Construction of Chimeric Catechol 2,3-Dioxygenase Exhibiting Improved Activity against the Suicide Inhibitor 4-Methylcatechol

Plasmid construction.The XbaI-HindIII fragment containing the entire operon encoding the meta cleavage enzymes of pWW0 (xylXYZLTEGFJQKIH), its regulation gene (xylS), and the 5′ end of xylR was isolated from pPL392 (11) and subcloned into the T7 expression vector, pT7-7, to produce pMETA21. A deletion extending between xylT and xylE was introduced into pMETA21 by the method outlined in Fig. 2A. First, four primers were designed. FxylL (5′-GCG CCA TCG TAA ATG TTT CCT-3′) corresponds to the middle sequence of xylL. The underlined sequence in RxylET (5′-ACC GGA CCA TCAATT CAT CCC AGA TAC ACT AAA-3′) is complementary to the underlined sequence in FxylTE, while the rest of the sequence is complementary to the initiation codon region of xylT. The sequence in bold letters in FxylTE (5′-TCT GGG ATG AATTGA TGG TCC GGT ACG ACT TAT-3′) is complementary to the sequence in bold letters in RxylET, while the rest of the sequence corresponds to the termination codon region of xylE. RxylG (5′-GGC GAC GAA CGC GTC GAA GAT-3′) is complementary to the middle sequence of xylG. In Fig. 2A, the boldface and underlined sequences in FxylTE and RxylET are indicated. With pPL392 as a template, a DNA segment upstream of xylT covering the 3′ half of xylL (′xylL) was amplified with the primer pair FxylL and RxylET, with the structure of the amplified sequence being 5′-(′xylL)-TCT GGG ATG AATTGA TGG TCC GGT. Another DNA segment downstream of xylE corresponding to the 5′ half of xylG (xylG′) was amplified with the primer pair FxylTE and RxylG, again with pPL392 as a template. The structure of the amplified sequence was 5′-TCT GGG ATG AATTGA TGG TCC GGT-(xylG′)-3′. The two DNA segments thus amplified were subsequently mixed together and subjected to a second PCR with the primers FxylL and RxylG. In this reaction, the 3′ end of 5′-(′xylL)-TCT GGG ATG AATTGA TGG TCC GGT-3′ and the 5′ end of 5′-TCT GGG ATG AATTGA TGG TCC GGT-(xylG′)-3′ were annealed to each other, and the DNA segment with the structure 5′-(′xylL)-TCT GGG ATG AATTGA TGG TCC GGT-(xylG′)-3′ was amplified. The product was excised from agarose gel, digested with BamHI (cleavage within xylL) and MluI (cleavage within xylG), and introduced into pMETA21 at the corresponding restriction enzyme sites to create pMETA213. The absence of any nucleotide substitutions and the successful introduction of the xylT-xylE deletion in this plasmid were confirmed by nucleotide sequencing.

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FIG. 2.

Plasmid vector pRGmeta. (A) Construction of a deletion extending between the xylT and xylE genes. Genes on TOL plasmid pWW0 (xylLTEG) are indicated by arrows. The open and gray-shaded boxes on primers FxylTE and RxylET are complementary to each other. The products of the first PCR were mixed and subjected to the second PCR with the FxylL and RxylG primers. Details are described in Materials and Methods. (B) The pRGmeta plasmid contains two origins of replication, one functional in Pseudomonas and the other functional in E. coli (pMB1). In addition, the genes of the meta cleavage operon (except xylT and xylE) and its promoter (Pm) on TOL plasmid pWW0 were cloned in the plasmid as the XbaI-HindIII fragments.

The pRG vector was constructed based on the broad-host-range pRO1614 vector, possessing two origins of replication, one derived from pMB1 and the other derived from an endogenous plasmid in Pseudomonas aeruginosa (34). The pRG vector had these two origins of replication, a gentamicin resistance gene, and the meta operon promoter Pm. The XbaI-HindIII fragment of pMETA213, which contained all of the meta cleavage pathway genes except xylT and xylE, the regulatory gene, xylS, and the 5′ part of xylR, was subcloned into the same sites of pRG. The resultant plasmid, pRGmeta, was used for cloning C23O genes into the NheI and HindIII sites (Fig. 2B).

Cassette PCR.DNA was isolated from phenol-acclimatized activated sludge harboring a variety of phenol-degrading bacteria (43). The central parts of the C23O genes were PCR amplified from DNA by using the primers CF and CR. Their respective sequences, 5′-GCC GAT GAG CCA GGT ATG GA(TC) TT(TC) ATG GG(TCAG) TT(TC) AA-3′ and 5′-ACT TCG TTG CGG TTA CCGGA(TCAG) TC(AG) AA(AG) AA(AG) TA(TCA) AT(TCAG) GT-3′, were based on the conserved sequences of known C23O genes (9). This primer pair amplified the C23O gene sequence corresponding to that of the 582-bp-long xylE at positions 4940 to 5522 of GenBank accession number M64747 (16). Boldface letters in the sequences of primers CF and CR were complementary to boldface letters in sequences of primers 5R and 3F, respectively, as shown below. Two primer pairs, 5F plus 5R and 3F plus 3R, were used for PCR amplification of the DNA segments corresponding to the 5′ and 3′ ends, respectively, of nahH and xylE. The sequences of these four primers were as follows: 5F, 5′-GGC GGC CGC TGA AGA GGT GAC GTC ATG AA-3′; 5R, 5′-CAT ACC TGG CTC ATC GGC TT-3′; 3R, 5′-GGG AAT TCT AAG TCG TAC CGG ACC ATC-3′; and 3F, 5′-TCC GGT AAC CGC AAC GAA GT-3′. Underlined sequences correspond to the recognition sites for NotI and EcoRI, respectively. The xylE segment was amplified from genomic DNA prepared from P. putida strain KT2440 containing TOL plasmid pWW0 (12), while the nahH segment was from pGSH2939 (13). Three PCR-amplified DNA segments, the 5′ end of xylE or nahH, the central parts of the C23O genes, and the 3′-end of xylE or nahH, were subsequently mixed together and subjected to a second PCR with the primers 5F and 3R. This PCR operation allowed the amplification of the hybrid genes to full length in the structure 5′-(xylE or nahH segment)-(central C23O gene segment)-(xylE or nahH segment)-3′. A full description of this method, called cassette PCR (Fig. 1), has previously been published (32).

Screening of C23Os with improved activity against 4-methylcatechol.The 1-kb final products of cassette PCR were separated by agarose gel electrophoresis, excised from agarose gel, and digested with NotI and EcoRI. The resulting fragment was ligated to pZErO-2 (Invitrogen) and introduced into Escherichia coli top10F′. Transformants were cultivated on Luria-Bertani plates each containing 20 μg of kanamycin/ml and 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG). Colonies that appeared on the plates were collected, and plasmids were prepared en masse from the collected cells. These plasmids were then digested with XbaI and HindIII and ligated with pRGmeta that had been digested with NheI and HindIII (Fig. 2B). The resulting plasmids were introduced into P. putida PaW94 (25) by electroporation (10), and the transformants were cultivated at 30°C on minimal M9 plates containing 10 μg of gentamicin/ml and 5 mM p-toluate. The transformants generally could not grow on the plates, since 4-methylcatechol, an intermediate formed from p-toluate, cannot be efficiently metabolized by C23O. However, colonies appeared on the plates at a low frequency, and they were replicated on fresh plates of the same type. After two replications, about 2,000 colonies remained. All of these colonies were spread for single-colony isolation on plates of the same composition except for a lower concentration of p-toluate (0.5 mM). After purification twice, 11 clones remained. These 11 clones were cultivated at 30°C with shaking on a minimal M9 medium containing 5 mM p-toluate. One clone which exhibited reproducible growth on the medium was retained for further analysis, and the plasmid in this clone was named pRGmeta-NY8.

Purification of C23Os.The C23O gene on pRGmeta-NY8 was subcloned into the pTrc99A vector (1) and introduced into E. coli BL21. The resulting transformant was grown in 200 ml of Luria-Bertani medium containing 50 μg of ampicillin/ml to an A₆₀₀ of 0.5 to 0.8. C23O was then induced by adding 1 mM IPTG, and the cells were subsequently cultivated for 5 h at 30°C. C23O encoded by pRGmeta-NY8 (hereinafter called NY8) was purified by a method described previously (32). XylE was similarly purified from P. putida PaW94 harboring TOL plasmid pWW0 (5).

Determination of the kinetic parameters of C23Os.Assays for C23O activity under the standard conditions were carried out with a 100 mM potassium phosphate buffer (pH 7.5) at 25°C with 330 μM catechol as a substrate, and the amount of the ring cleavage product of catechol (2-hydroxymuconic semialdehyde; ε = 33 mM⁻¹ cm⁻¹) was determined spectrophotometrically at 375 nm (5). The k_cat and K_m values of C23O for catechol, 3-methylcatechol, and 4-methylcatechol were determined under the standard conditions, except that the concentration of each substrate was varied in the range of 0.5 to 330 μM. The ring cleavage products of 3-methylcatechol (2-hydroxy-6-oxohepta-2,4-dinoate; ε = 19.4 mM⁻¹ cm⁻¹) and 4-methylcatechol (2-hydroxy-5-methyl-6-oxohexa-2,4-dienoate; ε = 28.1 mM⁻¹ cm⁻¹) were detected at 388 and 382 nm, respectively.

Determination of the kinetic constants for the inactivation of C23Os.The method used for determining the rate constants for the inactivation of C23O during catalysis (suicide inhibition or mechanism-based inactivation) was that of Cerdan et al. (5). Briefly, purified C23O (50 to 100 ng) was added to 1 ml of 100 mM potassium phosphate buffer (pH 7.5) containing catechol, 3-methylcatechol, or 4-methylcatechol at various concentrations, and the formation of the ring cleavage products was measured by the absorbance change at 375, 388, or 382 nm, respectively. After the initiation of the enzyme reaction, the absorbance increased. The rate of the absorbance change decreased as the enzyme became inactivated. The slope of the absorbance change, dA(t)/dt, was calculated at each point. When the logarithms of the dA(t)/dt values were plotted against t, a straight line was obtained. The slope represented the k_inactγ value. In this formula, k_inact and γ are the rate constant for enzyme inactivation and a constant, respectively (5, 6). The γ value is constant if the concentrations of the substrates are constant. The k_inactγ values were calculated at different substrate concentrations. When the concentrations of substrates, catechol or substituted catechols, and molecular oxygen are significantly higher than their respective K_m values, the γ value becomes 1. Curve fitting was carried out by using GraphPad Prism, version 3.03, for Windows (GraphPad Software) with nonlinear regression based on a one-site binding equation.

In vitro inactivation and reactivation of C23Os.Purified NY8 or XylE was diluted to 100 μM (14.8 μg/ml) in a 25 mM 3-(N-morpholino) propane sulfonic acid (MOPS) buffer (pH 7.5) containing 1% (vol/vol) isopropanol, and the solution was incubated with freshly prepared 200 μM 4-methylcatechol for 30 min at room temperature. As a control, an equivalent amount of each of these enzymes was similarly treated without 4-methylcatechol. After extensive dialysis twice against 1,000 volumes of a 25 mM MOPS buffer (pH 7.5) containing 1% (vol/vol) isopropanol, each enzyme sample was made anaerobic and kept under argon for 30 min at 4°C. The C23Os were then chemically reactivated in an anaerobic chamber in the presence of 1 mM FeSO₄ and 1 mM ascorbate (31). Samples were taken at different times, and their C23O activities were measured under the standard conditions.

Nucleotide sequence accession number.The nucleotide sequence data reported were deposited in the nucleotide sequence databases DDBJ, EMBL, GenBank, and GSDB under accession number AB074512 .

Plasmid construction.PaW94 is a P. putida mutant defective in benzoate 1,2-dioxygenase. This strain cannot, therefore, grow on benzoate (25). The pRGmeta plasmid (Fig. 2B) was constructed as described in Materials and Methods. This plasmid contained all of the genes for the meta cleavage pathway enzymes except xylT and xylE. When this plasmid was introduced into P. putida PaW94 by electroporation, the transformant could grow on benzoate but not on m-toluate or p-toluate. This strain could grow on benzoate because benzoate was transformed to catechol by toluate 1,2-dioxygenase (XylXYZ) and cis-1,2-dihydroxycyclohexa-3,5-diene dehydrogenase (XylL) encoded by pRGmeta, which was further metabolized by ortho cleavage pathway enzymes encoded by genes on the P. putida PaW94 chromosome. This strain could not grow on m-toluate or p-toluate because 3-methylcatechol and 4-methylcatechol that had been formed from the respective substrates could not be metabolized by the ortho pathway enzymes.

The xylE or xylT-xylE gene sequence was amplified by PCR and subcloned at the NheI and HindIII sites of pRGmeta. The cloned genes in these constructs were under the control of the constitutively expressed Pr promoter (13, 25, 26). When pRGmeta carrying xylE (pRGmeta-XylE) was introduced into PaW94, the transformant could grow on benzoate and m-toluate but not on p-toluate as the sole carbon source (Table 1). This growth phenotype of PaW94 harboring pRGmeta-XylE was exactly the same as the phenotype of P. putida harboring pWW0 defective in xylT (36). p-Toluate was not a growth substrate for these strains because 4-methylcatechol, a metabolite of p-toluate, is a suicide inhibitor of C23O; during the catalysis of 4-methylcatechol, catalytic Fe(II) of C23O is oxidized to Fe(III), and the enzyme is inactivated (2). When pRGmeta carrying both xylT and xylE (pRGmeta-XylTE) was introduced into PaW94, the transformant could grow on benzoate, m-toluate, and p-toluate like P. putida harboring wild-type pWW0 (Table 1). P. putida possessing xylT could grow on p-toluate because the gene product of xylT, a plant-type [2Fe-2S] ferredoxin, regenerates C23O inactivated by 4-methylcatechol (19, 39).

Screening of C23O with improved activity against 4-methylcatechol.Genomic DNA was isolated from phenol-acclimatized activated sludge. This sludge sample contained a variety of phenol-degrading bacteria, many of which were likely to harbor C23O genes (28). The central parts of the C23O genes were prepared by PCR with this DNA sample, while the 5′ and 3′ ends of the C23O genes were prepared by PCR with xylE of the pWW0 plasmid (12) or with nahH of the NAH7 plasmid (13) as a template. Primers for the amplification of the central parts of C23O genes were designed based on amino acid sequences that are highly conserved among extradiol dioxygenases (9, 32). The cassette PCR products were subcloned into pRGmeta and then introduced into P. putida PaW94 by electroporation (25), and the transformants capable of growing on p-toluate were screened. Although many transformants grew on M9 minimal agar plates containing 5 mM p-toluate as a sole carbon source, almost all colonies became brownish. This development indicated that the intermediate, 4-methylcatechol, was accumulated in the transformants without being utilized. When these brownish colonies were replicated on p-toluate at the lower concentration (0.5 mM), they did not grow and then were discarded. In contrast, nonbrownish colonies could grow even on the minimal agar plates with 0.5 mM p-toluate. These clones were cultivated in the liquid minimal medium with 0.5 mM p-toluate several times, and one clone that reproducibly grew in the liquid medium, PaW94(pRGmeta-NY8), was thus isolated. When the C23O gene from pRGmeta-NY8 was recloned into pRGmeta at the same restriction sites and introduced into P. putida PaW94, the resulting transformant was capable of growing on M9 minimal agar plates with 0.5 mM p-toluate as before, indicating that the isolated C23O gene, indeed, conferred to PaW94 the ability to grow on p-toluate (Table 1). PaW94(pRGmeta-NY8) grew twofold slower than PaW94(pRGmeta-XylTE) on p-toluate.

The nucleotide sequence of the C23O gene on pRGmeta-NY8 was determined. The deduced amino acid sequence of NY8 showed NahH and XylE sequences in the N-and C-terminal regions, respectively (Fig. 3), and the central region showed 86 and 84% similarity to those of C23Os from P. putida strain H (PhlH) (18) and Pseudomonas sp. strain CF600 (DmpB) (3).

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FIG. 3.

Alignment of the deduced amino acid sequences of C23Os. Dots indicate residues identical to those in NY8, and arrows indicate iron ligand residues. Gray-shaded boxes represent the PCR primer regions used for cassette PCR. The sequences are as follows: NY8, hybrid C23O obtained in this study; XylE, C23O of P. putida mt-2 (28); NahH, C23O of P. putida PpG7 (45); PhlH, C23O of P. putida strain H (18); DmpB, C23O of Pseudomonas sp. strain CF600 (3).

Kinetic parameters.Two C23Os, XylE and NY8, were purified, and their K_m and k_cat values for catechol, 3-methylcatechol, and 4-methylcatechol were determined (Table 2). The K_m and k_cat values of XylE for catechol, 3-methylcatechol, and 4-methylcatechol were almost identical to results obtained previously (6). The K_m values of NY8 for catechol, 3-methylcatechol, and 4-methylcatechol were the same as or even smaller than those of XylE. On the other hand, the k_cat values of NY8 for all of the substrates were much smaller than those of XylE. In our experience, the hybrid C23Os sometimes shows the lower catalytic activity, partly because the formation of a tetramer, which is absolutely necessary for enzyme activity, is insufficient (33).

During the catalysis of 3-methylcatechol and 4-methylcatechol, the catalytic speed of XylE and NY8 decreased with time as the enzymes were inactivated concomitantly by oxidation of the substrates. The rate of enzyme inactivation could be quantified by determining k_inactγ at different substrate concentrations. Since the K_m values of both enzymes for 4-methylcatechol were around 2 μM (Table 2), the value of γ was supposed to be close to 1 at a concentration of more than 300 μM, and the value of k_inactγ was taken as k_inact. The k_inact value for 4-methylcatechol was calculated by curve fitting (Fig. 4A) or with a double-reciprocal plot (Fig. 4B). The k_inact values of XylE and NY8 were not much different from each other (6.8 × 10⁻³ ± 0.3 × 10⁻³ and 6.5 × 10⁻³ ± 0.5 × 10⁻³ s⁻¹, respectively). On the other hand, the concentrations of 4-methylcatechol required for the half-maximum k_inact values of XylE and NY8 were 9.7 ± 2.9 and 30.8 ± 8.9 μM, respectively (Fig. 4). The k_inact values of NY8 and XylE for 3-methylcatechol were 6.3 × 10⁻³ and 3.3 × 10⁻³ s⁻¹, respectively. The k_inact values for catechol of NY8 and XylE were not very different from each other (1.0 × 10⁻³ and 0.8 × 10⁻³ s⁻¹, respectively).

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FIG. 4.

k _inactγ values for 4-methylcatechol. The k_inactγ values for 4-methylcatechol at various concentrations were determined as described in Materials and Methods by curve fitting (A) and with a double-reciprocal plot (B). For XylE (open circles), the dotted curve was calculated assuming a k_inact value of 6.8 × 10⁻³ ± 0.3 × 10⁻³ s⁻¹ and the concentration of 4-methylcatechol required for the half-maximum k_inact value was 9.7 ± 2.9 μM. For NY8 (closed circles), the solid curve was calculated assuming a k_inact value of 6.5 × 10⁻³ ± 0.5 × 10⁻³ s⁻¹ and the concentration of 4-methylcatechol required for the half-maximum k_inact value was 30.8 ± 8.9 μM.

In vitro reactivation of C23O.XylE and NY8 were inactivated by exposure to 200 μM 4-methylcatechol. After this treatment, XylE and NY8 both showed 1 to 3% of their initial activities. After dialysis of the inactivated enzymes against the 25 mM MOPS buffer (pH 7.5) containing 1% (vol/vol) isopropanol, the C23Os were chemically reactivated under the anaerobic condition in the presence of 1 mM FeSO₄ and 1 mM ascorbate (31). Samples were taken at different times, and their C23O activities were determined under the standard conditions. Under these conditions, the time required for XylE to recover the half-maximum activity was 2.8 min, while that required for NY8 was 0.5 min, showing that NY8 could be reactivated much faster than XylE without XylT ferredoxin (Fig. 5).

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FIG. 5.

In vitro reactivation of C23Os. XylE (open circles) or NY8 (closed circles) inactivated by 4-methylcatechol was incubated anaerobically in 25 mM MOPS buffer (pH 7.5) with 1 mM FeSO₄ and 1 buffer mM ascorbate, and the regenerated C23O activity was measured over an interval of 30 min. The C23O activity before inactivation was taken to be 100%. The averages of five measurements of C23O activity before inactivation were 208 units/mg (XylE) and 100 units/mg (NY8). The dotted (XylE) and the solid (NY8) curves were calculated assuming that the times to half-recovery were 2.8 min for XylE and 0.5 min for NY8.