Degradation of substituted indoles by an indole-degrading methanogenic consortium.

Degradation of indole by an indole-degrading methanogenic consortium enriched from sewage sludge proceeded through a two-step hydroxylation pathway yielding oxindole and isatin. The ability of this consortium to hydroxylate and subsequently degrade substituted indoles was investigated. Of the substituted indoles tested, the consortium was able to transform or degrade 3-methylindole and 3-indolyl acetate. Oxindole, 3-methyloxindole, and indoxyl were identified as metabolites of indole, 3-methylindole, and 3-indolyl acetate degradation, respectively. Isatin (indole-2,3-dione) was produced as an intermediate when the consortium was amended with oxindole, providing evidence that degradation of indole proceeded through successive hydroxylation of the 2- and 3-positions prior to ring cleavage between the C-2 and C-3 atoms on the pyrrole ring of indole. The presence of a methyl group (-CH3) at either the 1- or 2-position of indole inhibited the initial hydroxylation reaction. The substituted indole, 3-methylindole, was hydroxylated in the 2-position but not in the 3-position and could not be further metabolized through the oxindole-isatin pathway. Indoxyl (indole-3-one), the deacetylated product of 3-indolyl acetate, was not hydroxylated in the 2-position and thus was not further metabolized by the consortium. When an H atom or electron-donating group (i.e., -CH3) was present at the 3-position, hydroxylation proceeded at the 2-position, but the presence of electron-withdrawing substituent groups (i.e., -OH or -COOH) at the 3-position inhibited hydroxylation.     

Metabolism of 3-methylindole by a methanogenic consortium.

A methanogenic 3-methylindole (3-MI)-degrading consortium, enriched from wetland soil, completely mineralized 3-MI. Degradation proceeded through an initial hydroxylation reaction forming 3-methyloxindole. The consortium was unable to degrade oxindole or isatin, suggesting a new pathway for 3-MI fermentation.     

Metabolism of 3-methylindole by a methanogenic consortium.

A methanogenic 3-methylindole (3-MI)-degrading consortium, enriched from wetland soil, completely mineralized 3-MI. Degradation proceeded through an initial hydroxylation reaction forming 3-methyloxindole. The consortium was unable to degrade oxindole or isatin, suggesting a new pathway for 3-MI fermentation.     

Isolation and characterization of anaerobic indole- and skatole-degrading bacteria from composting animal wastes

Four species of indole-degrading Clostridium and 3 species of skatole-degrading Clostridium were isolated from piggery or chicken manure composting processes. Since type strains of respective isolates did not degrade these compounds, the degradability of the compounds was a novel characteristic. All isolates were mesophilic. The maximum growth allowance concentrations of these isolates were 300 to 800 mg/l in indole and 100 to 300 mg/l in skatole. All isolates showed better growth and utilization of indolic compounds in nutrient-rich medium than in minimal medium. Skatole-degrading isolates degraded some substituted indoles tested, 3-indoleacetic acid, indole and oxindole, but did not degrade 1-methylindole, 2-methylindole, isatin or anthranilic acid. On the other hand, indole-degrading isolates degraded only oxindole. The growth of Clostridium malenominatum A-3 was inhibited by a low concentration (0.005%) of indole or skatole, even when 200-fold excess glucose was present in the medium. When 0.03% indole or skatole was added to the medium, C. malenominatum A-3 showed a lag phase for about 10 and 70 h, respectively. When 0.01% of these compounds was added to the medium, the uptake of glucose was inhibited. C. malenominatum A-3 degraded these compounds under nutrient-rich and minimal conditions.     

Photomonomerization of pyrimidine dimers by indoles and proteins.

Model systems for the study of photoreactivation have been developed that utilize a variety of indole derivatives. These systems can split uracil cis-syn cyclobutadipyrimidine, either free or in RNA, when irradiated at wave-lengths absorbed only by the indole moiety. The ability of indole compounds to split dimers is closely related to their electronic properties. Those of high electron-donor capacity such as indole, 3-methylindole, indole-3-acetic acid, 5-hydroxytryptophan and tryptophan are good photosensitizers, with efficacy in that order. Indoles with electron-withdrawing substituents such as indole-3-carboxylic acid, indole-3-aldehyde and oxindole are inactive in the monomerization reaction. These findings support the proposed mechanism that the photosensitized monomerization occurs as a result of electron transfer from the excited indole molecules to the pyrimidine bases.Proteins containing fully exposed tryptophan residues (chicken egg white lysozyme and bovine diisopropylphosphoryltrypsin) also cause the splitting of the ¹⁴C-labeled dimers under the same conditions. In the case of lysozyme the quantum yield of monomerization is similar to that of free tryptophan. Much of the monomerization ability of lysozyme was lost after the solvent-available tryptophan had been oxidized by treatment with N-bromosuccinimide. Bovine pancreatic ribonuclease A, a protein devoid of tryptophan, failed to exhibit photosensitized monomerization of uracil dimers. The biological implication of these reactions involving a protein with an exposed tryptophan residue is discussed.Although indoles are able to split the dimers in RNA, they fail to photo-reactivate u.v.-damaged TMV-RNA. Indole-3-acetic acid, 3-methylindole and 5-hydroxytryptophan rapidly inactivate viral RNA when irradiated at 313 nm, possibly because of side reactions.     

Syntheses and biological activities of bis(3-indolyl)thiazoles, analogues of marine bis(indole)alkaloid nortopsentins

The thiazole analogues of the marine bis(indole)alkaloid nortopsentins, 2,4-bis(3-indolyl)thiazoles, were synthesized using Hantzsch reaction between indole-3-thioamides and 3-(alpha-bromoacetyl)indoles as the key step, and these analogues showed potent cytotoxic activities against a variety of human cancer cell lines in vitro.     

3-Methylindole (skatole) and indole production by mixed populations of pig fecal bacteria.

Pig fecal slurries converted added L-tryptophan either to indole without detectable intermediates or to 3-methylindole (skatole) via indole-3-acetate. The initial rate of production of 3-methylindole was greatest at pH 6.5 and less at pH 5.0 and 8.0; the initial rates of indole production were similar at pH 6.5 and 8.0. More than 80% of the tryptophan added was converted to 3-methylindole at pH 5.0; at pH 8.0 85% was converted to indole. Both pathways had similar Km values for tryptophan and similar maximum rates. Indole-3-carbinol and indole-3-acetonitrile completely inhibited the production of 3-methylindole from indole-3-acetate but had no effect on the reactions involving L-tryptophan.     

Characterization of a Novel Phenol Hydroxylase in Indoles Biotranformation from a Strain Arthrobacter sp. W1

Background

Indigoids, as popular dyes, can be produced by microbial strains or enzymes catalysis. However, the new valuable products with their transformation mechanisms, especially inter-conversion among the intermediates and products have not been clearly identified yet. Therefore, it is necessary to investigate novel microbial catalytic processes for indigoids production systematically.

Findings

A phenol hydroxylase gene cluster (4,606 bp) from Arthrobacter sp. W1 (PH_w1) was obtained. This cluster contains six components in the order of KLMNOP, which exhibit relatively low sequence identities (37–72%) with known genes. It was suggested that indole and all the tested indole derivatives except for 3-methylindole were transformed to various substituted indigoid pigments, and the predominant color products derived from indoles were identified by spectrum analysis. One new purple product from indole, 2-(7-oxo-1H-indol-6(7H)-ylidene) indolin-3-one, should be proposed as the dimerization of isatin and 7-hydroxylindole at the C-2 and C-6 positions. Tunnel entrance and docking studies were used to predict the important amino acids for indoles biotransformation, which were further proved by site-directed mutagenesis.

Conclusions/Significance

We showed that the phenol hydroxylase from genus Arthrobacter could transform indoles to indigoids with new chemical compounds being produced. Our work should show high insights into understanding the mechanism of indigoids bio-production.     

Peroxide oxidation of indole to oxindole by chloroperoxidase catalysis.

In the presence of chloroperoxidase, indole was oxidized by H2O2 to give oxindole as the major product. Under most conditions oxindole was the only product formed, and under optimal conditions the conversion was quantitative. This reaction displayed maximal activity at pH 4.6, although appreciable activity was observed throughout the entire pH range investigated, namely pH 2.5-6.0. Enzyme saturation by indole could not be demonstrated, up to the limit of indole solubility in the buffer. The oxidation kinetics were first-order with respect to indole up to 8 mM, which was the highest concentration of indole that could be investigated. On the other hand, 2-methylindole was not affected by H2O2 and chloroperoxidase, but was a strong inhibitor of indole oxidation. The isomer 1-methylindole was a poor substrate for chloroperoxidase oxidation, and a weak inhibitor of indole oxidation. These results suggest the possibility that chloroperoxidase oxidation of the carbon atom adjacent to the nitrogen atom in part results from hydrogen-bonding of the substrate N-H group to the enzyme active site.     

Metabolic pathways of quinoline, indole and their methylated analogs by Desulfobacterium indolicum (DSM 3383)

The transformation of quinoline, isoquinoline and 3-, 4-, 6- and 8-methylquinoline by Desulfobacterium indolicum was compared with that of the N-containing analogues indole and 1-, 2-, 3- and 7-methylindole. The metabolites were identified using high-performance liquid chromatography with UV detection, thin-layer chromatography, combined gas chromatography/mass spectrometry and proton NMR spectroscopy. All degraded compounds were initially hydroxylated at position 2 by D. indolicum. A new degradation product of quinoline was observed in the second transformation step, where 3,4-dihydro-2-quinolinone accumulated. This ring-reduced compound was further transformed into unidentified products. The transformation pathway of indole was characterized by well-known steps through oxindole, isatin, and anthranilic acid. No further transformation of the hydroxylated methyl analogues: 3- and 7-methyloxindole and 3- and 4-methyl-2-quinolinone, was observed within 162 days of incubation. These degradation products accumulated in stoichiometric amounts, while 6- and 8-methyl-2-quinolinone were further degraded to 6- and 8-methyl-3,4-dihydro-2-quinolinone in stoichiometric amounts. Isoquinoline, 2-methylquinoline and 1- and 2-methylindole were not degraded by D. indolicum. These observations indicate that a methyl group at or close to position 2 results in blockage of the microbial attack, and that transformation of hydroxyquinolines methylated at the heterocyclic ring also was blocked or sterically inhibited. An incomplete transformation of some methylated compounds was observed, e.g. for 3- and 6-methylquinoline and 3- and 7-methylindole, with residual concentrations of 0.5–4 mg/l in relation to initial concentrations of 10–15 mg/l. Received: 23 July 1996 / Received revision: 4 October 1996 / Accepted: 25 October 1996     

Metabolism of 17β-hydroxy-2α,3α-cyclopropano-5α-androstane in the rabbit

The neutral urinary excretion products of 17β-hydroxy-2α,3α-cyclopropano-5α-androstane from the rabbit, dosed orally, were investigated. Column chromatography yielded five crystalline metabolites which were identified by GLC and spectroscopic measurements. Three of these substances were hydroxylated in the 4α-position and one in the 6a-position with the cyclopropane ring intact. The fifth substance, 17β-hydroxy-3β-methyl-5α-androstan-2-one, can be derived from initial hydroxylation of the cyclopropane ring at C-2 followed by ring opening. The dosed substance and triol material was shown to be present by GLC and m.s. measurements. GLC determinations show that hydroxylation has occurred at C-4?C-6>C-2.     

Syntheses and anti-cancer activities of 2-[1-(indol-3-yl-/pyrimidin-5-yl-/pyridine-2-yl-/quinolin-2-yl)-but-3-enylamino]-2-phenyl-ethanols

Schiff bases prepared by the reactions of substituted amines with indole-/, pyrimidine-/, pyridine-/, and quinoline-aldehydes are made to undergo indium mediated allylation whereby a (substituted amine, allyl)methyl group has been introduced at C-3 of indole, C-5 of pyrimidine, and C-2 of pyridine and quinoline. Amongst the 16 compounds investigated for anti-cancer activities at 59 human tumor cell lines 3, 9-12, and 14 show appreciable activities. The structure-activity relationship studies point that the contribution of phenylglycinol moiety as a part of side chain at C-3 of indole and C-5 of pyrimidine seems to be crucial for exhibiting anti-cancer activities.     

Mutagen formation on nitrite treatment of indole compounds derived from indole-glucosinolate

The mutagenicities of 8 indole compounds (indole-3-acetonitrile, indole-3-carbinol, indole-3-acetamide, indole-3-acetic acid, 3-methylindole, indole-3-aldehyde, indole-3-carboxylic acid and indole) derived from indole glucosinolate were studied by mutation tests on Salmonella typhimurium TA98 and TA100 and Escherichia coli WP2 uvrA/pKM101 with and without S9 mix. None of the 8 indole compounds were mutagenic, but they became mutagenic on these 3 tester strains when treated with nitrite at pH 3. The nitrite-treated indole compounds were mutagenic without metabolic activation system (S9 mix), and their mutagenicities were decreased by the addition of S9 mix.     

Transformation of indole by methanogenic and sulfate-reducing microorganisms isolated from digested sludge

In the present study, mineralization of an aromaticN-heterocyclic molecule, indole, by microorganisms present in anaerobically digested sewage sludge was examined. The first step in indole mineralization was the formation of a hydroxylated intermediate, oxindole. The rate of transformation of indole to oxindole and its subsequent disappearance was dependent on the concentration of inoculum and indole and the incubation temperature. Methanogenesis appeared to be the dominant process in the mineralization of indole in 10% digested sludge even in the presence of high concentrations of sulfate. Enrichment of the digested sludge with sulfate as an electron acceptor allowed the isolation of a metabolically stable mixed culture of anaerobic bacteria which transformed indole to oxindole and acetate, and ultimately to methane and carbon dioxide. This mixed culture exhibited a predominance of sulfate-reducers over methanogens with more than 75% of the substrate mineralized to carbon dioxide. The investigation demonstrates that indole can be transformed by both methanogenic and sulfate-reducing microbial populations.     

Identification of the factors affecting the rate of deactivation of hypochlorous acid by melatonin

It has been found that melatonin reacts rapidly with hypochlorous acid in phosphate-buffered, ethanol-water solutions to produce 2-hydroxymelatonin. The rate law, d[2 - HOMel]/dt - kHOCl[Mel][HOCl] - kOCl-[Mel][OCl-], was obtained. At 37 degrees C and at a water concentration of 23.5 M, kOCl- = 6.0 x 10(2) L. mol-1. s-1, and kHOCl was found to be a function of the water concentration, kHOCl = 11 +/- 3 L3. mol-3. s-1. [H2O]2, indicating that the availability of water at the site of the reaction plays a significant role. The part that the structural components of melatonin play in determining the reaction pathway was examined by comparing the rate of deactivation of HOCl by melatonin to that of the model compounds indole, 5-methoxyindole, and 3-methylindole. The relative reactivity is explained in terms of steric and electronic effects, and it was found that the presence of the substituent at the 3-position influences the nature of the oxidation product. Melatonin and 3-methylindole yielded hydroxylated products, whereas indole and 5-methoxyindole produce chlorinated products.     

Novel reversible indole-3-carboxylate decarboxylase catalyzing nonoxidative decarboxylation

After enrichment culture with indole-3-carboxylate in static culture, a novel reversible decarboxylase, indole-3-carboxylate decarboxylase, was found in Arthrobacter nicotianae FI1612 and several molds. The enzyme reaction was examined in resting-cell reactions with A. nicotianae FI1612. The enzyme activity was induced specifically by indole-3-carboxylate, but not by indole. The indole-3-carboxylate decarboxylase of A. nicotianae FI1612 catalyzed the nonoxidative decarboxylation of indole-3-carboxylate into indole, and efficiently carboxylated indole and 2-methylindole by the reverse reaction. In the presence of 1 mM dithiothreitol, 50 mM Na2 S2O3, and 20% (v/v) glycerol, indole-3-carboxylate decarboxylase was partially purified from A. nicotianae FI1612. The purified enzyme had a molecular mass of approximately 258 kDa. The enzyme did not need any cofactor for the decarboxylating and carboxylating reactions.     

Oxygenation of indoles by a copper(I) chloride pyridine complex. a new tryptophan 2,3-dioxygenase model system

The copper(I) chloride, pyridine system has shown marked tryptophan 2,3-dioxygenase activity with tryptophan and indole derivatives. However, only in the cases of tryptophan and 3-methylindole was it possible to isolate the primary products N-formylkynurenine (～1%) and 2-formamidoacetophenone (70%), respectively. In other cases, such as 2-methylindole, methyl indole-3-acetate, tryptamine, indole-3-propionic acid, and acetyltryptophan methyl ester, only secundary products could be isolated or determined by GC-MS methods.     

Total synthesis of 0231B,an inhibitor of 3alpha-hydroxysteroid dehydrogenase produced by Streptomyces sp. HKI0231

The new inhibitors of 3alpha-hydroxysteroid dehydrogenase, 0231A 1 and 0231B 2, have a unique benz[c,d]indol-3(1H)-one structure in their molecules. In our advanced studies on indole chemistry, we have developed an efficient synthetic method for benz[c,d]indol-3(1H)-one derivatives. We report here its application to the synthesis of 0231B in 10 steps (8.1% overall yield) from 6-methylindole 8 by introducing an acyl group into the 3-position of the indole nucleus, cyclization of the side chain at the 3-position to the 4-position and subsequent elimination of the phenyl group, and conjugate addition of the substituted phenyl group.     

Efficient synthesis of 3,3-diheteroaromatic oxindole analogues and their in vitro evaluation for spermicidal potential

Syntheses of 3,3-diheteroaromatic oxindole derivatives has been achieved by coupling indole-2,3-dione (isatin) with differently substituted indoles and pyrrole in presence of I₂ in i-PrOH. The in vitro spermicidal potentials and the mode of spermicidal action of the synthesized analogues were evaluated and the derivative, 3,3-bis (5-methoxy-1H-indol-3-yl) indolin-2-one (3d) exhibited most significant activity.     

Transformation of indole and quinoline by Desulfobacterium indolicum (DSM 3383)

Degradation of indole and quinoline by Desulfobacterium␣indolicum was studied in batch cultures. The first step in the degradation pathway of indole and quinoline was a hydroxylation at the 2 position to oxindole and 2-hydroxyquinoline respectively. These hydroxylation reactions followed saturation kinetics. The kinetic parameters for indole were an apparent maximum specific transformation rate (V _Amax) of 263 μmol mg total protein⁻¹ day⁻¹ and an apparent half-saturation constant (K _Am) of 139 μM. The V _Amax for quinoline was 170 μmol mg total protein⁻¹ day⁻¹ and K _Am was 92 μM. Oxindole inhibited indole hydroxylation whereas 2-hydroxyquinoline stimulated quinoline hydroxylation. An adaptation period of approximately 20 days was required before transformation of 2-hydroxyquinoline in cultures previously grown on quinoline. Indole and quinoline were hydroxylated with a lag phase shorter than 4 h in a culture adapted to ethanol. Chloramphenicol inhibited the hydroxylation of indole and quinoline in ethanol-adapted cells, indicating an inducible enzyme system. Chloramphenicol had no effect on the hydroxylation of indole in quinoline-adapted cells or on the hydroxylation of quinoline in indole-adapted cells. This indicated that it was the same inducible enzyme system that hydroxylated indole and quinoline. Received: 16 July 1996 / Received revision: 23 September 1996 / Accepted: 29 September 1996