INTRODUCTION

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The microbial degradation of various chloroaromatics has been described to occur via chlorocatechols as central intermediates, which are further degraded through the modified ortho pathway (45, 49). Chlorocatechol 1,2-dioxygenase, chloromuconate cycloisomerase, dienelactone hydrolase, and maleylacetate reductase fulfill the convergence of the chlorocatechol and catechol degradative pathways.

Alternatively to the intradiol type of ring cleavage, the pathway is initiated by extradiol ring cleavage in some microorganisms. The catechol 2,3-dioxygenases of the meta pathway are able to convert catechol, both isomeric methylcatechols, and 4-chlorocatechol at respectable rates (10, 18, 27, 33, 38, 44, 47, 48). The further degradation of the ring cleavage product of 4-chlorocatechol seems to be a slow process, since all strains degrading a chloroaromatic compound via 4-chlorocatechol through the meta pathway grow slowly on these substrates (1, 2, 15, 16, 29).

However, when 3-chlorocatechol occurs in strains with a meta pathway, the catechol 2,3-dioxygenase is negatively influenced, either by 3-chlorocatechol itself, as a chelating compound resulting in a reversible inactivation (24), or by a reactive acylchloride, the product of the cleavage of 3-chlorocatechol, which causes irreversible inactivation of the enzyme (3). Auto-oxidation of accumulating 3-chlorocatechol leads to a general toxic effect on the cells; therefore, degradation of haloaromatics via meta cleavage of 3-chlorocatechol has been considered impossible.

Recently, we reported that Pseudomonas putida GJ31 degrades chlorobenzene with a generation time of 3 h via 3-chlorocatechol, using the meta pathway without any apparent toxic effects (26, 40). We now present data on the purification and characterization of the unusual meta-cleaving enzyme that converts 3-chlorocatechol productively. Comparison with various previously published catechol 2,3-dioxygenases was performed.

	MATERIALS AND METHODS

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Organism and culture conditions. P. putida GJ31 was grown at 30°C in five separate 0.5-liter cultures with mineral medium (9). The growth substrate chlorobenzene was added via the vapor phase. After the cultures had been grown to an optical density at 546 nm of 1.6, the cells were harvested.

Preparation of cell extracts. Cells were removed by centrifugation at 4,000 × g for 20 min at 4°C. The pellet was resuspended in Tris-HCl buffer (50 mM; pH 7.5) containing 1 mM ascorbate (buffer A). After another centrifugation at 4,000 × g for 20 min, the cells were suspended in 9 ml of the same buffer. Disruption took place at 4°C by one passage through a French pressure cell (140 MPa; Aminco, Silver Spring, Md.). Cell debris were removed by centrifugation at 100,000 × g for 60 min at 4°C.

Enzyme assays. Catechol 2,3-dioxygenase was measured by a modification of the method of Nozaki (37). The reaction mixture contained 50 µmol of phosphate buffer (pH 7.4) and 0.1 µmol of catechol in a total volume of 1 ml. After addition of enzyme, the increase at 375 nm (corresponding to the formation of 2-hydroxymuconic semialdehyde at  of 36,000 liters/mol · cm) was measured in a silica cuvette with a 1.0-cm light path. One unit of activity was defined as the amount of enzyme required to form 1 µmol of product per min under the conditions of the assay.

pH optimum. The pH optimum was determined by using a substrate concentration (catechol) of 0.1 mM in 50 mM NaH₂PO₄-Na₂HPO₄ buffer (pH 5.0 to 8.8) and 50 mM glycine-NaOH buffer (pH 8.4 to 10.0). Since the molar extinction coefficient of the reaction product of catechol was markedly increased by increasing the pH, it was determined at each pH.

Formation of chloride from 3-chlorocatechol. The liberation of chloride during turnover of 3-chlorocatechol was determined as follows. The assay mixture contained (in 1 ml) 50 µmol of phosphate buffer (pH 7.4), 0.05 to 0.2 µmol of 3-chlorocatechol, and 20 µl of purified enzyme (45 µg of protein). To remove any chloride from the enzyme stock solution, it was dialyzed against 100 mM Tris-H₂SO₄ (pH 7.5) containing 0.1 mM (NH₄)₂Fe(SO₄)₂. After addition of the enzyme, the assay mixture was incubated for 1 h. Chloride was quantitatively determined by the silver chloride method according to the method of Freier (11). To 800 µl of chloride-containing assay mixture, 100 µl of concentrated HNO₃ and 100 µl of 0.1 N AgNO₃ were added in sequence. After 10 min of incubation in the dark, the absorption at 546 nm was measured.

Protein determinations. Protein concentrations were determined by the Bradford method (7), with crystalline bovine serum albumin as the standard. During enzyme purification, the eluted protein was detected with a Pharmacia UV-1 monitor at 254 nm.

Enzyme purification. All enzyme purification steps were carried out at 4°C.

(i) Ammonium sulfate precipitation. A cold saturated aqueous solution of (NH₄)₂SO₄ (pH 7.0) containing 0.1 mM EDTA was added to crude extract (10 ml) with constant stirring to give 40% saturation. After 30 min of equilibration, the precipitate was collected by centrifugation at 8,000 × g for 20 min. The pellet was dissolved in buffer A to give a total volume of 2.5 ml, and the resulting protein solution was desalted by gel filtration through a PD10 column with buffer A.

(ii) Incubation with hydroxyapatite. The protein solution from step i was added to a suspension of 5 ml of preequilibrated hydroxyapatite gel in 5 ml of buffer A. After 30 min of incubation, the suspension was separated by centrifugation at 8,000 × g for 2 min. The supernatant containing catechol 2,3-dioxygenase was collected, and the pellet was washed with another 5-ml portion of buffer A. After centrifugation, both supernatants were combined. Because of the significant loss of activity due to complexation of Fe(II) ions, the protein solution was supplemented with (NH₄)₂Fe(SO₄)₂ to give a concentration of 0.1 mM.

(iii) Ion-exchange chromatography on Mono-Q. The pooled supernatants from step ii were applied at 0.5 ml/min to a Mono-Q column (5 by 10 mm) which had been preequilibrated with buffer A. After washing of the column with 30 ml of buffer A at 0.5 ml/min, catechol 2,3-dioxygenase was eluted with 20 ml of buffer A containing NaCl in a linear gradient from 0 to 1 M. Fractions of 0.5 ml were collected at a flow rate of 0.5 ml/min. Six fractions containing the highest level of activity were pooled.

(iv) Storage. The combined fractions from step iii were stored at 20°C in 100 mM Tris-HCl buffer, pH 7.5, containing 50% glycerol, 150 mM NaCl, and 1 mM ascorbate.

Determination of M_r values. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to determine the subunit M_r values and the purity of the catechol 2,3-dioxygenase. It was performed by the method of Laemmli (25) on 1-mm-thick vertical slab gels (13.5 by 15.5 cm) containing 12.3% (wt/vol) acrylamide in the resolving gels. Electrophoresis was performed at 100 V for 1 h followed by 200 V for 6 h. The apparatus was cooled to 4°C. Protein was detected by silver staining (30). The calibration proteins were bovine albumin (M_r, 66,000), chicken ovalbumin (M_r, 45,000), glyceraldehyde-3-phosphate dehydrogenase (M_r, 36,000), carbonic anhydrase (M_r, 29,000), trypsinogen (M_r, 24,000), and -lactalbumin (M_r, 14,200) (Sigma Chemical Co., St. Louis, Mo.).

Isoelectric focusing. Isoelectric focusing was carried out on 3.8% (wt/vol) polyacrylamide gels containing 2% (vol/vol) ampholytes by the method of O'Farrell (39) with carrier ampholytes in a pH range of 6 to 8.

Absorption spectra. Spectra were recorded on a Shimadzu Recording Spectrophotometer, model UV-240. Kinetic measurements at a single wavelength were carried out on a UVIKON 820 spectrophotometer (Fa. Kontron, Eching, Germany).

N-terminal amino acid sequencing and database search. One hundred thirty micrograms of the purified enzyme was applied to an SDS-polyacrylamide gel (1.5 mm thick). The subunits were blotted onto an Amersham Hybond polyvinylidene difluoride membrane according to the Amersham protocol (staining was done with amido black). The N-terminal amino acid sequence was determined with an Applied Biosystems model 477A protein sequencer and an Applied Biosystems model 120A on-line high-performance liquid chromatograph (HPLC).

HPLC. HPLC of substrates and metabolites was conducted as described previously by diode array detection, which allows a determination of the UV spectrum of the respective substrate or metabolite (19).

Chemicals. 2-Pyrone-6-carboxylic acid was prepared by cyclization of the condensation product obtained from diethyl oxalate and ethyl crotonate in the presence of potassium, according to the method of Wiley and Hart (51).

	RESULTS

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Purification of catechol 2,3-dioxygenase. Catechol 2,3-dioxygenase was purified to homogeneity from chlorobenzene-grown cells of P. putida GJ31. The results of a typical enzyme purification procedure are summarized in Table 1. During purification the specific activity of the enzyme increased to 6.48 U/mg of protein, indicating 21.6-fold purification with 72.5% recovery of activity. The specific activity of the GJ31 dioxygenase was very low compared to other catechol 2,3-dioxygenases.

View larger version (63K): [in this window] [in a new window]   FIG. 1.   Purification of the catechol 2,3-dioxygenase of P. putida GJ31 as monitored by SDS-PAGE. Lanes: A, crude extract (10 µg of protein); B, ammonium sulfate precipitate (10 µg of protein); C, hydroxyapatite supernatant (2 µg of protein); D, Mono-Q elution pool (2 µg of protein); E, molecular mass markers (ca. 1 µg each).

Structural properties. The molecular mass of each of the subunits of the denatured protein, as determined by SDS-PAGE, was found to be 33.4 kDa. Estimation of the native M_r value of the catechol 2,3-dioxygenase by gel filtration gave a molecular mass of 135 ± 10 kDa on a Superose 12 column. On the basis of these results, catechol 2,3-dioxygenase from strain GJ31 should be a tetramer.

Temperature and pH optima. The temperature optimum of the reaction rate of catechol 2,3-dioxygenase was estimated to be 50°C. However, denaturation of the enzyme was significant at this temperature.

N-terminal amino acid sequence. A single amino acid was obtained in each cycle of automated Edman degradation, indicating that the protein was homogeneous and consisted of identical subunits.

Inhibition and activation. The effect of various compounds on the activity of catechol 2,3-dioxygenase was tested. Strong Fe²⁺ chelators, such as o-phenanthroline and ,-dipyridyl, markedly inactivated catechol 2,3-dioxygenase at a 1 mM concentration (data not shown). The activation by ascorbate clearly showed that some part of the enzyme was in the oxidized form, i.e., inactive. The oxidizing agent H₂O₂ completely inactivated the enzyme. Some activity was restored if the sample was reduced with ascorbate. All of these results suggest that the active site is accessible to chelators and oxidizing and reducing agents.

Identification of 2-hydroxymuconate as the product of turnover of 3-chlorocatechol with the catechol 2,3-dioxygenase of P. putida GJ31. To identify the reaction product of enzymatic turnover of 3-chlorocatechol, several incubations with purified catechol 2,3-dioxygenase from P. putida GJ31 were done.

View larger version (58K): [in this window] [in a new window]   FIG. 2.   2-Hydroxymuconate and 4-oxalocrotonate as the products of enzymatic turnover of 3-chlorocatechol with the catechol 2,3-dioxygenase of P. putida GJ31. (A) Turnover of 3-chlorocatechol (0.2 µmol) with 8.4 µg of protein in a volume of 1 ml. Spectra were recorded at 0, 1, 3, 5, 7, 10, 15, 20, and 60 min. (B) Tautomerization of 2-hydroxymuconate (0.2 µmol per ml) to 4-oxalocrotonate in phosphate buffer (pH 7.4) in the absence of enzyme. Spectra were recorded at 2, 5, 10, 18, 28, 40, and 120 min. (C) Turnover of 3-chlorocatechol (0.1 µmol) in the presence of a large amount of enzyme (16.8 µg of protein) in a volume of 1 ml. Spectra were recorded at 0, 2, 5, 10, 20, and 30 min.

Stoichiometry of chloride elimination during turnover of 3-chlorocatechol. Chloride elimination during turnover of 3-chlorocatechol by catechol 2,3-dioxygenase from P. putida GJ31 was measured. Assay mixtures containing 3-chlorocatechol and the purified enzyme were incubated at room temperature until conversion was complete. Turnover of 3-chlorocatechol was accompanied by quantitative release of chloride (data not shown).

Substrate specificity. The substrate range of catechol 2,3-dioxygenase was determined by incubating the enzyme with various potential substrates and determining the rate of appearance of products (Table 2). Various substituted catechols were oxidized by the catechol 2,3-dioxygenase of strain GJ31. The absorbance maxima of the products were observed to be in the range of 365 to 387 nm (the exception was 3-chlorocatechol), suggesting the occurrence of substituted 2-hydroxymuconic semialdehydes (typical meta-cleavage products), which can be explained by the cleavage of the respective substrate in a proximal extradiol manner between C-2 and C-3. Distal extradiol cleavage seemed to occur with 3,5-dichlorocatechol, since a yellow compound was formed, which does not occur by cleavage between C-2 and C-3.

	DISCUSSION

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Structure of catechol 2,3-dioxygenase. We have described a purification procedure for the catechol 2,3-dioxygenase of strain GJ31. The isolation procedure allows purification of the enzyme to >95% homogeneity. After purification, the enzyme has a native molecular mass of 135 ± 10 kDa and consists of a single subunit type of 33.4 kDa, consistent with the enzyme existing as a tetramer of identical subunits. This is similar to the catechol 2,3-dioxygenase from P. putida PaW1 (mt-2), which has a molecular mass of 140 kDa and consists of four identical subunits (38). The N-terminal amino acid sequence of the catechol 2,3-dioxygenase of strain GJ31 shows around 70 to 42% identity to the published sequences of various catechol 2,3-dioxygenases (Fig. 3). The assignment of the metal as Fe²⁺ was based on chelation studies, the observation of inactivation by oxidants, and the requirement for Fe²⁺ in a reconstitution procedure. H₂O₂ inactivation at a concentration of 1 mM is typical for all iron-dependent extradiol dioxygenases, while manganese-dependent extradiol dioxygenases show only weak inactivation under these conditions (6, 43).

View larger version (40K): [in this window] [in a new window]   FIG. 3.   CLUSTAL V multiple-sequence alignment of the N-terminal amino acid sequence of the catechol 2,3-dioxygenase of P. putida GJ31 with those of other catechol 2,3-dioxygenases. The codes on the left are accession numbers. References are given at the end of each line.

Substrate specificity. It is shown here that the catechol 2,3-dioxygenase of strain GJ31 can convert a wide range of substrates. The substrate range of the enzyme is similar to that of XylE from strain PaW1, which can use methyl-substituted substrates and 4-chlorocatechol. However, XylE does not accept chlorine in the position immediately adjacent to the vicinal hydroxyl groups. The GJ31 dioxygenase is the only enzyme known to convert 3-chlorocatechol at a high rate, so it should be termed chlorocatechol 2,3-dioxygenase. The activities of the other listed catechol 2,3-dioxygenases indicate that some prefer 3-methylcatechol and others the analog substituted at the 4 position (Table 3). 3,5-Dichloro- and 4,5-dichlorocatechol were converted by the dioxygenase of strain GJ31 at a low rate, forming yellow products.

Products from 3-chlorocatechol. Surprisingly, P. putida GJ31 degraded chlorobenzene via 3-chlorocatechol and used a meta-cleavage pathway. 3-Halocatechols have been reported to be suicide substrates for the meta-fission enzyme catechol 2,3-dioxygenase of P. putida PaW1 (3) or to be inactivating chelators for the enzyme of P. putida F1 (24). In the first case, an acylhalide (5-chlorocarbonyl-2-hydroxy-penta-2,4-dienoic acid) has been proposed as the reactive intermediate which acylates the protein and destroys its enzymatic activity (Fig. 4, path 1).

View larger version (17K): [in this window] [in a new window]   FIG. 4.   Alternative reactions occurring when catechol 2,3-dioxygenases cleave 3-chlorocatechol. 1, Suicide inactivation when a nucleophilic group of the dioxygenases undergoes acylation; 2, Immediate and spontaneous cyclization occurring when the carbonyl group at C-6 of the ring fission product undergoes internal nucleophilic attack by the enolic hydroxyl at C-2 after configurational change; 3, Reaction of acylchloride with water to give 2-hydroxymuconate; 4, Distal extradiol cleavage between C-1 and C-6 to give a typical yellow meta-cleavage product of the 2-hydroxymuconic semialdehyde type.

Dechlorination mechanisms in the degradation of chloroaromatic compounds. The dechlorination mechanism used by strain GJ31 represents an alternative to the different dechlorination mechanisms which are normally used in the degradation of chlorobenzenes through the ortho pathway. These include the oxygenolytic removal of chlorine substituents at an early stage of the degradative pathway by the ring-activating dioxygenases prior to cleavage of the aromatic ring and, alternatively, degradation proceeding through chlorinated catechols as central metabolites. Chlorosubstituted aliphatic structures are then generated after ring cleavage, from which HCl is eliminated by chloromuconate cycloisomerases and maleylacetate reductases.