Formation of Catechols via Removal of Acid Side Chains from Ibuprofen and Related Aromatic Acids

Although ibuprofen [2-(4-isobutylphenyl)-propionic acid] is one of the most widely consumed drugs in the world, little is known regarding its degradation by environmental bacteria. Sphingomonas sp. strain Ibu-2 was isolated from a wastewater treatment plant based on its ability to use ibuprofen as a sole carbon and energy source. A slight preference toward the R enantiomer was observed, though both ibuprofen enantiomers were metabolized. A yellow color, indicative of meta-cleavage, accumulated transiently in the culture supernatant when Ibu-2 was grown on ibuprofen. When and only when 3-flurocatechol was used to poison the meta-cleavage system, isobutylcatechol was identified in the culture supernatant via gas chromatography-mass spectrometry analysis. Ibuprofen-induced washed-cell suspensions also metabolized phenylacetic acid and 2-phenylpropionic acid to catechol, while 3- and 4-tolylacetic acids and 2-(4-tolyl)-propionic acid were metabolized to the corresponding methyl catechols before ring cleavage. These data suggest that, in contrast to the widely distributed coenzyme A ligase, homogentisate, or homoprotocatechuate pathway for metabolism of phenylacetic acid and similar compounds, Ibu-2 removes the acidic side chain of ibuprofen and related compounds prior to ring cleavage.     

Metabolism of 2,2'-dihydroxybiphenyl by Pseudomonas sp. strain HBP1: production and consumption of 2,2',3-trihydroxybiphenyl.

Cells of Pseudomonas sp. strain HBP1 grown on 2-hydroxy- or 2,2'-dihydroxybiphenyl contain NADH-dependent monooxygenase activity that hydroxylates 2,2'-dihydroxybiphenyl. The product of this reaction was identified as 2,2',3-trihydroxybiphenyl by 1H nuclear magnetic resonance and mass spectrometry. Furthermore, the monooxygenase activity also hydroxylates 2,2',3-trihydroxybiphenyl at the C-3' position, yielding 2,2',3,3'-tetrahydroxybiphenyl as a product. An estradiol ring cleavage dioxygenase activity that acts on both 2,2',3-tri- and 2,2',3,3'-tetrahydroxybiphenyl was partially purified. Both substrates yielded yellow meta-cleavage compounds that were identified as 2-hydroxy-6-(2-hydroxyphenyl)-6-oxo-2,4-hexadienoic acid and 2-hydroxy-6-(2,3-dihydroxyphenyl)-6-oxo-2,4-hexadienoic acid, respectively, by gas chromatography-mass spectrometry analysis of their respective trimethylsilyl derivatives. The meta-cleavage products were not stable in aqueous incubation mixtures but gave rise to their cyclization products, 3-(chroman-4-on-2-yl)pyruvate and 3-(8-hydroxychroman-4-on-2-yl)pyruvate, respectively. In contrast to the meta-cleavage compounds, which were turned over to salicylic acid and 2,3-dihydroxybenzoic acid, the cyclization products are not substrates to the meta-cleavage product hydrolase activity. NADH-dependent salicylate monooxygenase activity catalyzed the conversions of salicylic acid and 2,3-dihydroxybenzoic acid to catechol and pyrogallol, respectively. The partially purified estradiol ring cleavage dioxygenase activity that acted on the hydroxybiphenyls also produced 2-hydroxymuconic semialdehyde and 2-hydroxymuconic acid from catechol and pyrogallol, respectively.     

Cometabolic ring fission of dibenzofuran by Gram-negative and Gram-positive biphenyl-utilizing bacteria

Thirty-five strains of soil bacteria were grown with biphenyl (BP) and tested for their capacity to cooxidize dibenzofuran (DBF). During metabolism of DBF, the culture medium of 17 strains changed from colorless to orange, indicating a meta-cleavage pathway of DBF degradation. The ring cleavage product of these isolates was shown to be 2-hydroxy-4-(3'-oxo-3' H-benzofuran-2'-yliden)but-2-enoic acid (HOBB). The strain SBUG 271, studied in detail and identified as Rhodococcus erythropolis, degraded DBF via 1,2-dihydroxydibenzofuran. The ensuing meta-cleavage yielded HOBB and salicylic acid. In addition, the four monohydroxylated monomers of DBF and two metabolites, which were not further characterized, were detected. Thus, our results demonstrate that the metabolic mechanism involves lateral dioxygenation of DBF followed by meta-cleavage and occurs in Gram-negative as well as in Gram-positive BP-degrading bacteria.     

Anaerobic naphthalene degradation by a sulfate-reducing enrichment culture

Anaerobic naphthalene degradation by a sulfate-reducing enrichment culture was studied by substrate utilization tests and identification of metabolites by gas chromatography-mass spectrometry. In substrate utilization tests, the culture was able to oxidize naphthalene, 2-methylnaphthalene, 1- and 2-naphthoic acids, phenylacetic acid, benzoic acid, cyclohexanecarboxylic acid, and cyclohex-1-ene-carboxylic acid with sulfate as the electron acceptor. Neither hydroxylated 1- or 2-naphthoic acid derivatives and 1- or 2-naphthol nor the monoaromatic compounds ortho-phthalic acid, 2-carboxy-1-phenylacetic acid, and salicylic acid were utilized by the culture within 100 days. 2-Naphthoic acid accumulated in all naphthalene-grown cultures. Reduced 2-naphthoic acid derivatives could be identified by comparison of mass spectra and coelution with commercial reference compounds such as 1,2,3, 4-tetrahydro-2-naphthoic acid and chemically synthesized decahydro-2-naphthoic acid. 5,6,7,8-Tetrahydro-2-naphthoic acid and octahydro-2-naphthoic acid were tentatively identified by their mass spectra. The metabolites identified suggest a stepwise reduction of the aromatic ring system before ring cleavage. In degradation experiments with [1-(13)C]naphthalene or deuterated D(8)-naphthalene, all metabolites mentioned derived from the introduced labeled naphthalene. When a [(13)C]bicarbonate-buffered growth medium was used in conjunction with unlabeled naphthalene, (13)C incorporation into the carboxylic group of 2-naphthoic acid was shown, indicating that activation of naphthalene by carboxylation was the initial degradation step. No ring fission products were identified.     

Colonic metabolism of dietary polyphenols: influence of structure on microbial fermentation products

The metabolism of chlorogenic acid, naringin, and rutin, representative members of three common families of dietary polyphenols, the hydroxycinnamates, the flavanones, and the flavonols, respectively, was studied in an in vitro mixed culture model of the human colonic microflora. Time- and concentration-dependent degradation of all three compounds was observed, which was associated with the following metabolic events after cleavage of the ester or glycosidic bond: reduction of the aliphatic double bond of the resulting hydroxycinnamate caffeic acid residue; dehydroxylation and ring fission of the heterocyclic C-ring of the resulting deglycosylated flavanone, naringenin, and of the deglycosylated flavonol, quercetin (which differed depending on the substitution). The metabolic events, their sequences, and major phenolic end products, as identified by GC-MS or LC-MS/MS, were elucidated from the structural characteristics of the investigated compounds. The major phenolic end products identified were 3-(3-hydroxyphenyl)-propionic acid for chlorogenic acid, 3-(4-hydroxyphenyl)-propionic acid and 3-phenylpropionic acid for naringin, and 3-hydroxyphenylacetic acid and 3-(3-hydroxyphenyl)-propionic acid for rutin. The degree of degradation of the compounds studied was significantly influenced by the substrate concentration as well as individual variations in the composition of the fecal flora. The results support extensive metabolism of dietary polyphenols in the colon, depending on substrate concentration and residence time, with resultant formation of simple phenolics, which can be considered biomarkers of colonic metabolism if subsequently absorbed. It is also apparent that a relatively small number of phenolic degradation products are formed in the colon from the diverse group of natural polyphenols.     

Identification of a serine hydrolase which cleaves the alicyclic ring of tetralin

A gene designated thnD, which is required for biodegradation of the organic solvent tetralin by Sphingomonas macrogoltabidus strain TFA, has been identified. Sequence comparison analysis indicated that thnD codes for a carbon-carbon bond serine hydrolase showing highest similarity to hydrolases involved in biodegradation of biphenyl. An insertion mutant defective in ThnD accumulates the ring fission product which results from the extradiol cleavage of the aromatic ring of dihydroxytetralin. The gene product has been purified and characterized. ThnD is an octameric thermostable enzyme with an optimum reaction temperature at 65 degrees C. ThnD efficiently hydrolyzes the ring fission intermediate of the tetralin pathway and also 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid, the ring fission product of the biphenyl meta-cleavage pathway. However, it is not active towards the equivalent intermediates of meta-cleavage pathways of monoaromatic compounds which have small substituents in C-6. When ThnD hydrolyzes the intermediate in the tetralin pathway, it cleaves a C-C bond comprised within the alicyclic ring of tetralin instead of cleaving a linear C-C bond, as all other known hydrolases of meta-cleavage pathways do. The significance of this activity of ThnD for the requirement of other activities to mineralize tetralin is discussed.     

Identification of 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid, 4-hydroxy-2-oxohexanoic acid, and 2-hydroxyhexa-2,4-dienoic acid and related enzymes involved in testosterone degradation in Comamonas testosteroni TA441

Comamonas testosteroni TA441 utilizes testosterone via aromatization of the A ring followed by meta-cleavage of the ring. The product of the meta-cleavage reaction, 4,5-9,10-diseco-3-hydroxy-5,9,17-trioxoandrosta-1(10),2-dien-4-oic acid, is degraded by a hydrolase, TesD. We directly isolated and identified two products of TesD as 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid and (2Z,4Z)-2-hydroxyhexa-2,4-dienoic acid. The latter was a pure 4Z isomer. 2-Hydroxyhexa-2,4-dienoic acid was converted by a hydratase, TesE, and the product isolated from the reaction solution was identified as 2-hydroxy-4-hex-2-enolactone, indicating the direct product of TesE to be 4-hydroxy-2-oxohexanoic acid.     

Metabolic breakdown of Kaneclors (polychlorobiphenyls) and their products by Acinetobacter sp.

Biodegradability of commercial polychlorobiphenyl mixtures (Kaneclors, KC 200 to KC 500) and their metabolic products by Acinetobacter sp. strain P6 were studied by gas chromatography-mass spectrometry analysis. KC 200 (primarily dichlorobiphenyls) rapidly degraded after 4 h of incubation with the P6 resting cells, showing predominant accumulation of monochlorobenzoic acids. KC 300 (primarily trichlorobiphenyls) were also degraded after 4 h of incubation, producing various metabolic intermediates such as mono- and dichlorobenzoic acids, dihydroxy biphenyl compounds with two and three chlorines, and the ring meta-cleavage compounds with two and three chlorines. KC 400 (primarily tetrachlorobiphenyls) were also susceptible to biodegradation by the same organism. Chlorobenzoic acids (chlorine number 1 to 3), dihydroxy compounds (chlorine number 2 to 4), and the ring meta-cleavage compounds (chlorine number 2 to 3) were observed as the products from KC 400. In addition to such products, a large amount of unknown compounds with two chlorines in the molecule, which can be derived from 2,3,2',3' - or 2,3,2',5'-tetrachlorobiphenyls or both, accumulated. In contrast to KC 200, KC 300, and KC 400, KC 500 (primarily pentachlorobiphenyls) were resistant to degradation and hardly metabolized. Only dihydroxy compounds of certain pentachlorobiphenyls were detected.     

Catabolism of aromatic acids in Trichosporon cutaneum.

Trichosporon cutaneum readily metabolized protocatechuate, homoprotocatechuate, and gentisate, but lacked ring fission dioxygenases for these compounds. Benzoic, salicylic, 2,3-dihydroxybenzoic, and gentisic acids were converted into beta-ketoadipic acid before entry into the Krebs cycle. Benzoic acid gave rise successively to 4-hydroxybenzoic acid, protocatechuic acid, and hydroxyquinol (1,3,4-trihydroxybenzene), which underwent ring fission to maleylacetic acid. Salicylate and 2,3-dihydroxybenzoate were both initially metabolized to give catechol. 2,3-Dihydroxybenzoate was the substrate for a specific nonoxidative decarboxylase induced by salicylate, although 2,3-dihydroxybenzoate was not a catabolite of salicylate. Gentisate was metabolized to maleylacetic acid and was also readily attacked by salicylate hydroxylase at each stage of a partial purification procedure. Phenylacetic acid was degraded through 3-hydroxyphenylacetic, homogentisic, and maleylacetoacetic acids to acetoacetic and fumaric acids. All the reactions of these catabolic sequences were catalyzed by cell extracts, supplemented with reduced pyridine nucleotide coenzymes where necessary, except for the hydroxylations of benzoic and phenylacetic acids which were demonstrated with cell suspensions and isotopically labeled substrates.     

Uptake of phenylacetic acid by two strains of Penicillium chrysogenum

Uptake of phenylacetic acid, the side-chain precursor of benzylpenicillin, was studied in Penicillium chrysogenum Wisconsin 54-1255 and in a strain yielding high levels of penicillin. In penicillin fermentations with the high-yielding strain, 100% recovery of phenylacetic acid in benzylpenicillin was found, whereas in the Wisconsin strain only 17% of the supplied phenylacetic acid was incorporated into benzylpenicillin while the rest was metabolized. Accumulation of total phenylacetic acid-derived carbon in the cells was nonsaturable in both strains at high external concentrations of phenylacetic acid (250-3500 microM), and in the high-yielding strain at low phenylacetic acid concentrations (2. 8-100 microM), indicating that phenylacetic acid enters the cells by simple diffusion, as concluded earlier for P. chrysogenum by other authors. However, at low external concentrations of phenylacetic acid saturable accumulation appeared in the Wisconsin strain. HPLC-analyses of cell extracts from the Wisconsin strain showed that phenylacetic acid was metabolized immediately after entry into the cells and different [14C]-labeled metabolites were detected in the cells. Up to approximately 50% of the accumulated phenylacetic acid was metabolized during the transport-assay period, the conversion having an impact on the uptake experiments. Nevertheless, accumulation of free unchanged phenylacetic acid in the cells showed saturation kinetics, suggesting the possible involvement of a high-affinity carrier in uptake of phenylacetic acid in P. chrysogenum Wisconsin 54-1255. At high concentrations of phenylacetic acid, contribution to uptake by this carrier is minor in comparison to simple diffusion and therefore, of no importance in the industrial production of penicillin.     

Anaerobic Naphthalene Degradation by a Sulfate-Reducing Enrichment Culture

Anaerobic naphthalene degradation by a sulfate-reducing enrichment culture was studied by substrate utilization tests and identification of metabolites by gas chromatography-mass spectrometry. In substrate utilization tests, the culture was able to oxidize naphthalene, 2-methylnaphthalene, 1- and 2-naphthoic acids, phenylacetic acid, benzoic acid, cyclohexanecarboxylic acid, and cyclohex-1-ene-carboxylic acid with sulfate as the electron acceptor. Neither hydroxylated 1- or 2-naphthoic acid derivatives and 1- or 2-naphthol nor the monoaromatic compounds ortho-phthalic acid, 2-carboxy-1-phenylacetic acid, and salicylic acid were utilized by the culture within 100 days. 2-Naphthoic acid accumulated in all naphthalene-grown cultures. Reduced 2-naphthoic acid derivatives could be identified by comparison of mass spectra and coelution with commercial reference compounds such as 1,2,3,4-tetrahydro-2-naphthoic acid and chemically synthesized decahydro-2-naphthoic acid. 5,6,7,8-Tetrahydro-2-naphthoic acid and octahydro-2-naphthoic acid were tentatively identified by their mass spectra. The metabolites identified suggest a stepwise reduction of the aromatic ring system before ring cleavage. In degradation experiments with [1-¹³C]naphthalene or deuterated D₈-naphthalene, all metabolites mentioned derived from the introduced labeled naphthalene. When a [¹³C]bicarbonate-buffered growth medium was used in conjunction with unlabeled naphthalene, ¹³C incorporation into the carboxylic group of 2-naphthoic acid was shown, indicating that activation of naphthalene by carboxylation was the initial degradation step. No ring fission products were identified.     

Bacterial catabolism of sulfanilic acid via catechol-4-sulfonic acid

Abstract A sulfanilic acid (4-aminobenzenesulfonic acid) degrading culture consisting of two strains (strain S1 and S2), was studied. Only strain S1 was able to attack sulfanilic acid. When strain S1 was cultavated in a mineral medium with sulfanilic acid an intensive violet colour was observed. The accumulating metabolite was isolated from the culture supernatant. By comparison with an authentic compound the metabolite was identified as catechol-4-sulfonic acid by thin layer and high performance liquid chromatography and by UV- and H-NMR spectroscopy. The occurrence of catechol-4-sulfonic acid indicates that there is no release of the sulfonic group before ring cleavage.     

Gene encoding the hydrolase for the product of the meta-cleavage reaction in testosterone degradation by Comamonas testosteroni

In a previous study we isolated the meta-cleavage enzyme gene, tesB, that encodes an enzyme that carries out a meta-cleavage reaction in the breakdown of testosterone by Comamonas testeroni TA441 (M. Horinouchi et al., Microbiology 147:3367-3375, 2001). Here we report the isolation of a gene, tesD, that encodes a hydrolase which acts on the product of the meta-cleavage reaction. We isolated tesD by using a Tn5 mutant of TA441 that showed limited growth on testosterone. TesD exhibited ca. 40% identity in amino acid sequence with BphDs, known hydrolases of biphenyl degradation in Pseudomonas spp. The TesD-disrupted mutant showed limited growth on testosterone, and the culture shows an intense yellow color. High-pressure liquid chromatography analysis of the culture of TesD-disrupted mutant incubated with testosterone detected five major intermediate compounds, one of which, showing yellow color under neutral conditions, was considered to be the product of the meta-cleavage reaction. The methylation product was analyzed and identified as methyl-4,5-9,10-diseco-3-methoxy-5,9,17-trioxoandrosta-1(10),2-dien-4-oate, indicating that the substrate of TesD in testosterone degradation is 4,5-9,10-diseco-3-hydroxy-5,9,17-trioxoandrosta-1(10),2-dien-4-oic acid. 4,5-9,10-Diseco-3-hydroxy-5,9,17-trioxoandrosta-1(10),2-dien-4-oic acid was transformed by Escherichia coli-expressed TesD. Downstream of tesD, we identified tesE, F, and G, which encode for enzymes that degrade one of the products of 4,5-9,10-diseco-3-hydroxy-5,9,17-trioxoandrosta-1(10),2-dien-4-oic acid converted by TesD.     

Aerobic degradation of 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (DDT) by Alcaligenes eutrophus A5.

Biotransformation of 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (DDT) by Alcaligenes eutrophus A5 was demonstrated by analysis of ethyl acetate-extracted products from resting cell cultures. Gas chromatography-mass spectrometry characterization of the neutral extracts revealed two hydroxy-DDT intermediates (m/z = 370) with retention times at 19.55 and 19.80 min that shared identical mass spectra. This result suggested that the hydroxylations occurred at the ortho and meta positions on the aromatic ring. UV-visible spectrum spectrophotometric analysis of a yellow metabolite in the culture supernatant showed a maximum A402 with, under acidic and basic conditions, spectrophotometric characteristics similar to those of the aromatic ring meta-cleavage products. 4-Chlorobenzoic acid was detected by thin-layer chromatography radiochemical scanning in samples from mineralization experiments by comparison of Rf values of [14C]DDT intermediates with that of an authentic standard. These results were further confirmed by gas chromatography-mass spectrometry analysis. This study indicates that DDT appears to be oxidized by a dioxygenase in A. eutrophus A5 and that the products of this oxidation are subsequently subjected to ring fission to eventually yield 4-chlorobenzoic acid as a major stable intermediate.     

Degradation of 4-chlorophenylacetic acid by a Pseudomonas species.

Pseudomonas sp. strain CBS3 was able to utilize 4-chlorophenylacetic acid as the sole source of carbon and energy. When this strain was grown with 4-chlorophenylacetic acid, homoprotocatechuic acid was found to be an intermediate which was further metabolized by the meta-cleavage pathway. Furthermore, three isomers of chlorohydroxyphenylacetic acid, two of them identified as 3-chloro-4-hydroxyphenylacetic acid and 4-chloro-3-hydroxyphenylacetic acid, were isolated from the culture medium. 4-Hydroxyphenylacetic acid was catabolized in a different manner by the glutathione-dependent homogentisate pathway. Degradation enzymes of both of these pathways were inducible.     

Effect of chlorine substitution on the bacterial metabolism of various polychlorinated biphenyls.

Of 36 pure isomers (chlorine numbers 1 to 5) of polychlorinated biphenyls examined, 23 compounds were metabolized by Alcaligenes sp. strain Y42, and 33 compounds were metabolized by Acinetobacter sp. strain P6. The major pathway of many polychlorinated biphenyl isomers examined was considered to proceed through 2',3'-dihydro-2',3'-diol compounds, concomitant dehydrogenated 2',3'-dihydroxy compounds, subsequently the 1',2'-meta-cleavage compounds (chlorinated derivatives of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acids), and then chlorobenzoic acids. The meta-cleavage products were usually converted to chlorobenzoic acids upon further incubation in many polychlorinated biphenyls, but they accumulated specifically in the metabolism of 2,4'-, 2,4,4'-, and 2,5,4'-chlorobiphenyls, which are all chlorinated at the 2,4'-position in the molecules in common. Dihydroxy compounds accumulated mainly in the metabolism of 2,6-, 2,3,6-, 2,4,2',5'-, 2,5,2',5'-, and 2,4,5,2',5'-chlorobiphenyls by Acinetobacter sp. P6. The 2,3,2',3'-, 2,3,2',5'-, and 2,4,5,2',3'-chlorobiphenyls, which are chlorinated at the 2,3-position of one of the rings, were metabolized in a different fashion. Two major metabolites of a chlorobenzoic acid and an unknown compound accumulated always in the metabolism of this group of polychlorinated biphenyls. 2,4,6-Trichlorobiphenyl was metabolized quite differently between the two organisms. Alcaligenes sp. Y42 metabolized this compound very slowly to trichlorobenzoic acid by the major oxidative route. In contrast, Acinetobacter sp. P6 metabolized it to a trihydroxy compound via a dihydroxy compound.     

Biotransformation of biphenyl by Paecilomyces lilacinus and characterization of ring cleavage products

We examined the pathway by which the fungicide biphenyl is metabolized in the imperfect fungus Paecilomyces lilacinus. The initial oxidation yielded the three monohydroxylated biphenyls. Further hydroxylation occurred on the first and the second aromatic ring systems, resulting in the formation of five di- and trihydroxylated metabolites. The fungus could cleave the aromatic structures, resulting in the transformation of biphenyl via ortho-substituted dihydroxybiphenyl to six-ring fission products. All compounds were characterized by gas chromatography-mass spectroscopy and proton nuclear magnetic resonance spectroscopy. These compounds include 2-hydroxy-4-phenylmuconic acid and 2-hydroxy-4-(4'-hydroxyphenyl)-muconic acid, which were produced from 3,4-dihydroxybiphenyl and further transformed to the corresponding lactones 4-phenyl-2-pyrone-6-carboxylic acid and 4-(4'-hydroxyphenyl)-2-pyrone-6-carboxylic acid, which accumulated in large amounts. Two additional ring cleavage products were identified as (5-oxo-3-phenyl-2,5-dihydrofuran-2-yl)-acetic acid and [5-oxo-3-(4'-hydroxyphenyl)-2,5-dihydrofuran-2-yl]-acetic acid. We found that P. lilacinus has a high transformation capacity for biphenyl, which could explain this organism's tolerance to this fungicide.     

Effect of chlorine substitution on the bacterial metabolism of various polychlorinated biphenyls.

Of 36 pure isomers (chlorine numbers 1 to 5) of polychlorinated biphenyls examined, 23 compounds were metabolized by Alcaligenes sp. strain Y42, and 33 compounds were metabolized by Acinetobacter sp. strain P6. The major pathway of many polychlorinated biphenyl isomers examined was considered to proceed through 2',3'-dihydro-2',3'-diol compounds, concomitant dehydrogenated 2',3'-dihydroxy compounds, subsequently the 1',2'-meta-cleavage compounds (chlorinated derivatives of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acids), and then chlorobenzoic acids. The meta-cleavage products were usually converted to chlorobenzoic acids upon further incubation in many polychlorinated biphenyls, but they accumulated specifically in the metabolism of 2,4'-, 2,4,4'-, and 2,5,4'-chlorobiphenyls, which are all chlorinated at the 2,4'-position in the molecules in common. Dihydroxy compounds accumulated mainly in the metabolism of 2,6-, 2,3,6-, 2,4,2',5'-, 2,5,2',5'-, and 2,4,5,2',5'-chlorobiphenyls by Acinetobacter sp. P6. The 2,3,2',3'-, 2,3,2',5'-, and 2,4,5,2',3'-chlorobiphenyls, which are chlorinated at the 2,3-position of one of the rings, were metabolized in a different fashion. Two major metabolites of a chlorobenzoic acid and an unknown compound accumulated always in the metabolism of this group of polychlorinated biphenyls. 2,4,6-Trichlorobiphenyl was metabolized quite differently between the two organisms. Alcaligenes sp. Y42 metabolized this compound very slowly to trichlorobenzoic acid by the major oxidative route. In contrast, Acinetobacter sp. P6 metabolized it to a trihydroxy compound via a dihydroxy compound.     

微生物降解对氯苯胺的一条新代谢途径

迄今为止的研究报道表明,对氯苯胺的生物降解只能以邻位途径或修饰邻位途径进行。采用HPLC、液相色谱质谱联用技术(LC/MS)对Diaphorobacter PCA039菌株降解对氯苯胺的中间代谢产物进行了分析和鉴定,结果表明,对氯苯胺经PCA039菌株的降解形成了氯代邻苯二酚,5-氯-4草酰巴豆酸,5-氯-2-氧戊烯酸,5-氯-2-氧-4-羟戊酸,氯代乙酸等中间代谢产物,这些都是典型的间位代谢途径(meta-pathway)的中间物质,说明Diaphorobacter PCA039菌株以间位裂解途径对对氯苯胺进行降解。这对于对氯代胺的生物降解代谢研究、代谢机理及其遗传表达调控研究具有意义。     

Identical ring cleavage products during anaerobic degradation of naphthalene, 2-methylnaphthalene, and tetralin indicate a new metabolic pathway

Anaerobic degradation of naphthalene, 2-methylnaphthalene, and tetralin (1,2,3,4-tetrahydronaphthalene) was investigated with a sulfate-reducing enrichment culture obtained from a contaminated aquifer. Degradation studies with tetralin revealed 5,6,7,8-tetrahydro-2-naphthoic acid as a major metabolite indicating activation by addition of a C(1) unit to tetralin, comparable to the formation of 2-naphthoic acid in anaerobic naphthalene degradation. The activation reaction was specific for the aromatic ring of tetralin; 1,2,3,4-tetrahydro-2-naphthoic acid was not detected. The reduced 2-naphthoic acid derivatives tetrahydro-, octahydro-, and decahydro-2-naphthoic acid were identified consistently in supernatants of cultures grown with either naphthalene, 2-methylnaphthalene, or tetralin. In addition, two common ring cleavage products were identified. Gas chromatography-mass spectrometry (GC-MS) and high-resolution GC-MS analyses revealed a compound with a cyclohexane ring and two carboxylic acid side chains as one of the first ring cleavage products. The elemental composition was C(11)H(16)O(4) (C(11)H(16)O(4)-diacid), indicating that all carbon atoms of the precursor 2-naphthoic acid structure were preserved in this ring cleavage product. According to the mass spectrum, the side chains could be either an acetic acid and a propenic acid, or a carboxy group and a butenic acid side chain. A further ring cleavage product was identified as 2-carboxycyclohexylacetic acid and was assumed to be formed by beta-oxidation of one of the side chains of the C(11)H(16)O(4)-diacid. Stable isotope-labeling growth experiments with either (13)C-labeled naphthalene, per-deuterated naphthalene-d(8), or a (13)C-bicarbonate-buffered medium showed that the ring cleavage products derived from the introduced carbon source naphthalene. The series of identified metabolites suggests that anaerobic degradation of naphthalenes proceeds via reduction of the aromatic ring system of 2-naphthoic acid to initiate ring cleavage in analogy to the benzoyl-coenzyme A pathway for monoaromatic hydrocarbons. Our findings provide strong indications that further degradation goes through saturated compounds with a cyclohexane ring structure and not through monoaromatic compounds. A metabolic pathway for anaerobic degradation of bicyclic aromatic hydrocarbons with 2-naphthoic acid as the central intermediate is proposed.