INTRODUCTION

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Many chlorinated aromatic compounds are of environmental concern because of their potential toxicity and persistence. Nevertheless, several microorganisms with the ability to degrade chlorinated aromatics have been described (9, 27, 28). The aerobic bacterial degradation of chlorinated aromatic compounds usually proceeds via the intermediate formation of chlorinated catechols. The further metabolism of these chlorocatechols is generally catalyzed by dioxygenases which cleave the aromatic ring between the two hydroxy groups (intradiol, ortho, or 1,2-cleavage) (35). In contrast, only a few dioxygenases are known which cleave chlorocatechols at a bond adjacent to the two hydroxy groups (extradiol, meta, or 2,3-cleavage) (14, 24, 30, 32, 40). Moreover, the degradation of certain chlorinated aromatics (e.g., 3-chlorobenzoate) results in the intermediate formation of (substituted) 3-chlorocatechols, and most extradiol dioxygenases are inactivated very rapidly during the conversion of 3-chlorinated catechols (13, 21, 33). For the catechol 2,3-dioxygenase from Pseudomonas putida strain mt-2 (C23O-mt2), it was demonstrated that the inactivation of the enzyme by 3-chlorocatechol was irreversible (2). The inactivation of the extradiol dioxygenases by 3-chlorinated catechols appears to be one major reason why many strains which are able to degrade nonchlorinated (methyl-) aromatics are unable to convert mixtures of chlorinated and methylated aromatics (31, 39).

There are very few extradiol dioxygenases which are able to efficiently transform (substituted) 3-chlorocatechols. Thus, the catechol 2,3-dioxygenase from P. putida strain GJ31 transforms 3-chlorocatechol by a proximal cleavage to 2-hydroxymuconic acid (17, 24) (Fig. 1).

View larger version (14K): [in this window] [in a new window]   FIG. 1.   Distal and proximal extradiol ring cleavage of 3-chlorocatechol and formation of possible reaction products.

A distal extradiol ring cleavage of 3-chlorocatechol resulting in the formation of 3-chloro-2-hydroxymuconic semialdehyde was demonstrated for the 2,3-dihydroxybiphenyl (2,3-DHBP) 1,2-dioxygenase BphC1 from Sphingomonas xenophaga BN6 (29, 30) (Fig. 1). However, during the conversion of 3-chlorocatechol and all other substrates tested, the activity of BphC1 decreased rapidly, presumably due to the loss of the catalytically active Fe(II) ions. This resulted in a relatively low turnover capacity of the enzyme for the oxidation of 3-chlorocatechol. In a previous study (29), an attempt was made to improve the efficiency of this reaction by site-directed mutagenesis of bphC1, but no significant improvement was achieved, basically due to the lack of structural information about BphC1.

In contrast to site-directed mutagenesis, directed evolution offers the possibility of altering an enzyme feature significantly, also in the absence of structural information about the corresponding enzyme (11, 22, 38). In the present study, an attempt was made to generate variant forms of BphC1 with improved efficiency of 3-chlorocatechol conversion. Therefore, bphC1 was randomly mutated using error-prone PCR (6, 23). The catalytic properties of the variants obtained were analyzed in order to obtain a better understanding of the interrelation between the substrate specificity, enzyme stability, and the rather unique ability of BphC1 to cleave 3-chlorocatechol by a distal cleavage mechanism.

	MATERIALS AND METHODS

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Bacterial strains and plasmids. The 2,3-DHBP 1,2-dioxygenase from strain BN6 (BphC1) was produced in Escherichia coli DH5(pGHS118). Plasmid pGHS118 is a derivative of pJOE 2783.1. In pGHS118, bphC1 is under the control of the lac promoter (30). The source of catechol 2,3-dioxygenase from P. putida mt-2 (C23O-mt2) was E. coli BL21(DE3)(pJF150) (30). All E. coli strains were routinely cultured in Luria-Bertani (LB) medium with the appropriate antibiotics.

Random mutagenesis. Plasmid pGHS118 harboring bphC1 was used as a template for the error-prone PCR. In this plasmid, bphC1 (489 bp) is flanked by an Ndel restriction site 3 bp upstream of the start codon and a BamHI restriction site 4 bp downstream from the stop codon. Using the two oligonucleotide primers 5'-CCC CAG GCT TTA CAC TTT-3' and 5'-GCT TCT GCG TTC TGA TTT-3', a DNA fragment was amplified which contained the entire bphC1 gene, 95 bp upstream of the NdeI restriction site and 86 bp downstream of the BamHI site. The following PCR program was used for the amplification of the 677-bp DNA fragment. After 3 min of denaturing at 95°C, 40 cycles of annealing (1.5 min at 48°C), polymerization (3 min at 72°C), and denaturing (40 s at 95°C) followed. The program was completed by an additional annealing (1.5 min at 48°C) and polymerization (10 min at 72°C) step.

Cloning of the mutated forms of bphC1. The success of the PCR amplification reaction was verified by agarose gel electrophoresis. Amplified DNA fragments were cut out from the gel and extracted using an Easy-Pure kit (Biozym, Hessisch Oldendorf, Germany). The DNA fragments containing the mutated forms of bphC1 were digested with NdeI and BamHI. After purification by gel electrophoresis, these DNA fragments were ligated with the linearized vector DNA generated by digestion of pJOE 2783.1 with NdeI and BamHI. E. coli DH5 was transformed with the ligation mixture and plated onto LB plates containing 100 µg of ampicillin ml¹.

Screening for clones with an improved activity for 3-chlorocatechol. The transformants obtained on LB plates with ampicillin were replicated to sterile filter discs (Schleicher und Schuell, Dassel, Germany), which were subsequently sprayed with a solution of 3-chlorocatechol (5 mM concentration in 100 mM Na- K phosphate buffer [pH 7.5]). After this treatment, the clones which were able to convert 3-chlorocatechol by a distal extradiol ring cleavage mechanism turned pale yellow within about 5 min due to the formation of the distal ring cleavage product (_max = 378 nm). Unfortunately, it was not possible by this method to detect differences in the ring cleavage activity of different clones. In order to make the formation of the distal ring cleavage product more visible, the filter discs were placed under a UV lamp ( = 366 nm; Desaga, Heidelberg, Germany). Under these conditions, the conversion of 3-chlorocatechol to the distal ring cleavage product resulted in a dark coloration of the respective colonies. To further intensify the UV and visible (UV/Vis) absorption of the distal ring cleavage product, polyethyleneglycol 4000 (10% in methanol) was sprayed on these filters (15) immediately after the treatment with 3-chlorocatechol. The colonies that showed the highest efficiency for 3-chlorocatechol conversion were restreaked on LB plates containing 100 µg of ampicillin ml¹ to obtain single colonies for further investigations.

DNA sequencing and sequence analysis. The DNA sequences were determined by the method of Sanger et al. (34). Cycle sequencing was performed using the Pharmacia ALFexpress DNA sequencer and a Thermo Sequenase Cy 5 Dye Terminator kit (both from Amersham Pharmacia Biotech, Uppsala, Sweden). Computer-based sequence analysis was performed with the PC/GENE program package (Intelligenetics Inc., Mountain View, Calif.).

Recombination and site-directed mutagenesis. The wild-type gene (bphC1) and the mutated forms harboring two or three nucleotide exchanges were digested at the unique AluI or MslI restriction sites at positions 163 and 261 of bphC1, respectively. After ligation of mutated and unmutated gene fragments, variant enzymes with single amino acid exchanges were generated.

Preparation of cell extracts. Single colonies were used to inoculate 200 ml of LB medium containing ampicillin (100 µg ml¹). These cultures were grown overnight at 37°C. The cells were harvested by centrifugation (7,000 × g; 15 min; 4°C) and washed with Na-K phosphate buffer (100 mM; pH 7.5). The cells were finally suspended in 2 ml of the same buffer and disrupted by using a French press (Aminco, Silver Spring, Md.) at 80 MPa. Cell debris was removed by centrifugation (100,000 × g; 45 min; 4°C). The protein concentration was determined by the method of Bradford (3), with bovine serum albumin as the standard.

Enzyme assays and analysis of enzyme kinetics. In general, enzyme assays were performed in 1 ml of Na-K phosphate buffer (100 mM; pH 7.5) with the respective substrate, started by the addition of 1 µl of cell extract (with a protein concentration of about 50 mg ml¹) containing the corresponding enzyme, and measured for 30 s.

Determination of the different ratios of distal to proximal ring cleavage products. Cell extracts containing the wild type or a variant enzyme (R3, R6, or R9) were repeatedly added to different concentrations of 2,3-DHBP, 3-methyl-, or 3-chlorocatechol (0.01, 0.05, 0.1, and 0.3 mM concentrations, in 1 ml of 100 mM Na-K phosphate buffer [pH 7.5]) until no further formation of the ring cleavage products could be detected spectrophotometrically. The concentrations of the ring cleavage products were determined by high-performance liquid chromatography (HPLC) (see below). The solvent system A was used for the analysis of the ring cleavage products of 2,3-DHBP, and the solvent system B was used for the products of 3-methylcatechol. For the analysis of the distal and proximal products of 3-chlorocatechol, the solvent systems B and C (see below), respectively, were used.

Preparation of substituted picolinic acids from the ring cleavage products of 3-methylcatechol. 3-Methylcatechol (1 mM) was dissolved in 1 ml of Na-K phosphate buffer (50 mM; pH 7.5) and completely converted by the repeated addition of cell extracts with extradiol dioxygenase activities (C23O-mt2 or the variant enzyme R9). The reaction mixtures were filtered through a 10,000-molecular-weight cutoff membrane (Centricon; Millipore, Bedford, Mass.) in order to separate macromolecular contaminants. The filtrates containing the ring cleavage products were incubated with NH₄Cl (1.2 M) and shaken overnight at 30°C (25, 30). Subsequently, these solutions were analyzed by HPLC using solvent system A (see below).

HPLC. All ring cleavage products and the picolinic acids were monitored by HPLC (HPLC Millenium Chromatography Manager 2.0, equipped with a photodiode array detector, model 486, and HPLC pumps, model 510; Waters Associates, Milford, Mass.). The following solvent systems were used.

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Chemicals. 3-Methylpicolinic acid was purchased from TCI (Tokyo, Japan). 2-Hydroxymuconic acid and 2-pyrone-6-carboxylic acid were kindly donated by S. Kaschabek and W. Reineke (Universität-Gesamtschule Wuppertal, Wuppertal, Germany). The sources of all other chemicals have been described previously (29, 30).

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Random mutagenesis for improved conversion of 3-chlorocatechol. To improve the catalytic efficiency of BphC1 from strain BN6 for the extradiol cleavage of 3-chlorocatechol, bphC1 was randomly mutated using error-prone PCR (see Materials and Methods). The amplification products obtained were cloned into the expression vector pJOE 2783.1 (30), and cells of E. coli DH5 were transformed with these plasmids. Using a spray test, about 5,000 colonies were screened on agar plates for the improved formation of the distal ring cleavage product of 3-chlorocatechol (see Materials and Methods). For 15 colonies an improvement in this reaction in comparison to results for the wild-type enzyme was supposed. These clones were cultivated in liquid medium, cell extracts were prepared, and the formation of the distal ring cleavage product from 3-chlorocatechol (_max = 378 nm) was analyzed spectrophotometrically for different substrate concentrations (0.1, 0.5, and 1.0 mM). Only three cell extracts showed increased activities compared to the wild-type enzyme (Table 2). The most significant improvement for the formation of the distal ring cleavage product was observed with the mutant enzyme 1, which revealed about fivefold-higher relative activities than the wild-type enzyme for all substrate concentrations tested. The mutant enzyme 3 showed higher relative activities than the wild-type enzyme only for substrate concentrations of 0.5 mM.

Recombination and site-directed mutagenesis. In order to identify the mutations responsible for the changed catalytic properties, the following variant enzymes were constructed by combination of mutated and unmutated gene fragments (see Materials and Methods): R1 (M37T, F66L, N96T, and K106R); R2 (M37T); R3 (F66L); R4 (N96T and K106R); R5 (K22E); and R6 (D155Y).

Distal and proximal cleavage of 3-chlorocatechol by the different forms of BphC1-BN6. 3-Chlorocatechol (0.05 mM concentration in 100 mM Na-K phosphate buffer [pH 7.5]) was completely converted by the repeated addition of cell extracts containing the wild-type enzyme or the variant enzymes R3 (F66L), R6 (D155Y), or R9 (F66L and P67A) until no further formation of the distal ring cleavage product could be detected spectrophotometrically at 378 nm. Surprisingsly, the reaction mixtures containing the variant enzyme R3 or R9 showed an about 1.6-fold-higher rate of final absorption at 378 nm than the assay mixtures containing the wild-type enzyme or the variant enzyme R6. This indicated that the different forms of BphC1 produced different amounts of the distal ring cleavage product during the conversion of identical amounts of 3-chlorocatechol. This suggested that some of the enzymes oxidized 3-chlorocatechol not only to the distal ring cleavage product but also to another product(s), presumably by a proximal ring cleavage mechanism.

Conversion of 3-methylcatechol and 2,3-DHBP by the variant enzymes R3, R6, and R9. The experiments with 3-chlorocatechol demonstrated that the mutations in the variant forms R3 and R9 of BphC1 resulted in significant changes in the regioselectivity of the enzyme. These variant enzymes showed an enhanced tendency to perform a distal ring cleavage reaction. Therefore, the conversion of 3-methylcatechol and 2,3-DHBP by the wild-type enzyme and the variant enzymes R3, R6, and R9 also was investigated with regard to the regioselectivity.

View larger version (18K): [in this window] [in a new window]   FIG. 2.   UV/Vis spectra (at pH 10) of the reaction products formed during the conversion of 3-chlorocatechol (A), 3-methylcatechol (B), or 2,3-DHBP (C). The substrates (0.1 mM concentrations in 100 mM Na-K phosphate buffer [pH 7.5]) were converted with cell extracts of recombinant E. coli strains containing the variant form R9 of BphC1. The products were analyzed by HPLC using a photodiode array detector (see Materials and Methods). The analysis of the product formed from 3-chlorocatechol (Rt = 10.8 min) and also of the products from 3-methylcatechol (for product 1, Rt = 4.9 min; for product 2, Rt = 5.9 min) was performed using solvent system B. The oxidation products of 2,3-DHBP were analyzed with solvent system A (for product 1, Rt = 1.8 min; for product 2, Rt = 3.1 min).

View larger version (15K): [in this window] [in a new window]   FIG. 3.   Possible directions for the extradiol ring cleavage of 3-methylcatechol and transformation of the ring cleavage products into different substituted picolinic acids.

Kinetic data for the conversion of different substrates by the variant enzymes and the wild-type enzyme. Various concentrations of 2,3-DHBP, catechol, 3-methyl-, or 3-chlorocatechol were converted by cell extracts containing the wild-type enzyme or the variant enzymes R3, R6, or R9. The different forms of BphC1 produced different ratios of proximal and distal ring cleavage products during conversion of the same substrate. Therefore, reaction coefficients had to be determined for the respective reactions (see Materials and Methods).

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	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

Until now, the crystal structures of three extradiol dioxygenases have been resolved. These are the 2,3-dihydroxybiphenyl 1,2-dioxygenases originating from Burkholderia (Pseudomonas) cepacia LB400 and Pseudomonas sp. KKS102 and the catechol 2,3-dioxygenase from P. putida mt-2 (C23O-mt2). These crystal structures showed very similar active centers and indicated a similar reaction mechanism for this class of dioxygenases (10, 20, 36).

However, sequence alignments of various extradiol dioxygenases showed that these enzymes can be separated into different groups with very low total sequence homology (7). This was very evident for BphC1, which showed less than 20% sequence identity to the most related enzymes, two 2,3-DHBP 1,2-dioxygenases from Rhodococcus globerulus P6 (12). Furthermore, BphC1 from strain BN6 and the 2,3-DHBP dioxygenases from strain P6 belong to a specific group of small extradiol dioxygenases which show significantly lower molecular weights of the single subunits compared to almost all other extradiol dioxygenases. For this group of extradiol dioxygenases, no crystal structure is currently available. Thus, although the catalytic mechanism of BphC1 is probably similar to that of most other extradiol dioxygenases, it is difficult to rationalize the connection between the amino acid sequence and the catalytic function of this enzyme.

In the present study, basically two different types of variant enzymes were obtained. The variant enzyme R6 (D155Y) demonstrated a lower substrate affinity and a higher stability during substrate turnover. In contrast, the variant enzymes R3 (F66L) and R9 (F66L and P67A) showed a changed regioselectivity of ring cleavage and a higher catalytic activity for 3-chlorocatechol compared to the wild-type enzyme.

In the variant enzyme R6, an aspartate residue at the C-terminal part was replaced with a tyrosine residue. From the crystal structure of the C23O-mt2, it was proposed that the catechols must enter the active site of the enzyme through a channel (barrel structure) and that some of the amino acid residues at the C terminus narrow the substrate entrance (20). Furthermore, it may be deduced from the structures obtained for the C23O-mt2 that the carboxy-terminal region of the enzyme is located rather near to the catalytic center. Thus it is possible that the reduced substrate affinity of the variant enzyme R6 is caused by a reduced accessibility of the active center to the organic substrate or by subtle changes in the active center caused by the mutation in the C-terminal region.

The major aim of this study was to obtain variant forms of BphC1 with improved efficiency for 3-chlorocatechol oxidation. This was achieved by the isolation of the variant enzymes R3 and R9, which showed about fivefold-higher reaction rates for the conversion of 3-chlorocatechol than the wild-type enzyme. This is the first example showing that the catalytic activity of an extradiol dioxygenase could be increased by random mutagenesis, and this demonstrated that error-prone PCR combined with an appropriate screening method is a simple and effective method for altering the catalytic properties of an enzyme in the desired way. A different strategy of directed evolution of extradiol dioxygenases was performed by Kikuchi et al. (18, 19) using family shuffling in order to construct a catechol 2,3-dioxygenase with increased thermostability.

The variant enzymes R3 and R9 were selected for the improved oxidation of 3-chlorocatechol. Therefore, the observation that these variant enzymes also showed an altered regioselectivity for the ring cleavage of 3-methylcatechol was rather astonishing. The variant enzymes R3 and R9 oxidized about half of the 3-methylcatechol by a distal cleavage mechanism. This finding was very unexpected, because until now only a single extradiol dioxygenase has been known (from an Achromobacter sp.), which was claimed to convert 3-methylcatechol by a distal ring cleavage mechanism (14). Furthermore, the HPLC analysis and the UV/Vis spectra obtained for the conversion of 2,3-DHBP by these variant enzymes also suggested that this substrate was oxidized to significant amounts of a distal cleavage product. This indicates that the variant enzymes R3 and R9 have an enhanced tendency for a distal cleavage of all 3-substituted catechols. This demonstrates that the substitution of only a single amino acid can change the catalytic properties of the enzyme significantly. Moreover, this is the first time that a change in the direction of ring cleavage was described for an extradiol dioxygenase.

The altered catalytic properties of the variant enzymes R3 and R9 were caused by the replacement of the amino acids at positions 66 and 67 of BphC1 by smaller ones. These residues probably belong to a region of the enzyme which is involved in substrate binding. A possible explanation for the changed regioselectivity in the mutant enzymes may be deduced from the reaction mechanism of extradiol dioxygenases. For the catechol 2,3-dioxygenase from P. putida mt-2, the catalytic cycle is supposed to involve a complexation of the catalytically active Fe(II) ion by a monoanionic catecholate as a bidentate ligand. The ring cleavage reaction is proposed to proceed via an attack of the iron-bound activated oxygen species on the nonhydroxylated position vicinal to the carbon atom bearing the phenolate anion (20, 36, 37). A substituted catechol can in principle bind in two different modes as a bidentate ligand to the catalytically active ferrous iron. This would finally result in a different regioselectivity of the substrate cleavage. The mutation F66L may therefore result in an altered sterical situation in the catalytic center which could allow binding of 3-methylcatechol and 2,3-DHBP to the catalytically active ferrous iron in a way which finally results in a distal cleavage of the substrates. However, preliminary experiments which were performed in order to detect differences in the kinetic parameters for the formation of the proximal and distal ring fission products from 2,3-DHBP by the variant enzymes did not show significant differences in the substrate affinities for the formation of the proximal and distal ring fission products. Thus, probably only the crystal structure of BphC1 and its variant forms could completely explain the unique ability of the mutant enzymes to perform a distal ring cleavage of different substituted catechols.

The variant forms of BphC1 may be used for the production of novel picolinic acid derivatives or for the construction of strains with an improved capacity to degrade chlorinated aromatic compounds.