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1.
The biochemical characterization of the muconate and the chloromuconate cycloisomerases of the chlorophenol-utilizing Rhodococcus erythropolis strain 1CP previously indicated that efficient chloromuconate conversion among the gram-positive bacteria might have evolved independently of that among gram-negative bacteria. Based on sequences of the N terminus and of tryptic peptides of the muconate cycloisomerase, a fragment of the corresponding gene has now been amplified and used as a probe for the cloning of catechol catabolic genes from R. erythropolis. The clone thus obtained expressed catechol 1,2-dioxygenase, muconate cycloisomerase, and muconolactone isomerase activities. Sequencing of the insert on the recombinant plasmid pRER1 revealed that the genes are transcribed in the order catA catB catC. Open reading frames downstream of catC may have a function in carbohydrate metabolism. The predicted protein sequence of the catechol 1,2-dioxygenase was identical to the one from Arthrobacter sp. strain mA3 in 59% of the positions. The chlorocatechol 1,2-dioxygenases and the chloromuconate cycloisomerases of gram-negative bacteria appear to be more closely related to the catechol 1,2-dioxygenases and muconate cycloisomerases of the gram-positive strains than to the corresponding enzymes of gram-negative bacteria.  相似文献   

2.
3.
Pseudomonas sp. strain P51 is able to use 1,2-dichlorobenzene, 1,4-dichlorobenzene, and 1,2,4-trichlorobenzene as sole carbon and energy sources. Two gene clusters involved in the degradation of these compounds were identified on a catabolic plasmid, pP51, with a size of 110 kb by using hybridization. They were further characterized by cloning in Escherichia coli, Pseudomonas putida KT2442, and Alcaligenes eutrophus JMP222. Expression studies in these organisms showed that the upper-pathway genes (tcbA and tcbB) code for the conversion of 1,2-dichlorobenzene and 1,2,4-trichlorobenzene to 3,4-dichlorocatechol and 3,4,6-trichlorocatechol, respectively, by means of a dioxygenase system and a dehydrogenase. The lower-pathway genes have the order tcbC-tcbD-tcbE and encode a catechol 1,2-dioxygenase II, a cycloisomerase II, and a hydrolase II, respectively. The combined action of these enzymes degrades 3,4-dichlorocatechol and 3,4,6-trichlorocatechol to a chloromaleylacetic acid. The release of one chlorine atom from 3,4-dichlorocatechol takes place during lactonization of 2,3-dichloromuconic acid.  相似文献   

4.
Degradation of para-toluate by Rhodococcus opacus 1cp was investigated. Activities of the key enzymes of this process, catechol 1,2-dioxygenase and muconate cycloisomerase, are detected in this microorganism. Growth on p-toluate was accompanied by induction of two catechol 1,2-dioxygenases. The substrate specificity and physicochemical properties of one enzyme are identical to those of chlorocatechol 1,2-dioxygenase; induction of the latter enzyme was observed during R. opacus 1cp growth on 4-chlorophenol. The other enzyme isolated from the biomass grown on p-toluate exhibited lower rate of chlorinated substrate cleavage compared to the catechol substrate. However, this enzyme is not identical to the catechol 1,2-dioxygenase cloned in this strain within the benzoate catabolism operon. This supports the hypothesis on the existence of multiple forms of dioxygenases as adaptive reactions of microorganisms in response to environmental stress.  相似文献   

5.
6.
During cultivation in a liquid medium, the bacterium Rhodococcus opacus 1G was capable of growing on phenol at a concentration of up to 0.75 g/l. Immobilization of Rhodococcus opacus 1G had a positive effect on cell growth in the presence of phenol at high concentrations. The substrate at concentrations of 1.0 and 1.5 g/l was completely utilized over 24 and 48 h, respectively. The key enzymes of phenol degradation (two catechol 1,2-dioxygenases and muconate cycloisomerase) were isolated. One of the dioxygenases was very unstable. By substrate specificity, another enzyme belonged to catechol 1,2-dioxygenases of the classical ortho-pathway. Chlorocatechols and chlorophenols served as competitive inhibitors of catechol 1,2-dioxygenases. The inhibitory effect of other aromatic compounds was less significant. Our results suggest that this strain holds promise for bioremediation of phenol wastewater.  相似文献   

7.
Cell extracts were prepared from Trichosporon cutaneum grown with phenol or p-cresol, and activities were assayed for enzymes catalyzing conversion of these two carbon sources into 3-ketoadipate (beta-ketoadipate) and 3-keto-4-methyladipate, respectively. When activities of each enzyme were expressed as a ratio, the rate for methyl-substituted substrate being divided by that for the unsubstituted substrate, it was apparent that p-cresol-grown cells elaborated pairs of enzymes for hydroxylation, dioxygenation, and delactonization. One enzyme of each pair was more active against its methyl-substituted substrate, and the other was more active against its unsubstituted substrate. Column chromatography was used to separate two hydroxylase activities and also 1,2-dioxygenase activities; the catechol 1,2-dioxygenases were further purified to electrophoretic homogeneity. Extracts of phenol-grown cells contained only those enzymes in this group that were more active against unsubstituted substrates. In contrast, whether cells were grown with phenol or p-cresol, only one muconate cycloisomerase (lactonizing enzyme) was elaborated which was more active against 3-methyl-cis,cis-muconate than against cis,cis-muconate; in this respect it differed from a cycloisomerase of another strain of T. cutaneum which has been characterized. The cycloisomerase was purified from both phenol-grown and p-cresol-grown cells, and some characteristics were determined.  相似文献   

8.
Abstract A Pseudomonas sp. strain JWS was isolated from an enrichment culture with 3,5-dichlorosalicylate as the sole source of carbon and energy. Additionally, 3-chloro-, 5-chloro-, and 3,5-dibromosalicylate, but not 4-chlorosalicylate were mineralized by the organism. During growth on the chlorosalicylates, stoichiometric amounts of chloride were released into the culture medium. In the presence of both salicylate and 3,5-dichlorosalicylate, high activities were induced for the turnover of non-halogenated as well as halogenated salicylates. Enzyme activities assayed in crude cell extracts which are responsible for the oxidation of catechol and its halogenated derivatives as well as those for cycloisomerization of cis,cis -muconate and its 2,4-dichloro derivative provided indications for the involvement of inducible type II catechol 1,2-dioxygenase and muconate cycloisomerase in biodegradation of halogenated salicylates.  相似文献   

9.
Induction of modified ortho-pathway enzymes (catechol 1.2-dioxygenase II, muconate cycloisomerase II, dienelactone hydrolase, and maleylacetate reductase) was found in Pseudomonas putida 87, when 3-chlorobenzoic acid was used as a sole carbon and energy source. Catechol 1.2-dioxygenase II, the key chlorocatechol cleaving enzyme, was purified and characterized. The enzyme molecular mass as determined by gel filtration was 65,000 Da; the minimum molecular mass upon SDS electrophoresis was 33,000 Da. The pH and temperature optima for the enzyme were 7.2-7.8 and 35 degrees C, respectively. The highest stability of catechol 1.2-dioxygenase II upon storage was observed in 50 mM Tris-HCl buffer pH 7.8 at 4 degrees C. The relative values of Vmax for catechol 1.2-dioxygenase II with 3-chloro-, 4-chloro-, and 3.5-dichlorocatechols were 28%, 50%, and 41% of those for catechol. The enzyme affinity for chlorocatechols was 3-9 times higher than for methylcatechols and 10-20 times higher than for unsubstituted catechol.  相似文献   

10.
A pure bacterial culture was isolated by its ability to utilize 3-nitrotoluene (3NT) as the sole source of carbon, nitrogen, and energy for growth. Analysis of its 16S rRNA gene showed that the organism (strain ZWL3NT) belongs to the genus Rhodococcus. A rapid disappearance of 3NT with concomitant release of nitrite was observed when strain ZWL3NT was grown on 3NT. The isolate also grew on 2-nitrotoluene, 3-methylcatechol and catechol. Two metabolites, 3-methylcatechol and 2-methyl-cis,cis-muconate, in the reaction mixture were detected after incubation of cells of strain ZWL3NT with 3NT. Enzyme assays showed the presence of both catechol 1,2-dioxygenase and catechol 2,3-dioxygenase in strain ZWL3NT. In addition, a catechol degradation gene cluster (catRABC cluster) for catechol ortho-cleavage pathway was cloned from this strain and cell extracts of Escherichia coli expressing CatA and CatB exhibited catechol 1,2-dioxygenase activity and cis,cis-muconate cycloisomerase activity, respectively. These experimental evidences suggest a novel pathway for 3NT degradation with 3-methylcatechol as a key metabolite by Rhodococcus sp. strain ZWL3NT.  相似文献   

11.
Streptomyces setonii (ATCC 39116) is a thermophilic soil actinomycete capable of degrading single aromatic compounds including phenol and benzoate via the ortho-cleavage pathway. Previously, a 6.3-kb S. setonii DNA fragment containing a thermophilic catechol 1,2-dioxygenase (C12O) gene was isolated and functionally overexpressed in Escherichia coli (An et al., FEMS Microbiol. Lett. 195 (2001) 17-22). Here the 6.3-kb S. setonii DNA fragment was shown to be organized into two putative divergently transcribed gene clusters with six complete and one incomplete open reading frames (ORFs). The first cluster with three ORFs showed homologies to previously known benA, benB, and benC, implying it is a part of the benzoate catabolic operon. The second cluster revealed an ortho-cleavage catechol catabolic operon with three translationally coupled ORFs (in order): catR, a putative LysR-type regulatory gene; catB, a muconate cycloisomerase gene; catA, a C12O gene. Each of these individually cloned ORFs was expressed in E. coli and identified as a distinct protein. The expression of the cloned S. setonii catechol operon was induced in Streptomyces lividans by specific single aromatic compounds including catechol, phenol, and 4-chlorophenol. A similar induction pattern was also observed using a luciferase gene-fused reporter system.  相似文献   

12.
13.
Pseudomonas sp. strain MT1 is capable of degrading 4- and 5-chlorosalicylates via 4-chlorocatechol, 3-chloromuconate, and maleylacetate by a novel pathway. 3-Chloromuconate is transformed by muconate cycloisomerase of MT1 into protoanemonin, a dominant reaction product, as previously shown for other muconate cycloisomerases. However, kinetic data indicate that the muconate cycloisomerase of MT1 is specialized for 3-chloromuconate conversion and is not able to form cis-dienelactone. Protoanemonin is obviously a dead-end product of the pathway. A trans-dienelactone hydrolase (trans-DLH) was induced during growth on chlorosalicylates. Even though the purified enzyme did not act on either 3-chloromuconate or protoanemonin, the presence of muconate cylcoisomerase and trans-DLH together resulted in considerably lower protoanemonin concentrations but larger amounts of maleylacetate formed from 3-chloromuconate than the presence of muconate cycloisomerase alone resulted in. As trans-DLH also acts on 4-fluoromuconolactone, forming maleylacetate, we suggest that this enzyme acts on 4-chloromuconolactone as an intermediate in the muconate cycloisomerase-catalyzed transformation of 3-chloromuconate, thus preventing protoanemonin formation and favoring maleylacetate formation. The maleylacetate formed in this way is reduced by maleylacetate reductase. Chlorosalicylate degradation in MT1 thus occurs by a new pathway consisting of a patchwork of reactions catalyzed by enzymes from the 3-oxoadipate pathway (catechol 1,2-dioxygenase, muconate cycloisomerase) and the chlorocatechol pathway (maleylacetate reductase) and a trans-DLH.  相似文献   

14.
The growth of Rhodococcus opacus 1CP in medium with different concentrations of p-cresol as the sole source of carbon and energy was studied. It was shown that the optimal concentration of p-cresol was 600 mg/L. The ability of this strain to transform practically all amounts of p-cresol to 4-methylcatechol followed by its utilization through ortho-pathway was shown. New enzymes (4-methylcatechol 1,2-dioxygenase, catechol 1,2-dioxygenase, and methylmuconate cycloisomerase) were purified to homogeneity and characterized. Based on the data obtained on p-cresol degradation, formation of intermediates, and the enzymes participating in this pathway, we suggest an ortho-pathway of p-cresol degradation by R. opacus 1CP through 4-methylcatechol and 3-methyl-cis, cis-muconate.  相似文献   

15.
Pseudomonas sp. strain MT1 has recently been reported to degrade 4- and 5-chlorosalicylate by a pathway assumed to consist of a patchwork of reactions comprising enzymes of the 3-oxoadipate pathway. Genes encoding the initial steps in the degradation of salicylate and substituted derivatives were now localized and sequenced. One of the gene clusters characterized (sal) showed a novel gene arrangement, with salA, encoding a salicylate 1-hydroxylase, being clustered with salCD genes, encoding muconate cycloisomerase and catechol 1,2-dioxygenase, respectively, and was expressed during growth on salicylate and chlorosalicylate. A second gene cluster (cat), exhibiting the typical catRBCA arrangement of genes of the catechol branch of the 3-oxoadipate pathway in Pseudomonas strains, was expressed during growth on salicylate. Despite their high sequence similarities with isoenzymes encoded by the cat gene cluster, the catechol 1,2-dioxygenase and muconate cycloisomerase encoded by the sal cluster showed unusual kinetic properties. Enzymes were adapted for turnover of 4-chlorocatechol and 3-chloromuconate; however, 4-methylcatechol and 3-methylmuconate were identified as the preferred substrates. Investigation of the substrate spectrum identified 4- and 5-methylsalicylate as growth substrates, which were effectively converted by enzymes of the sal cluster into 4-methylmuconolactone, followed by isomerization to 3-methylmuconolactone. The function of the sal gene cluster is therefore to channel both chlorosubstituted and methylsubstituted salicylates into a catechol ortho cleavage pathway, followed by dismantling of the formed substituted muconolactones through specific pathways.  相似文献   

16.
M Shoda  S Udaka 《Applied microbiology》1980,39(6):1129-1133
A phenol-utilizing yeast, Trichosporon cutaneum POB 14, which has a partially constitutive activity of catechol 1,2-oxygenase, utilized phenol in preference to glucose in a medium containing both phenol (200 mg/liter) and glucose (0.15%) as carbon sources. The glucose consumption was not observed until the concentration of phenol decreased to around 10 mg/liter. This phenomenon was confirmed by [U-14C]glucose uptake experiments. The intracellular activities of hexokinase (EC 2.7.1.1) and catechol 1,2-oxygenase (EC 1.13.1.1) changed inversely when phenol was added during growth in the glucose medium.  相似文献   

17.
3-Carboxy-cis,cis-muconate lactonizing enzyme (CMLE; EC 5.5.1.5) from Neurospora crassa catalyzes the reversible gamma-lactonization of 3-carboxy-cis,cis-muconate by a syn-1,2 addition-elimination reaction. The stereochemical and regiochemical course of the reaction is (i) opposite that of CMLE from Pseudomonas putida (EC 5.5.1.2) and (ii) identical to that of cis,cis-muconate lactonizing enzyme (MLE; EC 5.5.1.1) from P. putida. In order to determine the mechanistic and evolutionary relationships between N. crassa CMLE and the procaryotic cycloisomerases, we have purified CMLE from N. crassa to homogeneity and determined its nucleotide sequence from a cDNA clone isolated from a p-hydroxybenzoate-induced N. crassa cDNA library. The deduced amino acid sequence predicts a protein of 41.2 kDa (365 residues) which does not exhibit sequence similarity with any of the bacterial cycloisomerases. The cDNA encoding N. crassa CMLE was expressed in Escherichia coli, and the purified recombinant protein exhibits physical and kinetic properties equivalent to those found for the isolated N. crassa enzyme. We also report that N. crassa CMLE possesses substantially reduced yet significant levels of MLE activity with cis,cis-muconate and, furthermore, does not appear to be dependent on divalent metals for activity. These data suggest that the N. crassa CMLE may represent a novel eucaryotic motif in the cycloisomerase enzyme family.  相似文献   

18.
Catechol 1,2-dioxygenase [catechol: oxygen 1,2-oxidoreductase (decyclizing); EC 1.13.11.1], the aromatic intradiol ring-cleaving enzyme of Nocardia sp. NCIB 10503 prepared by freeze-drying cell-free extracts, was covalently attached to cyanogen bromide-activated Agarose. The properties of the immobilized enzyme were compared to those of the free enzyme preparation. Immobilization was shown to increase the thermal stability of the enzyme. The pH-activity profile was altered by immobilization. Various explanations for this phenomenon are discussed. The Vmax and Km of the enzyme were not significantly affected on immobilization. The enzyme had a broader substrate specificity than any previously reported catechol 1,2-dioxygenase, and this was largely unaltered by immobilization. The properties of the preparations are compared to those of other (free) catechol 1,2-dioxygenases. The results presented show that the immobilization of catechol 1,2-dioxygenase offers an attractive means for the production of cis,cis-muconate and novel substituted analogues.  相似文献   

19.
Enzymatic conversion of 4-fluorocatechol in the simultaneous presence of partially purified preparations of catechol 1,2-dioxygenase from Pseudomonas cepacia and muconate cycloisomerase from Alcaligenes eutrophus 335 yielded a product that was unambiguously identified as (+)-4-fluoromuconolactone [(+)-4-carboxymethyl-4-fluoro-but-2-en-4-olide]. This compound was shown to be the only major product formed from 3-fluoro-cis,cis-muconate by the action of muconate cycloisomerases from A. eutrophus 335, A. eutrophus JMP134, and P. cepacia as well as by the action of dichloromuconate cycloisomerase from A. eutrophus JMP134. This finding implies that dichloromuconate cycloisomerase, like the muconate cycloisomerases, catalyzes primarily a cycloisomerization reaction, which only in the case of chloro- and bromo-substituted substrates is connected to a dehalogenation. 4-Fluoromuconolactone at pH 7 decomposes by spontaneous reactions mainly to maleylacetate, which then decarboxylates to give cis-acetylacrylate. Although significant amounts of an unidentified compound are also formed from the fluorolactone, HF elimination to the two isomeric dienelactones (4-carboxymethylenebut-2-en-4-olides) is negligible. However, all spontaneous reactions proceed so slowly that an enzymatic conversion of 4-fluoromuconolactone must be assumed. Participation of dienelactone hydrolases in this reaction is indicated by their induction during growth of various strains with 4-fluorobenzoate. However, experiments with cell extracts of P. putida A3.12 suggest that at least one other hydrolytic enzyme is able to contribute to 4-fluoromuconolactone conversion. In light of these observations, earlier proposals for a 4-fluorobenzoate degradative pathway are discussed.  相似文献   

20.
When Frateuria species ANA-18 was grown on aniline, two catechol 1,2-dioxygenases (CD I and CD II, EC 1.13.11.1) were found in a cell-free extract of the strain. CD I and CD II were separated from each other by DE-52 chromatography and purified to homogeneity by successive column chromatography. The molecular weights of CD I and CD II were 38,000 and 36,000, respectively, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. CD I contained 2 mol of sulfhydryl groups per mol of protein, but CD II did not have cysteine or cystine residues. CD I was more stable on the acidic side and against heating than CD II. The activity of CD I was inhibited by AgN03, HgCl2, iodoacetic acid and p-chloromercuribenzoic acid, but that of CD II was not inhibited or was less affected by them because of the lack of sulfhydryl groups. CD I exhibited a high activity for the extradiol cleavage of 3-methylcatechol, and the ratio of intradiol to extradiol activity was 100 :120.  相似文献   

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