Undescribed Metabolites Research Articles

Catabolism of the Last Two Steroid Rings in Mycobacterium tuberculosis and Other Bacteria.

ABSTRACTMost mycolic acid-containing actinobacteria and some proteobacteria use steroids as growth substrates, but the catabolism of the last two steroid rings has yet to be elucidated. In Mycobacterium tuberculosis, this pathway includes virulence determinants and has been proposed to be encoded by the KstR2-regulated genes, which include a predicted coenzyme A (CoA) transferase gene (ipdAB) and an acyl-CoA reductase gene (ipdC). In the presence of cholesterol, ΔipdC and ΔipdAB mutants of either M. tuberculosis or Rhodococcus jostii strain RHA1 accumulated previously undescribed metabolites: 3aα-H-4α(carboxyl-CoA)-5-hydroxy-7aβ-methylhexahydro-1-indanone (5-OH HIC-CoA) and (R)-2-(2-carboxyethyl)-3-methyl-6-oxocyclohex-1-ene-1-carboxyl-CoA (COCHEA-CoA), respectively. A ΔfadE32 mutant of Mycobacterium smegmatis accumulated 4-methyl-5-oxo-octanedioic acid (MOODA). Incubation of synthetic 5-OH HIC-CoA with purified IpdF, IpdC, and enoyl-CoA hydratase 20 (EchA20), a crotonase superfamily member, yielded COCHEA-CoA and, upon further incubation with IpdAB and a CoA thiolase, yielded MOODA-CoA. Based on these studies, we propose a pathway for the final steps of steroid catabolism in which the 5-member ring is hydrolyzed by EchA20, followed by hydrolysis of the 6-member ring by IpdAB. Metabolites accumulated by ΔipdF and ΔechA20 mutants support the model. The conservation of these genes in known steroid-degrading bacteria suggests that the pathway is shared. This pathway further predicts that cholesterol catabolism yields four propionyl-CoAs, four acetyl-CoAs, one pyruvate, and one succinyl-CoA. Finally, a ΔipdAB M. tuberculosis mutant did not survive in macrophages and displayed severely depleted CoASH levels that correlated with a cholesterol-dependent toxicity. Our results together with the developed tools provide a basis for further elucidating bacterial steroid catabolism and virulence determinants in M. tuberculosis.

Regio- and stereo-selective hydroxylations of ingenane diterpenoids by Mortierella ramanniana and Gibberella fujikuroi

The regio- and stereo-selective hydroxylations of two ingenane diterpenoids, 20-deoxyingenol (1) and 13-oxyingenol dodecanoat (2), by the filamentous fungi Mortierella ramanniana and Gibberella fujikuroi were investigated in the present study. Four undescribed metabolites (3–6) of substrate 1 and two undescribed metabolites (7 and 8) of substrate 2 were isolated. All the metabolites were identified as hydroxylated ingenane derivatives by extensive NMR and HR-ESI-MS data analyses. All the biotransformed compounds and the substrates were evaluated for their cytotoxicities against three human cancer cell lines, including human colon cancer Caco-2, breast cancer MCF-7, and adriamycin (ADM)-resistant MCF-7/ADM cell lines. All ingenane alcohols (1, and 3–6) displayed no significant cytotoxic activities. The substrate 13-oxyingenol dodecanoat (2) showed moderate cytotoxicity with IC50 values being 35.59 ± 5.37 μmol·L−1 (Caco-2), 24.04 ± 4.70 μmol·L−1 (MCF-7), and 22.24 ± 5.19 μmol·L−1 (MCF-7/ADM). However, metabolites 7 and 8 displayed no significant cytotoxicity. These results indicated that the hydroxylation at the C-13 aliphatic acid ester of substrate 2 can significantly reduce the cytotoxic activity.

Is the lower atmosphere a readily accessible reservoir of culturable, antimicrobial compound-producing Actinomycetales?

Recent metagenomic studies have revealed that microbial diversity in the atmosphere rivals that of surface environments. This indicates that the atmosphere may be worth bioprospecting in for novel microorganisms, especially those selected for by harsh atmospheric conditions. This is interesting in light of the antibiotic resistance crisis and renewed interests in bioprospecting for members of the Actinomycetales, which harbor novel secondary metabolite-producing pathways and produce spores that make them well suited for atmospheric travel. The latter leads to the hypothesis that the atmosphere may be a promising environment in which to search for novel Actinomycetales. Although ubiquitous in soils, where bioprospecting efforts for Actinomycetales have been and are largely still focused, we present novel data indicating that culturable members of this taxonomic order are 3–5.6 times more abundant in air samples collected at 1.5, 4.5, 7.5, and 18 m above the ground, than in the underlying soil. These results support the hypothesis that mining the vast and readily accessible lower atmosphere for novel Actinomycetales in the search for undescribed secondary metabolites could prove fruitful.

Gulypyrones A and B and Phomentrioloxins B and C Produced by Diaporthe gulyae, a Potential Mycoherbicide for Saffron Thistle (Carthamus lanatus).

A virulent strain of Diaporthe gulyae, isolated from stem cankers of sunflower and known to be pathogenic to saffron thistle, has been shown to produce both known and previously undescribed metabolites when grown in either static liquid culture or a bioreactor. Together with phomentrioloxin, a phytotoxic geranylcyclohexenetriol recently isolated from a strain of Phomopsis sp., two new phytotoxic trisubstituted α-pyrones, named gulypyrones A and B (1 and 2), and two new 1,O- and 2,O-dehydro derivatives of phomentrioloxin, named phomentrioloxins B and C (3 and 4), were isolated from the liquid culture filtrates of D. gulyae. These four metabolites were characterized as 6-[(2S)2-hydroxy-1-methylpropyl]-4-methoxy-5-methylpyran-2-one (1), 6-[(1E)-3-hydroxy-1-methylpropenyl]-4-methoxy-3-methylpyran-2-one (2), 4,6-dihydroxy-5-methoxy-2-(7-methyl-3-methyleneoct-6-en-1-ynyl)cyclohex-2-enone (3), and 2,5-dihydroxy-6-methoxy-3-(7-methyl-3-methyleneoct-6-en-1-ynyl)cyclohex-3-enone (4) using spectroscopic and chemical methods. The absolute configuration of the hydroxylated secondary carbon of the 2-hydroxy-1-methylpropyl side chain at C-6 of gulypyrone A was determined as S by applying a modified Mosher's method. Other well-known metabolites were also isolated including 3-nitropropionic, succinic, and p-hydroxy- and p-methylbenzoic acids, p-hydroxybenzaldehyde, and nectriapyrone. When assayed using a 5 mM concentration on punctured leaf disks of weedy and crop plants, apart from 3-nitropropionic acid (the main metabolite responsible for the strong phytotoxicity of the culture filtrate), phomentrioloxin B caused small, but clear, necrotic spots on a number of plant species, whereas gulypyrone A caused leaf necrosis on Helianthus annuus plantlets. All other compounds were weakly active or inactive.

Production of fungal and bacterial growth modulating secondary metabolites is widespread among mycorrhiza-associated streptomycetes.

BackgroundStudies on mycorrhiza associated bacteria suggest that bacterial-fungal interactions play important roles during mycorrhiza formation and affect plant health. We surveyed Streptomyces Actinobacteria, known as antibiotic producers and antagonists of fungi, from Norway spruce mycorrhizas with predominantly Piloderma species as the fungal partner.ResultsFifteen Streptomyces isolates exhibited substantial variation in inhibition of tested mycorrhizal and plant pathogenic fungi (Amanita muscaria, Fusarium oxysporum, Hebeloma cylindrosporum, Heterobasidion abietinum, Heterobasidion annosum, Laccaria bicolor, Piloderma croceum). The growth of the mycorrhiza-forming fungus Laccaria bicolor was stimulated by some of the streptomycetes, and Piloderma croceum was only moderately affected. Bacteria responded to the streptomycetes differently than the fungi. For instance the strain Streptomyces sp. AcM11, which inhibited most tested fungi, was less inhibitory to bacteria than other tested streptomycetes. The determined patterns of Streptomyces-microbe interactions were associated with distinct patterns of secondary metabolite production. Notably, potentially novel metabolites were produced by strains that were less antagonistic to fungi. Most of the identified metabolites were antibiotics (e.g. cycloheximide, actiphenol) and siderophores (e.g. ferulic acid, desferroxiamines). Plant disease resistance was activated by a single streptomycete strain only.ConclusionsMycorrhiza associated streptomycetes appear to have an important role in inhibiting the growth of fungi and bacteria. Additionally, our study indicates that the Streptomyces strains, which are not general antagonists of fungi, may produce still un-described metabolites.

Identification of Finasteride Metabolites in Human Bile and Urine by High-Performance Liquid Chromatography/Tandem Mass Spectrometry

The objective of this study was to further investigate the metabolism of the 5alpha-reductase inhibitor, finasteride, and to identify previously unknown phase I and phase II metabolites in vitro and in vivo in human bile and urine. Healthy volunteers were given 5 mg of finasteride, directly to the intestine, and bile and urine were collected for 3 and 24 h, respectively. A single-pass perfusion technique, Loc-I-Gut, was used for drug administration and bile collection from the proximal jejunum, distal to papilla of Vater. Incubations with human liver microsomes/S9 fractions and different cofactors were performed with finasteride and the previously known metabolites, omega-hydroxy finasteride (M1) and finasteride-omega-oic acid (M3). Liquid chromatography coupled to triple quadrupole mass spectrometry (MS) with positive/negative electrospray ionization and ion trap with MS(n) measurements were used for structural investigations and identification of metabolites. Two hydroxy metabolites of finasteride, other than M1, and one intact hydroxy finasteride glucuronide were identified in vitro and in bile and urine. The glucuronide and at least one of the hydroxy metabolites were previously unidentified. M1 and M3 were glucuronidated in vitro by specific human UDP-glucuronosyltransferases, UGT1A4 and UGT1A3, respectively. M1 glucuronide was not identified in vivo, and M3 glucuronide, an acyl glucuronide, was present in low amounts in bile from a few individuals. In conclusion, previously undescribed metabolites were identified, in vitro and in human urine and bile. Bile collection using the Loc-I-Gut technique followed by sensitive mass spectrometry analysis led to the discovery of novel, both phase I and phase II, finasteride metabolites in human bile.

Activated Protein C-Resistance Induced by a Low Molecular Weight Inhibitor.

Activated protein C (APC) inactivates factor Va (fVa) following three cleavages in the heavy chain at R506, R306 and R679. Cleavage at R506 precedes cleavage at R306. Cleavage at Arg306 is strictly lipid-dependent and results in total inactivation of the factor Va molecule with dissociation of fragments from the A2 domain from the rest of the molecule. Factor VLeiden is associated with an R→Q substitution at position 506 and is present at approximately 8% of the Caucasian population. The heterozygous presentation of factor VLeiden results in delayed inactivation of factor Va and “APC-resistance” with attendant increased risk of venous thrombosis. However, not all cases of “APC-resistance” are explained by factor VLeiden. We observed “APC-resistance” in a patient displaying heterozygous factor VLeiden, Waldenstrom's macroglobulinenemia, systemic lupus erythrematosus (anticoagulant) and a history of coronary artery disease. The patient's plasma resistance to APC inactivation was not repaired by immunodepletion of his factor VLeiden and replacement by normal plasma factor V. Conversely when the patient's fVa was returned to factor V immunodepleted normal plasma it did not display APC-resistance. Cleavage of the patient's plasma fVa at R306 was not detected following prolonged incubation of his clotted plasma at 37°C even when 2 nM APC was added following clotting. These data suggested that the APC-resistance observed in the patient was not due to the presence of factor VLeiden, but due to some property which inhibited the lipid dependent cleavage at Arg306. The patient's plasma was depleted of IgG/IgM and the purified immunoglobulin fraction assessed for inhibition of APC cleavage and inactivation of fVa in a system using purified reagents. The data were compared with the inhibition of fVa inactivation by APC by an IgG/IgM fraction obtained from normal plasma under similar experimental conditions. No inhibition of APC cleavage and inactivation of fVa by the IgM/IgG fraction obtained from either plasma were observed. In addition, the fVa molecule contained in the IgM/IgG-depleted patient plasma was still resistant to cleavage and inactivation by APC. Following dialysis the patient's plasma lost its ability to inhibit fVa cleavage and inactivation by APC. Overall these studies indicate that inhibition of fVa cleavage and inactivation by APC in the patient's plasma is caused by a hitherto undescribed metabolite of low molecular weight. The mechanism of action of this metabolite is not yet known, but the evidence suggests that the metabolite interferes with the lipid-dependent cleavage and inactivation of fVa by APC at R306. These data demonstrate the existence of an as yet unknown APC inhibitor of low molecular weight in the plasma of a patient with lupus anticoagulant and severe thrombotic symptoms.

Deoxyscytalidin and Lignicol: New Metabolites from Scytalidium Species

As part of a chemosystematic study of the genus Scytalidium several strains belonging to the closely related species Scytalidium album, Scytalidium lignicola, Scytalidium aurantiacum and Scytalidium circinatum have been grown in liquid shake culture. The various strains examined did not show a consistent pattern of metabolites. Scytalidin [1], the major metabolite of Sc. album was produced by two of three tested strains but not demonstrated by any strain of Sc. lignicola, Sc. aurantiacum or Sc. circinatum. The scytalidin-producing strains also produced a closely related but undescribed metabolite, deoxyscytalidin [2]. One strain of Sc. lignicola produced a new compound which we call lignicol [3] [...]

Microsomal metabolism of cyclopenta[cd]pyrene. Characterization of new metabolites and their mechanism of formation.

Oxidation of cyclopenta[cd]pyrene (CPP) by mouse and human liver microsomes was used to produce several previously undescribed metabolites, which were separated and isolated by reversed-phase HPLC. Three of these, 3,4-dihydroCPP-c-3,4-diol, 4-hydroxy-3,4-dihydroCPP, and 4-oxo-3,4-dihydroCPP, were fully characterized by GC-MS and UV spectroscopic analysis as well as by total synthesis. Two additional pairs of metabolites were identified as isomeric tetrahydrotetrols and dihydrotriols by GC-MS analysis of their trimethylsilyl derivatives. Their UV spectra were recorded and found to agree with the structure assignments. The tetrahydrotetrols were further characterized by the fact that either 3,4- or 9,10-trans-dihydrodiol could serve as their precursor, indicating that they are the two diastomeric 3,4,9,10-tetrahydroCPP-t-3,4-t-9,10-tetrols. The dihydrotriols were shown to possess t-3,4-dihydrodiol functionality. As found previously using rat liver microsomes, the most abundant metabolite was 3,4-dihydroCPP-t-3,4-diol. It was produced with one enantiomer in severalfold excess over the other, and the major enantiomer was shown to have 3R,4R configuration by exciton chirality circular dichroism. Microsomal oxidation of [4-2H]CPP, which was synthesized for this study, was used to determine the mechanisms of formation of 4-oxo- and 4-hydroxy-3,4-dihydroCPP. The ketone was produced without detectable retention of deuterium label, eliminating the NIH shift as a possible mechanism. The alcohol was shown to arise by NADPH-dependent reduction of both the ketone and another intermediate presumed to be the 3,4-epoxide.(ABSTRACT TRUNCATED AT 250 WORDS)

Transformation of arachidonic acid in leukocytes. isolation and structural analysis of a novel dihydroxy derivative

A suspension of mixed peripheral blood leukocytes was incubated with arachidonic acid. After ether extraction and silicic acid fractionation of the products, the fraction containing the mono- and dihydroxy derivatives of arachidonic acid was further analyzed by high pressure liquid chromatography on silica gel and octadecyl silica (reversed-phase) columns. A previously undescribed metabolite was detected and isolated in pure form. The compound co-chromatographed with leukotriene B 4 on octadecyl silica but was eluted earlier than leukotriene B 4 from silica gel columns. Ultraviolet spectrophotometry, gas chromatography-mass spectrometry, ozonolysis and steric analysis indicated that the new metabolite was the 5S,12S-dihydroxy-6,8,10,14-icosatetraenoic acid. The yield of the novel dihydroxy acid was 1 to 4% of the added substrate. The new metabolite showed less than 1% of the myotropic activity of leukotriene B4 on the guinea pig lung parenchymal strip.

ChemInform Abstract: MINOR LANKAMYCIN‐RELATED ANTIBIOTICS FROM STREPTOMYCES VIOLACEONIGER

AbstractDie Strukturen der beiden Titelantibiotica (I) und (II) werden 1H‐NMR‐ und massenspektroskopisch bestimmt.

Minor lankamycin-related antibiotics from Streptomyces violaceoniger.

AbstractSeveral previously undescribed metabolites related to lankamycin have been isolated from fermentation broths of the lankamycin‐producing organism Streptomyces violaceoniger. Two of these metabolites, of weak antibacterical activity, have been characterized as 15‐O‐(α‐L‐4‐O‐acetyl‐arcanosyl)‐lankamycin and 15‐deoxy‐15‐oxo‐lankamycin. The latter antibiotic is prepared in high yield by Jones oxidation of lankamycin.

Identification of a new metabolite of l-homocitrulline

Following the intraperitoneal injection of l-homocitrulline into rats a previously undescribed metabolite of this amino acid was detected in the urine. A compound with identical properties could also be prepared with l-amino acid oxidase. Mass spectrometric studies indicated that both of these derivatives had a molecular weight of 170.0690 and an empirical formula of C 7H 10O 3N 2. Upon exposure to acidic conditions the compound was converted a second form which had a molecular weight of 152.0585 and an empirical formula of C 7H 8O 2N 2. Nuclear magnetic resonance studies confirmed the cyclic structure of the unknown.

The identification of 3,3', 5,5',-tetraiodothyroformic acid within the rat liver.

Homogenized rat livers were extracted with organic solvents and the extracts converted into more readily volatile derivatives (trimethylsilylmethyl esters and permethyl esters). Combined g.l.c.-mass-spectrometric analysis identified the presence of tetraiodothyroformate. It is postulated that mitochondrial beta-oxidation of tetraiodothyropyruvate results in the formation of this apparently undescribed metabolite.

The metabolism of 25-hydroxycholecalciferol by isolated renal tubules in vitro as studied by a new chromatographic technique

The metabolism of 25-hydroxycholecalciferol by isolated chick renal tubules has been investigated by the use of a new chromatographic method employing silica-treated paper as the supporting phase. This method has been found to be superior to Sephadex LH20 column chromatography in resolving the metabolites of 25-hydroxycholecalciferol, particularly in separating 1,25-dyhydroxycholecalciferol from other metabolites. With this method several heretofore undescribed metabolites of 25-hydroxycholecalciferol have been recognized and partially characterized.

6-Methylthioguanylic acid, a metabolite of 6-thioguanine

Human epidermoid carcinoma (H. Ep. No. 2) cells, grown in culture in the presence of 6-thioguanine- 35S or methionine[methyl- 14C] plus unlabeled 6-thioguanine, contained a previously undescribed metabolite that was characterized by Chromatographic and electrophoretic migration and by chemical and enzymatic hydrolysis as 6-methylthioguanylic acid. Two other nucleotides present in these cells had the properties expected of the di- and triphosphates of 6-methylthioguanosine. In contrast to previously reported results with 6-mercaptopurine ribonucleotide and its S-methyl derivative, 6-methylthioguanylic acid was inferior to 6-thioguanylic acid as an inhibitor of PP-ribose-P amidotransferase. Thus, the methylated derivatives of 6-thioguanylic acid probably do not contribute significantly to the marked inhibition of purine synthesis de novo produced in mammalian cells by 6-thioguanine

(-)-Sclerotiorin, A Major Metabolite of Penicillium hirayamae UDAGAWA.

From the mycelium of Penicillium hirayamae UDAGAWA two hitherto undescribed metabolites have been isolated. The major product is a levorotatory isomer of the known sclerotiorin. The minor component, rubrotiorin, C21H21∼23O4Cl, m. p. 172°, is given as red needles and appears to be a new azaphilone compound.