹⁴C]naphthoquinone and [Me-¹⁴C]menadione is not incorporated into phylloquinone to any significant extent.     

Studies on l-ascorbic acid biosynthesis and metabolism in Parthenocissus quinquefolia L. (vitaceae)

Parthenocissus quinquefolia (L.) Planch., commonly known as Virginia Creeper, is a vitaceous tartrate-accumulating vine that exhibits C-4/C-5 cleavage of l-ascorbic acid (AA) to produce l-tartaric acid (TA) from the C₄ fragment and carbohydrate pool material from the C₂ fragment. Experiments in which detached leaves were supplied d-[5-³H,1-¹⁴C]glucose or d-[5-³H,6-¹⁴C]glucose yielded AA devoid of ³H whereas the l-threonic acid (ThA) and TA recovered from the same tissues still retained some ³H. These comparative experiments also indicated that the ThA was derived from carbons 3 through 6 of d-glucose. ThA was shown to be a natural constituent of P. quinquefolia but apparently not an intermediate between AA and TA. Results are consistent with a biosynthetic pathway from d-glucose to AA that involves a hydrogen-exchanging epimerization at C-5 as reported earlier for the geraniaceous plant Pelargonium crispum, but differing from P.crispum in biosynthesis and metabolism of ThA.When l-[6-¹⁴C]idonate or its lactone was supplied to P. quinquefolia leaves, about 80% of the ¹⁴C appeared in the carbohydrates, an observation remarkably similar to previous observations with [6-¹⁴C]AA-labeled leaves. l-Idonate and its lactone appear to have an intermediate role in AA metabolism in vitaceous plants.