Purification and Characterization of Catechol 1,2-Dioxygenase from <Emphasis Type="Italic">Acinetobacter</Emphasis> sp. DS002 and Cloning,Sequencing of Partial <Emphasis Type="Italic">catA</Emphasis> Gene

Catechol 1,2-dioxygenase (C12O) was purified to electrophoretic homogeneity from Acinetobacter sp. DS002. The pure enzyme appears to be a homodimer with a molecular mass of 66 kDa. The apparent K_m and V_max for intradiol cleavage of catechol were 1.58 μM and 2 units per mg of protein respectively. Unlike other C12Os reported in the literature, the catechol 1,2-dioxygenase of Acinetobacter showed neither intradiol nor extradiol cleavage activity when substituted catechols were used as substrates. However, it has shown mild intradiol cleavage activity when benzenetriol was used as substrate. As determined by two dimensional electrophoresis (2DE) followed MALDI-TOF/TOF analyses and gel permeation chromatography, no isoforms of C12O was observed in Acinetobacter sp. DS002. Further, the C12O was seen only in cultures grown in benzoate and it was completely absent in succinate grown cultures. Based on the sequence information obtained from MS/MS data, degenerate primers were designed to amplify catA gene from the genomic DNA of Acinetobacter sp. DS002. The sequence of the PCR amplicon and deduced amino acid sequence showed 97% similarity with a catA gene of Acinetobacter baumannii AYE (YP_001713609).     

Extradiol Cleavage of 3-Methylcatechol by Catechol 1,2-Dioxygenase from Various Microorganisms

The isofunctional enzymes of catechol 1,2-dioxygenase from species of Acinetobacter, Pseudomonas, Nocardia, Alcaligenes, and Corynebacterium oxidize 3-methylcatechol according to both the intradiol and extradiol cleavage patterns. However, the enzyme preparations from Brevibacterium and Arthrobacter have only the intradiol cleavage activity. Comparison of substrate specificity among these isofunctional dioxygenases shows striking differences in the oxidation of 3-methylcatechol, 4-methylcatechol and pyrogallol.     

Purification of hydroxyquinol 1,2-dioxygenase and maleylacetate reductase: the lower pathway of 2,4,5-trichlorophenoxyacetic acid metabolism by Burkholderia cepacia AC1100.

The enzyme hydroxyquinol 1,2-dioxygenase, which catalyzes ortho cleavage of hydroxyquinol (1,2,4-trihydroxybenzene) to produce maleylacetate, was purified from Escherichia coli cells containing the tftH gene from Burkholderia cepacia AC1100. Reduction of the double bond in maleylacetate is catalyzed by the enzyme maleylacetate reductase, which was also purified from E. coli cells, these cells containing the tftE gene from B. cepacia AC1100. The two enzymes together catalyzed the conversion of hydroxyquinol to 3-oxoadipate. The purified hydroxyquinol 1,2-dioxygenase was specific for hydroxyquinol and was not able to use catechol, tetrahydroxybenzene, 6-chlorohydroxyquinol, or 5-chlorohydroxyquinol as its substrate. The native molecular mass of hydroxyquinol 1,2-dioxygenase was 68 kDa, and the subunit size of the protein was 36 kDa, suggesting a dimeric protein of identical subunits.     

Bacterial aromatic ring-cleavage enzymes are classified into two different gene families

Dioxygenases that catalyze the cleavage of the aromatic ring are classified into two groups according to their mode of ring fission. Substrates of ring-cleavage dioxygenases usually contain hydroxyl groups on adjacent aromatic carbons, and intradiol enzymes cleave the ring between these two hydroxyl groups. Extradiol enzymes in contrast cleave the ring between one hydroxylated carbon and its adjacent nonhydroxylated carbon. In this study, we determined the complete nucleotide sequence of nahC, the structural gene for 1,2-dihydroxynaphthalene dioxygenase encoded in the NAH7 plasmid of Pseudomonas putida. This enzyme is an extradiol ring-cleavage enzyme that cleaves the first ring of 1,2-dihydroxynaphthalene. The amino acid sequence of the dioxygenase deduced from the DNA sequence demonstrated that the molecular weight of the enzyme is 33,882. This result was in agreement with those of maxicell analyses that showed that the nahC product was a 36-kDa protein. Interestingly, the amino acid sequence of 1,2-dihydroxynaphthalene dioxygenase was 50% homologous with that of 2,3-dihydroxybiphenyl dioxygenase, which catalyzes extradiol cleavage of the first ring of 2,3-dihydroxybiphenyl (Furukawa, K., Arimura, N., and Miyazaki, T. (1987) J. Bacteriol. 169, 427-429). The amino acid sequence similarity of 1,2-dihydroxynaphthalene dioxygenase with catechol 2,3-dioxygenase, which is an authentic extradiol dioxygenase, was rather low (16%). However, a statistical analysis by the method of S. B. Needleman and C. D. Wunsch [1970) J. Mol. Biol. 48, 443-453) clearly showed that these two dioxygenases are evolutionarily related. Therefore, these extradiol enzymes are considered as products of the same gene superfamily. From the significant sequence similarity between intradiol enzymes, it has been shown (Neidle, E. L., Harnett, C., Bonitz, S., and Ornston, L. N. (1988) J. Bacteriol. 170, 4874-4880) that intradiol enzymes evolved from a common ancestor. Comparison of the amino acid sequence of extradiol enzymes with those of intradiol dioxygenases did not show any significant global or localized similarity.     

An archetypical extradiol-cleaving catecholic dioxygenase: the crystal structure of catechol 2,3-dioxygenase (metapyrocatechase) from Ppseudomonas putida mt-2.

BACKGROUND: Catechol dioxygenases catalyze the ring cleavage of catechol and its derivatives in either an intradiol or extradiol manner. These enzymes have a key role in the degradation of aromatic molecules in the environment by soil bacteria. Catechol 2, 3-dioxygenase catalyzes the incorporation of dioxygen into catechol and the extradiol ring cleavage to form 2-hydroxymuconate semialdehyde. Catechol 2,3-dioxygenase (metapyrocatechase, MPC) from Pseudomonas putida mt-2 was the first extradiol dioxygenase to be obtained in a pure form and has been studied extensively. The lack of an MPC structure has hampered the understanding of the general mechanism of extradiol dioxygenases. RESULTS: The three-dimensional structure of MPC has been determined at 2.8 A resolution by the multiple isomorphous replacement method. The enzyme is a homotetramer with each subunit folded into two similar domains. The structure of the MPC subunit resembles that of 2,3-dihydroxybiphenyl 1,2-dioxygenase, although there is low amino acid sequence identity between these enzymes. The active-site structure reveals a distorted tetrahedral Fe(II) site with three endogenous ligands (His153, His214 and Glu265), and an additional molecule that is most probably acetone. CONCLUSIONS: The present structure of MPC, combined with those of two 2,3-dihydroxybiphenyl 1,2-dioxygenases, reveals a conserved core region of the active site comprising three Fe(II) ligands (His153, His214 and Glu265), one tyrosine (Tyr255) and two histidine (His199 and His246) residues. The results suggest that extradiol dioxygenases employ a common mechanism to recognize the catechol ring moiety of various substrates and to activate dioxygen. One of the conserved histidine residues (His199) seems to have important roles in the catalytic cycle.     

Purification and Characterization of Hydroxyquinol 1,2-Dioxygenase from Azotobacter sp. Strain GP1

Hydroxyquinol 1,2-dioxygenase was purified from cells of the soil bacterium Azotobacter sp. strain GP1 grown with 2,4,6-trichlorophenol as the sole source of carbon. The presumable function of this dioxygenase enzyme in the degradative pathway of 2,4,6-trichlorophenol is discussed. The enzyme was highly specific for 6-chlorohydroxyquinol (6-chloro-1,2,4-trihydroxybenzene) and hydroxyquinol (1,2,4-trihydroxybenzene) and was found to perform ortho cleavage of the hydroxyquinol compounds, yielding chloromaleylacetate and maleylacetate, respectively. With the conversion of 1 mol of 6-chlorohydroxyquinol, the consumption of 1 mol of O(inf2) and the formation of 1 mol of chloromaleylacetate were observed. Catechol was not accepted as a substrate. The enzyme has to be induced, and no activity was found in cells grown on succinate. The molecular weight of native hydroxyquinol 1,2-dioxygenase was estimated to 58,000, with a sedimentation coefficient of 4.32. The subunit molecular weight of 34,250 indicates a dimeric structure of the dioxygenase enzyme. The addition of Fe(sup2+) ions significantly activated enzyme activity, and metal-chelating agents inhibited it. Electron paramagnetic resonance data are consistent with high-spin iron(III) in a rhombic environment. The NH(inf2)-terminal amino acid sequence was determined for up to 40 amino acid residues and compared with sequences from literature data for other catechol and chlorocatechol dioxygenases.     

Extradiol cleavage of 3-substituted catechols by an intradiol dioxygenase, pyrocatechase, from a Pseudomonad.

Pyrocatechase (catechol 1,2-oxidoreductase (decyclizing), EC 1.13.11.1), a ferric ion-containing dioxygenase from Pseudomonas arvilla C-1, catalyzes the intradiol cleavage of catechol with insertion of 2 atoms of molecular oxygen to form cis,cis-muconic acid. The enzyme also catalyzed the oxidation of various catechol derivatives, including 4-methylcatechol, 4-chlorocatechol, 4-formylcatechol (protocatechualdehyde), 4,5-dichlorocatechol, 3,5-dichlorocatechol, 3-methylcatechol, 3-methoxycatechol, and 3-hydroxycatechol (pyrogallol). All of these substrates gave products having an absorption maximum at around 260 nm, which is characteristic of cis,cis-muconic acid derivatives. However, when 3-methylcatechol was used as substrate, the product formed showed two absorption maxima at 390 and 260 nm. These two absorption maxima were found to be attributable to two different products, 2-hydroxy-6-oxo-2,4-heptadienoic acid and 5-carboxy-2-methyl-2,4-pentadienoic acid (2-methylmuconic acid). The former was produced by the extradiol cleavage between the carbon atom carrying the hydroxyl group and the carbon atom carrying the hydroxyl group and the carbon atom carrying the methyl group; the latter by an intradiol cleavage between two hydroxyl groups. Since these products were unstable, they were converted to and identified as 6-methylpyridine-2-carboxylic acid and 2-methylmuconic acid dimethylester, respectively. Similarly, 3-methoxycatechol gave two products, namely, 2-hydroxy-5-methoxycarbonyl-2,4-pentadienoic acid and 5-carboxy-2-methoxy-2,4-pentadienoic acid (2-methoxymuconic acid). With 3-methylcatechol as substrate, the ratio of intradiol and extradiol cleavage activities of Pseudomonas pyrocatechase during purification was almost constant and was about 17. The final preparation of the enzyme was homogeneous when examined by disc gel electrophoresis and catalyzed both reactions simultaneously with the same ratio as during purification. All attempts to resolve the enzyme into two components with separate activities, including inactivation of the enzyme with urea or heat, treatment with sulfhydryl-blocking reagents or chelating agents, and inhibition of the enzyme with various inhibitors, proved unsuccessful. These results strongly suggest that Pseudomonas pyrocatechase is a single enzyme, which catalyzes simultaneously both intradiol and extradiol cleavages of some 3-substituted catechols.     

Catechol 1,2-dioxygenase from the new aromatic compounds - Degrading Pseudomonas putida strain N6

This study aimed to characterization of catechol 1,2-dioxygenase from a Gram-negative bacterium, being able to utilize a wide spectrum of aromatic substrates as a sole carbon and energy source. Strain designated as N6, was isolated from the activated sludge samples of a sewage treatment plant at Bentwood Furniture Factory Jasienica, Poland. Morphology, physio-biochemical characteristics and phylogenetic analysis based on 16S rDNA sequence indicate that strain belongs to Pseudomonas putida. When cells of strain N6 grown on protocatechuate or 4-hydroxybenzoic acid mainly protocatechuate 3,4-dioxygenase was induced. The activity of catechol 1,2-dioxygenase was rather small. The cells grown on benzoic acid, catechol or phenol showed high activity of only catechol 1,2-dioxygenase. This enzyme was optimally active at 35 °C and pH 7.4. Kinetic studies showed that the value of K_m and V_max was 85.19 ??M and 14.54 ??M min⁻¹ respectively. Nucleotide sequence of gene encoding catechol 1,2-dioxygenase in strain N6 has 100% identity with catA genes from two P. putida strains. The deduced 301-residue sequence of enzyme corresponds to a protein of molecular mass 33.1 kDa. The deduced molecular structure of the catechol 1,2-dioxygenase from P. putida N6 was very similar and characteristic for the other intradiol dioxygenases.     

A comparison of biodegradation of phenol and homologous compounds by Pseudomonas vesicularis and Staphylococcus sciuri strains

Pseudomonas vesicularis and Staphylococcus sciuri were isolated as dominant strains from phenol-acclimated activated sludge. P. vesicularis was an efficient degrader of phenol, catechol, p-cresol, sodium benzoate and sodium salicylate in a single substrate system. Under similar conditions S. sciuri degraded only phenol and catechol from among aromatic compounds that were tested. Cell-free extracts of P. vesicularis grown on phenol (376 mg l(-1)), sodium benzoate (576 mg l(-1)) and sodium salicylate (640 mg l(-1)) showed catechol 2,3-dioxygenase activity initiating an extradiol (meta) splitting pathway. The degradative intradiol (ortho) pathway as a result of catechol 1,2-dioxygenase synthesis was induced in P. vesicularis cells grown on catechol (440 mg l(-1)) orp-cresol (432 mg l(-1)). Catechol 1,2-dioxygenase and the ortho-cleavage has been also reported in S. sciuri cells capable of degrading phenol (376 mg l(-1)) or catechol (440 mg l(-1)). In cell-free extracts of S. sciuri no meta-cleavage enzyme activity was detected. These results demonstrated that gram-positive S. sciuri strain was able to effectively metabolize some phenols as do many bacteria of the genus Pseudomonas but have a different capacity for degrading of these compounds.     

Extradiol cleavage of o-aminophenol by pyrocatechase

o-Aminophenol is cleaved by the intradiol dioxygenase, pyrocatechase, in an extradiol manner to give picolinic acid as the major product. Inhibition of o-aminophenol cleavage with various reagents was comparable to that observed for catechol cleavage, indicating that both reactions are catalyzed by the same enzyme. Though other substrate analogues have been shown to yield some extradiol cleavage products, this is the first case wherein >95% of the products characterized derived solely from the extradiol cleavage of the ring.     

Catechol dioxygenases from Escherichia coli (MhpB) and Alcaligenes eutrophus (MpcI): sequence analysis and biochemical properties of a third family of extradiol dioxygenases.

The nucleotide sequence of the Escherichia coli mhpB gene, encoding 2,3-dihydroxyphenylpropionate 1,2-dioxygenase, was determined by sequencing of a 3.1-kb fragment of DNA from Kohara phage 139. The inferred amino acid sequence showed 58% sequence identity with the sequence of an extradiol dioxygenase, MpcI, from Alcaligenes eutrophus and 10 to 20% sequence identity with protocatechuate 4,5-dioxygenase from Pseudomonas paucimobilis, with 3,4-dihydroxyphenylacetate 2,3-dioxygenase from E. coli, and with human 3-hydroxyanthranilate dioxygenase. Sequence similarity between the N- and C-terminal halves of this new family of dioxygenases was detected, with conserved histidine residues in the N-terminal domain. A model is proposed to account for the relationship between this family of enzymes and other extradiol dioxygenases. The A. eutrophus MpcI enzyme was expressed in E. coli, purified, and characterized as a protein with a subunit size of 33.8 kDa. Purified MhpB and MpcI showed similar substrate specificities for a range of 3-substituted catechols, and evidence for essential histidine and cysteine residues in both enzymes was obtained.     

Cleavage of pyrogallol by non-heme iron-containing dioxygenases

Both intradiol and proximal extradiol dioxygenases are thought to produce the same product, alpha-hydroxymuconic acid, when pyrogallol (3-hydroxycatechol) is used as a substrate. However, when these enzymes were reacted with pyrogallol, they gave different products. A proximal extradiol dioxygenase, metapyrocatechase (catechol:oxygen 2,3-d-oxidoreductase (decyclizing), EC 1.13.11.2), gave a product having an absorption maximum at 290 nm, which was gradually converted to a more stable compound having an absorption maximum at 239 nm. On the other hand, an intradiol dioxygenase, protocatechuate 3,4-dioxygenase (protocatechuate:oxygen 3,4-oxidoreductase (decyclizing), EC 1.13.11.3), gave a product having an absorption maximum at 300 nm. Based on the spectral data and direct comparison with authentic samples, the primary products obtained by the action of the former and the latter enzymes were identified as alpha-hydroxymuconic acid and 2-pyrone-6-carboxylic acid, respectively. While another intradiol dioxygenase, pyrocatechase (catechol:oxygen 1,2-oxidoreductase (decyclizing), EC 1.13.11.1), gave a mixture of nearly equimolar amounts of these two compounds. Isotope labeling experiments indicated that 1 atom of oxygen was incorporated in 2-pyrone-6-carboxylic acid from the atmosphere. Based on these findings, the reaction mechanism for the formation of 2-pyrone-6-carboxylic acid is discussed. This may be the first experimental evidence indicating the presence of a seven-membered lactone intermediate during the oxygenative cleavage of catechols, proposed by Hamilton (Hamilton, G.A. (1974) in Molecular Mechanisms of Oxygen Activation (Hayaishi, O., ed) pp. 405-451, Academic Press, New York).     

Purification and characterization of 6-chlorohydroxyquinol 1,2-dioxygenase from Streptomyces rochei 303: comparison with an analogous enzyme from Azotobacter sp. strain GP1.

The enzyme which cleaves the benzene ring of 6-chlorohydroxyquinol was purified to apparent homogeneity from an extract of 2,4,6-trichlorophenol-grown cells of Streptomyces rochei 303. Like the analogous enzyme from Azotobacter sp. strain GP1, it exhibited a highly restricted substrate specificity and was able to cleave only 6-chlorohydroxyquinol and hydroxyquinol and not catechol, chlorinated catechols, or pyrogallol. No extradiol-cleaving activity was observed. In contrast to 6-chlorohydroxyquinol 1,2-dioxygenase from Azotobacter sp. strain GP1, the S. rochei enzyme had a distinct preference for 6-chlorohydroxyquinol over hydroxyquinol (kcat/Km = 1.2 and 0.57 s-1.microM-1, respectively). The enzyme from S. rochei appears to be a dimer of two identical 31-kDa subunits. It is a colored protein and was found to contain 1 mol of iron per mol of enzyme. The NH2-terminal amino acid sequences of 6-chlorohydroxyquinol 1,2-dioxygenase from S. rochei 303 and from Azotobacter sp. strain GP1 showed a high degree of similarity.     

DNA sequence of the Acinetobacter calcoaceticus catechol 1,2-dioxygenase I structural gene catA: evidence for evolutionary divergence of intradiol dioxygenases by acquisition of DNA sequence repetitions.

The DNA sequence of a 1.6-kilobase-pair SalI-KpnI Acinetobacter calcoaceticus restriction fragment carrying catA, the structural gene for catechol 1,2-dioxygenase I, was determined. The 933-nucleotide gene encodes a protein product with a deduced molecular weight of 34,351. The similarly sized Pseudomonas clcA gene encodes catechol 1,2-dioxygenase II, an enzyme with relatively broad substrate specificity and relatively low catalytic efficiency. Comparison of the catA and clcA sequences demonstrated their common ancestry and suggested that acquisitions of direct and inverted sequence repetitions of 6 to 10 base pairs were frequent events in their evolutionary divergence. The catechol 1,2-dioxygenases proved to be evolutionarily homologous with the alpha and beta subunits of Pseudomonas protocatechuate 3,4-dioxygenase, and analysis of conserved residues in the intradiol dioxygenases revealed conserved histidyl and tyrosyl residues that are probably involved in the ligation of ferric ion in their active sites.     

Functional identification of the gene locus (ncg12319 and characterization of catechol 1,2-dioxygenase in Corynebacterium glutamicum

Corynebacterium glutamicum assimilated phenol, benzoate, 4-hydroxybenzoate p-cresol and 3,4-dihydroxybenzoate. Ring cleavage was by catechol 1,2-dioxygenase when phenol or benzoate was used and by protocatechuate 3,4-dioxygenase when the others were used as substrate. The locus ncg12319 of its genome was cloned and expressed in Escherichia coli. Enzyme assays showed that ncg12319 encodes a catechol 1,2-dioxygenase. This catechol 1,2-dioxygenase was purified and accepted catechol, 3-, or 4-methylcatechols, but not chlorinated catechols, as substrates. The optimal temperature and pH for catechol cleavage catalyzed by the enzyme were 30 degrees C and 9, respectively, and the Km and Vmax were determined to be 4.24 micromol l(-1) and 3.7 micromol l(-1) min(-1) mg(-1) protein, respectively.     

Cloning,expression, and characterization of catechol 1,2-dioxygenase from a phenol-degrading Candida tropicalis JH8 strain

The sequence cato encoding catechol 1,2-dioxygenase from Candida tropicalis JH8 was cloned, sequenced, and expressed in Escherichia coli. The sequence cato contained an ORF of 858?bp encoding a polypeptide of 285?amino acid residues. The recombinant catechol 1,2-dioxygenase exists as a homodimer structure with a subunit molecular mass of 32 KD. Recombinant catechol 1,2-dioxygenase was unstable below pH 5.0 and stable from pH 7.0 to 9.0; its optimum pH was at 7.5. The optimum temperature for the enzyme was 30°C, and it possessed a thermophilic activity within a broad temperature range. Under the optimal conditions with catechol as substrate, the K_m and V_max of recombinant catechol 1,2-dioxygenase were 9.2?µM and 0.987?µM/min, respectively. This is the first article presenting cloning and expressing in E. coli of catechol 1,2-dioxygenase from C. tropicalis and characterization of the recombinant catechol 1,2-dioxygenase.     

Properties of cyanogen bromide-activated, Agarose-immobilized catechol 1,2-dioxygenase from freeze-dried extracts of Nocardia sp. NCIB 10503

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 V_max and K_m 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.     

Reactions of 3-ethylcatechol and 3-(methylthio)catechol with catechol dioxygenases

The reactions of 3-ethylcatechol and 3-(methylthio)catechol with catechol 1,2-dioxygenase and catechol 2,3-dioxygenase from Pseudomonas putida were examined. Both 3-substituted catechols are oxidized by catechol 2,3-dioxygenase at approximately 30% of the rate observed for catechol oxidation by this enzyme. Analysis of the products of the reactions showed that ring cleavage occurs in a normal fashion between carbons 2 and 3 of the alternate substrates. 3-Ethylcatechol is oxidized by catechol 1,2-dioxygenase at about 6% of the rate of catechol oxidation; ring cleavage occurs between carbons 1 and 2 to give 2-ethyl-cis,cis-muconic acid. However, 3-(methylthio)catechol is a very poor substrate for catechol 1,2-dioxygenase (0.8% of the rate of catechol), but it is a potent competitive inhibitor (Ki = 0.6 microM). The effects of 3-(methylthio)catechol and 3-ethylcatechol on the visible and EPR spectra of catechol 1,2-dioxygenase are also reported.     

Molecular cloning of genenahH encoding extradiol-type dioxygenase from the NAH plasmid ofPseudomonas stutzeri NA1

Naphthalene-degradingPseudomonas stutzeri NA1 was found to harbour the NAH plasmid, which contains the classical upper and lower catabolic genes required for naphthalene mineralization. The lower pathway inP. stutzeri NA1 was found to proceedviameta-ring cleavage of catechol due to the presence of thenahH gene encoding extradiol catechol 2,3-dioxygenase. Naphthalene-induced cells were able to mineralise both salicylate and catechol. Absorption spectra and gas chromatography/mass spectrometry analysis ofritermediate metabolites of salicylate or catechol degradation by a crude extract ofP. stutzeri NA1 revealed the presence of themeta-ring cleavage product 2-hydroxymuconate semialdehyde as a major constituent. The extradiol ring cleavage genenahH was amplified successfully from the NAH plasmid ofP. stutzeri NA1 with catechol 2,3-dioxygenase-specific primers and cloned inEscherichia coli JM109 The complete nucleotide sequence of cloned PCR fragment was determined. Sequence analysis of cloned PCR fragment revealed an open reading frame with similarity to other extradiol dioxygenases. The deduced amino acid sequence ofnahH fromP. stutzeri NA1 showed 96% sequence identity with the catechol 2,3-dioxygenase gene fromPseudomonas putida strain H. However, when compared to othernahH genes from different pseudomonads, it was in a separate phylogenetic branch, indicating a degree of speciation among the extradiol dioxygenase family.     

Catechol 1,2-dioxygenase from α-naphthol degrading thermophilic Geobacillus sp. strain: purification and properties

The purpose of this study was purification and characterization of catechol 1,2-dioxygenase from Geobacillus sp. G27 strain, which degrades α-naphthol by the β-ketoadipate pathway. The catechol 1,2-dioxygenase (C1,2O) was purified using four steps of ammonium sulfate precipitation, DEAE-celullose, Sephadex G-150 and hydroxylapatite chromatographies. The enzyme was purified about 18-fold with a specific activity of 7.42 U mg of protein⁻¹. The relative molecular mass of the native enzyme estimated on gel chromatography of Sephadex G-150 was 96 kDa. The pH and temperature optima for enzyme activity were 7 and 60°C, respectively. A half-life of the catechol 1,2-dioxygenase at the optimum temperature was 40 min. The kinetic parameters of the Geobacillus sp. G27 strain catechol 1,2-dioxygenase were determined. The enzyme had apparent K_m of 29 μM for catechol and the cleavage activities for methylcatechols were much less than for catechol and no activity with gentisate or protocatechuate was detected.