Catechol Pathway Research Articles

2-chloromuconate and ClcR-mediated activation of the clcABD operon: in vitro transcriptional and DNase I footprint analyses.

In Pseudomonas putida, the plasmid-borne clcABD operon encodes enzymes involved in 3-chlorocatechol degradation. Previous studies have demonstrated that these enzymes are induced when P. putida is grown in the presence of 3-chlorobenzoate, which is converted to 3-chlorocatechol, and that ClcR, a LysR-type regulator, is required for this induction. The clcABD operon is believed to have evolved from the chromosomal catBCA operon, which encodes enzymes that utilize catechol and is regulated by CatR. The inducer for the catBCA operon is an intermediate of the catechol pathway, cis,cis-muconate. In this study, we demonstrate by the use of in vitro transcription assays and lacZ transcription fusions in vivo that the analogous intermediate of the 3-chlorocatechol pathway, 2-chloromuconate, is the inducer of the clcABD operon. The DNase I footprints of ClcR with and without 2-chloromuconate were also determined. An extended region of the promoter from -79 to -25 was occupied in the absence of inducer, but the -35 region was unprotected. When 2-chloromuconate was added to the binding assays, the footprint contracted approximately 4 bp at the proximal end of the promoter, and the -35 region was contacted. It is interesting to note that CatR actually extends its footprint 14 bp on the catBCA promoter in response to its inducer. Although CatR and ClcR change their nucleotide protection patterns in different manners when exposed to their respective inducers, their final footprints resemble each other. Therefore, it is possible that their transcriptional activation mechanisms may be evolutionarily conserved.

Effects of pesticides on the ratio of 16 alpha/2-hydroxyestrone: a biologic marker of breast cancer risk.

Xenobiotic estrogens are external compounds with estrogenic activity that may thereby affect the risk of breast cancer. This paper describes a mechanism by which xeno-estrogens may affect the development of breast cancer. Estradiol metabolism proceeds by hydroxylation at one of two mutually exclusive sites at C-2 and C-16 alpha. The catechol pathway yields the weakly estrogenic 2-hydroxyestrone (2-OHE1), which inhibits breast cell proliferation. In contrast, the alternative pathway yields the genotoxic 16 alpha-hydroxyestrone (16 alpha-OHE1), which enhances breast cell growth, increases unscheduled DNA synthesis, and oncogene and virus expression, and increases anchorage-independent growth. Using a radiometric assay that measures the relative formation of 16 alpha-OHE1 versus 2-OHE1 from specifically tritiated estradiol in (ER+) MCF-7 cells, we compared the ratio of 16 alpha-OHE1/2-OHE1 observed after treatment with the known rodent carcinogen 7,12-dimethylbenz[a]anthracene (DMBA) with the ratios after treatment with DDT, atrazine, gamma-benzene hexachloride, kepone, coplanar PCBs, endosulfans I and II, linoleic and eicosapentenoic acids, and indole-3-carbinol (I3C). These pesticides significantly increase the ratio of 16 alpha-OHE1/2-OHE1 metabolites to values comparable to or greater than those observed after DMBA. In contrast, the antitumor agent I3C increased 2-OHE1 formation and yielded ratios that are 1/3 of those found in unexposed control cells and 1/10th of those found in DMBA-treated cells. Thus the ratio of 16 alpha-OHE1/2-OHE1 may provide a marker for the risk of breast cancer. Assays of this ratio, which can be measured in spot urines, may prove useful for a variety of in vitro and in vivo studies bearing on breast cancer risk.

Sequence of the hpcC and hpcG genes of the meta-fission homoprotocatechuic acid pathway of Escherichia coli C: nearly 40% amino-acid identity with the analogous enzymes of the catechol pathway

The meta-fission pathway for homoprotocatechuic acid (HPC) catabolism is chemically analogous to the oxidative meta-fission pathway for catechol degradation and so provides an opportunity to investigate how the enzymes of chemically similar, but specific, pathways might have arisen. Two more genes of the HPC pathway from Escherichia coli C, hpcC, encoding 5-carboxymethyl-2-hydroxymuconic acid semialdehyde (CHMS) dehydrogenase, and hpcG, encoding 2-oxohept-3-ene-1,7-dioic acid (OHED) hydratase, have now been sequenced to aid this analysis. The CHMS dehydrogenase showed 40% amino acid (aa) sequence identity with the corresponding enzyme of the catechol pathway, and the OHED hydratase showed 36% as sequence identity with the catechol pathway hydratase. The CHMS dehydrogenase is a member of the aldehyde dehydrogenase superfamily that includes enzymes from animal, plant and microbial sources. Since it appears that the dioxygenase, isomerase and decarboxylase enzymes of the two pathways are not closely related, it is proposed that the two sets of enzymes have arisen separately, but with the muconic acid semialdehyde dehydrogenases and the hydratases being recruited, respectively, from the same ancestral sources.

The Influence of Glucose Supplement on the Degradation of Catechol

A series of batch Biochemical Methane Potential(BMP) tests and two parallel Upflow Anaerobic Sludge Bed(UASB) processes were conducted to investigate the influence of glucose supplement en the degradation of catechol. The substrate inhibition of catechol caused by thermal shock, restart or other operating problems was also studied. The results showed that the glucose supplement could accelerate the degradation rate of catechol, and the effect was more pronounced in the higher concentration range. The mechanism so-called cosubstrate utilization and the anaerobic metabolic pathway of catechol were proposed to explain these symptoms. Data of both batch tests and UASB processes indicated that high concentration of catechol did inhibit the bioactivity of anaerobic microbes. Operation strategy of effluent recirculation could maintain appropriate in-tank concentration to promote the high bioactivity in the reactors. The critical concentration of the highest degradation rate was estimated with a modified Haldane equation derived from the batch BMP data. Low temperature could also contribute inhibition to the microbes, the temperature factor θ with a value of 1.093 estimated from the batch data indicated that anaerobic microbes were very sensitive to the temperature change. The low bioactivity and catechol accumulation were observed during the restart period. It was suggested that lower volumetric loading should be applied in order to resume the bioactivity and to restart the reactor.

Metabolism of Dibenzofuran by Pseudomonas sp. Strain HH69 and the Mixed Culture HH27

A Pseudomonas sp. strain, HH69, and a mixed culture, designated HH27, were isolated by selective enrichment from soil samples. The pure strain and the mixed culture grew aerobically on dibenzofuran as the sole source of carbon and energy. Degradation proceeded via salicylic acid which was branched into the gentisic acid and the catechol pathway. Both salicylic acid and gentisic acid accumulated in the culture medium of strain HH69. The acids were slowly metabolized after growth ceased. The enzymes responsible for their metabolism showed relatively low activities. Besides the above-mentioned acids, 2-hydroxyacetophenone, benzopyran-4-one (chromone), several 2-substituted chroman-4-ones, and traces of the four isomeric monohydroxydiben-zofurans were identified in the culture medium. 2,2',3-Trihydroxybiphenyl was isolated from the medium of a dibenzofuran-converting mutant derived from parent strain HH69, which can no longer grow on dibenzofuran. This gives evidence for a novel type of dioxygenases responsible for the attack on the biarylether structure of the dibenzofuran molecule. A meta-fission mechanism for cleavage of the dihydroxylated aromatic nucleus of 2,2',3-trihydroxybiphenyl is suggested as the next enzymatic step in the degradative pathway.

Physically associated enzymes produce and metabolize 2-hydroxy-2,4-dienoate, a chemically unstable intermediate formed in catechol metabolism via meta cleavage in Pseudomonas putida.

The meta-cleavage pathway of catechol is a major mechanism for degradation of aromatic compounds. In this pathway, the aromatic ring of catechol is cleaved by catechol 2,3-dioxygenase and its product, 2-hydroxymuconic semialdehyde, is further metabolized by either a hydrolytic or dehydrogenative route. In the dehydrogenative route, 2-hydroxymuconic semialdehyde is oxidized to the enol form of 4-oxalocrotonate by a dehydrogenase and then further metabolized to acetaldehyde and pyruvate by the actions of 4-oxalocrotonate isomerase, 4-oxalocrotonate decarboxylase, 2-oxopent-4-enoate hydratase, and 4-hydroxy-2-oxovalerate aldolase. In this study, the isomerase, decarboxylase, and hydratase encoded in the TOL plasmid pWW0 of Pseudomonas putida mt-2 were purified and characterized. The 28-kilodalton isomerase was formed by association of extremely small identical protein subunits with an apparent molecular weight of 3,500. The decarboxylase and the hydratase were 27- and 28-kilodalton polypeptides, respectively, and were copurified by high-performance-liquid chromatography with anion-exchange, hydrophobic interaction, and gel filtration columns. The structural genes for the decarboxylase (xylI) and the hydratase (xylJ) were cloned into Escherichia coli. The elution profile in anion-exchange chromatography of the decarboxylase and the hydratase isolated from E. coli XylI+XylJ- and XylI-XylJ+ clones, respectively, were different from those isolated from XylI+ XylJ+ bacteria. This suggests that the carboxylase and the hydratase form a complex in vivo. The keto but not the enol form of 4-oxalocrotonate was a substrate for the decarboxylase. The product of decarboxylation was 2-hydroxypent-2,4-dienoate rather than its keto form, 2-oxopent-4-enoate. The hydratase acts on the former but not the latter isomer. Because 2-hydroxypent-2,4-dienoate is chemically unstable, formation of a complex between the decarboxylase and the hydratase may assure efficient transformation of this unstable intermediate in vivo.

Role of melanin in susceptibility and resistance of Rhizoctonia solani to microbial lysis

Melanin was detected in self-anastomosing isolates (SAIs) but not in non-self-anastomosing isolates (NSAIs) of anastomosis group (AG) 2-2. Melanin was also detected in isolates of AG 2-1, AG 3 and AG 5, but not of AG 1 and AG 4, The known melanin precursors, catechol and ( + )-scytalone, and inhibitors, Na 2 EDTA, tricyclazole and chlobenthiazone, did not affect hyphal melanization of SAIs and NSAIs. This indicates that the pentaketide or catechol pathway of melanin synthesis may be absent in R. solani . In SAIs, the hyphal cells were composed of extremely thickened walls possessing electron-dense multilayered and microfibrillar structures, whereas in NSAIs the hyphal cell wall was usually very thin, and there was no electron-dense layer even if the cell wall was thick. The hyphal cell wall of AG 1 was also very thin and was transparent. In soil, the longevity of mycelium of SAI was much longer than that of NSAIs. Longevities of isolates of AG 2-1, AG 2-2 and AG 3 were longer than those of AG 1 and AG 4. It seems that there is a correlation between melanin content in the hyphal cell wall and susceptibility or resistance to microbial lysis.

Indirect evidence for the metabolic dehalogenation of tetrafluoro-diethylstilbestrol by rat and hamster liver and kidney microsomes: Species- and organ-dependent differences

In order to assess the significance of the catechol pathway for the carcinogenic activity of diethylstilbestrol (DES), the stability of 3′,5′,3″,5″-tetrafluoro-DES (TF-DES) against metabolic catechol formation was examined in vitro. A radioenzymatic assay was used for determining the estrogen hydroxylase activity of liver and kidney microsomes from male and female Syrian golden hamsters and from male Wistar rats for the substrates TF-DES, DES, estradiol-17 β and 2-fluoro-estradiol-17 β. With all microsomes tested, catechols were formed from TF-DES to an extent similar to or, in some cases, even exceeding that observed with DES and the steroidal estrogens. The estrogen hydroxylase activity measured for the various microsomes depended on the species, organ and substrate. Analysis by high performance liquid chromatography showed that four products were formed in the radioenzymatic assay with DES and TF-DES. These data demonstrate that the fluorine substitution present in TF-DES does not prevent catechol formation and imply that the catechol pathway must be taken into account as a putative pathway for the metabolic activation of DES.

Pathways for the degradation of m-cresol and p-cresol by Pseudomonas putida.

A comparison of the oxidation rates of various compounds by whole cells of Pseudomonas putida 3, 5 indicated that m-cresol is metabolized by oxidation to 3-hydroxybenzoate followed by hydroxylation to gentisate, the ring-fission substrate, when grown with 3, 5-xylenol. However, when m-cresol was the growth substrate, similar experiments suggested a different pathway involving a methyl-substituted catechol, and ring-fission by meta cleavage. Assays of ring-fission enzymes in cell-free extracts confirmed that different pathways are induced by the two growth substrates. 3, 5-Xylenol-grown cells contained high levels of gentisate oxygenase and only very small amounts of catechol oxygenase, whereas gentisate ocygenase could not be detected in m-cresol-grown cells, but levels of catechol oxygenase were greatly increased. Extracts of m-cresol-grown cells also contained 2-hydroxymuconic semialdehyde dehydrogenase and hydrolase, whose specificities enable them to metabolize the ring-fission products from catechol, 3-methylcatechol, and 4-methylcatechol. This catechol pathway is also used by m-cresol-grown cells for p-cresol metabolism. In contrast, the results for cells grown with p-cresol point to an alternative pathway involving oxidation to 4-hydroxybenzoate and hydrosylation to protocatechuate as ring-fission substrate. Extracts of these cells contained high levels of protocatechuate oxygenase and only small amounts of catechol oxygenase.

The metabolism of aromatic acids by micro-organisms. Metabolic pathways in the fungi

1. The metabolic pathways of aromatic-ring fission were examined in a range of fungal genera that utilize several compounds related to lignin. 2. Most of the genera, after growth on p-hydroxybenzoate, protocatechuate or compounds that are degraded to the latter (e.g. caffeate, ferulate or vanillate), rapidly oxidized these compounds, but not catechol. 3. Such genera possessed a protocatechuate 3,4-oxygenase and accumulated beta-carboxymuconate as the product of protocatechuate oxidation. This enzyme had a high pH optimum in most organisms; the Rhodotorula enzyme was competitively inhibited by catechol. 4. beta-Carboxymuconate was converted by all competent fungi into beta-carboxymuconolactone, which was isolated and characterized. None of the fungi produced or utilized at significant rates the corresponding bacterial intermediate gamma-carboxymuconolactone. 5. The lactonizing enzymes of Rhodotorula and Neurospora crassa had a pH optimum near 5.5 and approximate molecular weights of 19000 and 190000 respectively. 6. The fungi did not degrade the isomeric (+)-muconolactone, gamma-carboxymethylenebutanolide or beta-oxoadipate enol lactone at significant rates, and thus differ radically from bacteria, where beta-oxoadipate enol lactone is the precursor of beta-oxoadipate in all strains examined. 7. The end product of beta-carboxymuconolactone metabolism by extracts was beta-oxoadipate. 8. Evidence for a coenzyme A derivative of beta-oxoadipate was found during further metabolism of this keto acid. 9. A few anomalous fungi, after growth on p-hydroxybenzoate, had no protocatechuate 3,4-oxygenase, but possessed all the enzymes of the catechol pathway. Catechol was detected in the growth medium in one instance. 10. A strain of Penicillium sp. formed pyruvate but no beta-oxoadipate from protocatechuate, suggesting the existence also of a ;meta' type of ring cleavage among fungi.

The Conversion of Catechol and Protocatechuate to β-Ketoadipate by Pseudomonas putida: IV. REGULATION

Abstract Study of the regulation of the syntheses of enzymes of the catechol and protocatechuate pathways in Pseudomonas putida has shown that two groups of enzymes are subject to coordinate control. cis,cis-Muconate-lactonizing enzyme and muconolactone isomerase, which are uniquely associated with the catechol pathway, constitute the first coordinate block of enzymes. The synthesis of these enzymes, as well as that of catechol oxygenase (which is regulated independently), seems to be induced by cis,cis-muconate. The second coordinate block of enzymes comprises β-carboxy-cis,cis-muconate-lactonizing enzyme and γ-carboxymuconolactone decarboxylase, which are uniquely associated with the protocatechuate pathway, and β-ketoadipate enol-lactone hydrolase, which is functional in both the protocatechuate and the catechol pathways. This group of enzymes seems to be induced by β-ketoadipate or β-ketoadipyl coenzyme A. Moraxella lwoffii, which degrades protocatechuate and catechol through identical step-reactions, regulates the synthesis of the enzymes mediating these conversions by a different mechanism.

The Conversion of Catechol and Protocatechuate to β-Ketoadipate by Pseudomonas putida: I. BIOCHEMISTRY

Two new intermediates were identified in the protocatechuate pathway of Pseudomonas putida. The first of these, γ-carboxymuconolactone (γ-carboxy-γ-carboxymethyl-Δα-butenolide), is the product of the enzymic lactonization of β-carboxy-cis, cis-muconate. Enzymic decarboxylation of γ-carboxymuconolactone gives rise to β-ketoadipate enollactone (γ-carboxymethyl-Δβ-butenolide), the second newly discovered intermediate in the protocatechuate pathway. β-Ketoadipate enol-lactone, which was isolated and physically characterized, is also an intermediate in the catechol pathway; the catechol and protocatechuate pathways converge at this point. β-Ketoadipate enol-lactone is hydrolyzed to β-ketoadipate by an enzyme which is essential for utilization of either catechol or protocatechuate. Studies with Moraxella lwoffii showed that this organism also degrades protocatechuate and catechol by the pathways characteristic of P. putida. Elucidation of the bacterial pathway for the dissimilation of protocatechuate revealed that the three step-reactions responsible for the conversion of this compound to β-ketoadipate enol-lactone are analogous with the step-reactions responsible for the conversion of catechol to β-ketoadipate enol-lactone.

The Conversion of Catechol and Protocatechuate to β-Ketoadipate by Pseudomonas putida: II. ENZYMES OF THE PROTOCATECHUATE PATHWAY

Extracts of benzoate-grown Pseudomonas putida have high levels of β-carboxy-cis,cis-muconate-lactonizing enzyme and γ-carboxymuconolactone decarboxylase, even though these enzymes are uniquely associated with the protocate-chuate pathway. Since these activities might result from nonspecific catalysis by the enzymes which catalyze the analogous reactions in the catechol pathway, cis,cis-muconate-lactonizing enzyme and muconolactone isomerase were purified extensively and their specificities were examined. Crystalline preparations had no detectable activity on the carboxylated substrate analogues of the parallel convergent pathway. The enzymes of the catechol pathway do, however, share some physical characteristics with the enzymes that catalyze the analogous reactions in the protocatechuate pathway.

The Conversion of Catechol and Protocatechuate to β-Ketoadipate by Pseudomonas putida: III. ENZYMES OF THE CATECHOL PATHWAY

The Conversion of Catechol and Protocatechuate to β-Ketoadipate by Pseudomonas putida: III. ENZYMES OF THE CATECHOL PATHWAY

A New Metabolic Pathway of Catechol

A New Metabolic Pathway of Catechol