Characterization of Extradiol Dioxygenases from a Polychlorinated Biphenyl-Degrading Strain That Possess Higher Specificities for Chlorinated Metabolites

Frédéric H Vaillancourt; María-Amparo Haro; Nathalie M Drouin; Zamil Karim; Halim Maaroufi; Lindsay D Eltis

doi:10.1128/JB.185.4.1253-1260.2003

. 2003 Feb;185(4):1253–1260. doi: 10.1128/JB.185.4.1253-1260.2003

Characterization of Extradiol Dioxygenases from a Polychlorinated Biphenyl-Degrading Strain That Possess Higher Specificities for Chlorinated Metabolites

Frédéric H Vaillancourt ^1,2,^†, María-Amparo Haro ^2,^‡, Nathalie M Drouin ^2,^§, Zamil Karim ¹, Halim Maaroufi ², Lindsay D Eltis ^1,2,^*

PMCID: PMC142886 PMID: 12562795

Abstract

Recent studies demonstrated that 2,3-dihydroxybiphenyl 1,2-dioxygenase from Burkholderia sp. strain LB400 (DHBD_LB400; EC 1.13.11.39) cleaves chlorinated 2,3-dihydroxybiphenyls (DHBs) less specifically than unchlorinated DHB and is competitively inhibited by 2′,6′-dichloro-2,3-dihydroxybiphenyl (2′,6′-diCl DHB). To determine whether these are general characteristics of DHBDs, we characterized DHBD_P6-I and DHBD_P6-III, two evolutionarily divergent isozymes from Rhodococcus globerulus strain P6, another good polychlorinated biphenyl (PCB) degrader. In contrast to DHBD_LB400, both rhodococcal enzymes had higher specificities for some chlorinated DHBs in air-saturated buffer. Thus, DHBD_P6-I cleaved the DHBs in the following order of specificity: 6-Cl DHB > 3′-Cl DHB ∼ DHB ∼ 4′-Cl DHB > 2′-Cl DHB > 4-Cl DHB > 5-Cl DHB. It also cleaved its preferred substrate, 6-Cl DHB, three times more specifically than DHB. Interestingly, some of the worst substrates for DHBD_P6-I were among the best for DHBD_P6-III (4-Cl DHB > 5-Cl DHB ∼ 6-Cl DHB ∼ 3′-Cl DHB > DHB > 2′-Cl DHB ∼ 4′-Cl DHB; DHBD_P6-III cleaved 4-Cl DHB two times more specifically than DHB). Generally, each of the monochlorinated DHBs inactivated the enzymes more rapidly than DHB. The exceptions were 4-Cl DHB for DHBD_P6-I and 2′-Cl DHB for DHBD_P6-III. As observed in DHBD_LB400, chloro substituents influenced the reactivity of the dioxygenases with O₂. For example, the apparent specificities of DHBD_P6-I and DHBD_P6-III for O₂ in the presence of 2′-Cl DHB were lower than those in the presence of DHB by factors of >60 and 4, respectively. DHBD_P6-I and DHBD_P6-III shared the relative inability of DHBD_LB400 to cleave 2′,6′-diCl DHB (apparent catalytic constants of 0.088 ± 0.004 and 0.069 ± 0.002 s⁻¹, respectively). However, these isozymes had remarkably different apparent K_m values for this compound (0.007 ± 0.001, 0.14 ± 0.01, and 3.9 ± 0.4 μM for DHBD_LB400, DHBD_P6-I, and DHBD_P6-III, respectively). The markedly different reactivities of DHBD_P6-I and DHBD_P6-III with chlorinated DHBs undoubtedly contribute to the PCB-degrading activity of R. globerulus P6.

The microbial degradation of polychlorinated biphenyls (PCBs) has been intensively studied as a possible means to destroy these toxic, persistent environmental pollutants. Particular attention has focused on the bph pathway, which is responsible for the aerobic degradation of biphenyl in a wide range of microorganisms (1, 21). The upper bph pathway consists of four enzymatic activities which together transform biphenyl to benzoate and 2-hydroxypenta-2,4-dienoate. These enzymes also transform some of the approximately 100 different congeners of chlorinated biphenyl that can occur in commercial mixtures of PCBs. The range of PCBs that is transformed by the bph pathway is highly dependent upon the bacterial strain. For example, Burkholderia sp. strain LB400 (Burkholderia sp. strain LB400 was originally identified as Pseudomonas sp. strain LB400 [13]) and Rhodococcus globerulus strain P6 (R. globerulus strain P6 has variously been identified as Acinetobacter sp. strain P6, Corynebacterium sp. strain MB1, and Arthrobacter sp. strain M5 [7, 22]) transform exceptionally broad, but different, ranges of PCBs, including some hexachlorinated congeners (13, 29). Burkholderia sp. strain LB400 transforms ortho-chlorinated or non-dioxin-like congeners relatively well. In contrast, R. globerulus P6 preferentially transforms more coplanar or dioxin-like congeners exemplified by 3,3′-dichloro (3,3′-diCl) and 4,4′-diCl biphenyls (11, 12).

The optimization of microbial catabolic activities for the biodegradation of PCBs necessitates the determination of the specificity of bph pathway enzymes for chlorinated metabolites. PCB transformation has been studied largely as a function of biphenyl dioxygenase, the first enzyme of the bph pathway (for recent examples, see references 10 and 42). A few studies have focused on the second enzyme of the pathway (15, 39). However, its role in PCB degradation remains to be fully elucidated. More recently, the fourth enzyme of the pathway was identified as an important determinant of PCB degradation, being competitively inhibited by some chlorinated metabolites (36, 37).

2,3-Dihydroxybiphenyl 1,2-dioxygenase (DHBD; EC 1.13.11.39) is the third enzyme of the bph degradation pathway. This enzyme utilizes a mononuclear nonheme iron(II) center to cleave 2,3-dihydroxybiphenyl (DHB) in an extradiol fashion (Fig. 1). Whole-cell studies indicate that DHBD is of particular significance to the degradation of PCBs, as it is incapable of transforming certain chlorinated DHBs (23, 38). Recent studies demonstrated that DHBD from Burkholderia sp. strain LB400 (DHBD_LB400) cleaves chlorinated DHBs less specifically than unchlorinated DHB and is competitively inhibited by 2′,6′-diCl DHB (17). DHBD is also inhibited by 3-chlorocatechol (2, 5, 8, 41, 44), a compound that is formed during the degradation of PCBs. In the DHBD-catalyzed reaction, the catechol binds to the enzyme first, activating the active-site Fe(II) for O₂ binding (3, 4, 27, 43). During steady-state cleavage, DHBD is subject to two types of inhibition: reversible substrate inhibition and an irreversible mechanism-based inactivation, or suicide inhibition (43). The suicide inhibition of DHBD by DHB and 3-chlorocatechol involves the release of superoxide from the DHBD:DHB:O₂ ternary complex and the oxidation of the active-site Fe(II) (44). It is unclear whether the reactivity of DHBD_LB400 with PCB metabolites is representative of that of other DHBDs.

R. globerulus strain P6 contains three different enzymes that have been identified as DHBDs based on their abilities to cleave DHB (6, 8). Of the three isozymes, DHBD_P6-I, encoded by bphC1, appears to be the functional homologue of DHBD_LB400: both are two-domain enzymes (25), sharing 52% sequence identity, and the genes encoding these enzymes occur in the bph operon. The physiological function of DHBD_P6-II and DHBD_P6-III, encoded by bphC2 and bphC3, respectively, is less clear. These isozymes are single-domain enzymes that share 58% sequence identity with each other and approximately 17% sequence identity with the C-terminal domain of the two-domain enzymes (19, 25). Analysis of the DNA sequence surrounding bphC2 and bphC3 reveals no significant similarity to other bph genes. Gel filtration results indicate that R. globerulus P6 expresses DHBD_P6-I and DHBD_P6-II and/or DHBD_P6-III when grown on biphenyl as the sole carbon source (6). Interestingly, several other gram-positive actinomycetes also possess multiple genes coding for DHBDs (30, 32, 35, 40). Most of these organisms are able to transform a wide range of PCBs and other aromatic compounds. However, the relationship between these catabolic capabilities and the occurrence of multiple DHB-cleaving enzymes in a single organism is unclear.

Herein, we report the reactivity of DHBD_P6-I and DHBD_P6-III towards each of the six monochlorinated DHBs. Steady-state kinetic studies based on the general mechanism of DHBD (Fig. 2) were conducted to determine the apparent specificities of the enzyme for DHBs and O₂. The reactivity of these two DHBDs towards the recalcitrant 2′,6′-diCl DHB was also investigated. Finally, the suicide inhibition of these enzymes by each of the DHBs was studied. The results are discussed with respect to the reactivity of DHBD_LB400 with these compounds as well as the physiological significance of the occurrence of evolutionarily divergent DHBDs in a single bacterium.

FIG. 2. — General mechanism of DHBD during steady-state turnover. Only DHBD_P6-I is subject to substrate inhibition. E* denotes inactivated forms of the enzyme. The rate constants j₁ and j₃ are associated with reactions that result in enzyme inactivation.

MATERIALS AND METHODS

Chemicals.

DHB, 4-Cl DHB, 5-Cl DHB, 6-Cl DHB, 2′-Cl DHB, 3′-Cl DHB, 4′-Cl DHB, and 2′,6′-diCl DHB were synthesized according to standard procedures (33) and were a gift from Victor Snieckus. Ferene S was from ICN Biomedicals, Inc. (Costa Mesa, Calif.). All other chemicals were of analytical grade and used without further purification.

Strains, media, and growth.

Chromosomal DNA of R. globerulus strain P6 was a kind gift from Michel Sylvestre. Escherichia coli strain DH5α was used for DNA propagation and was cultured at 37°C and 200 rpm in Luria-Bertani broth with the appropriate antibiotics. Pseudomonas putida strain KT2442 (26), transformed with pVLT31 (18) derivatives, was used for overexpression and was cultured at 30°C and 250 rpm in Luria-Bertani broth with 20 μg of tetracycline/ml supplemented with phosphate buffer and mineral salts as previously described (43).

Construction of plasmids and overexpression of protein.

DNA was purified, restricted, ligated, and transformed according to standard protocols (9). The bphC1 and bphC3 genes were amplified by PCR from the chromosomal DNA of R. globerulus strain P6 that had been partially digested with BamHI. The bphC1 gene was amplified with two primers: C1for (5′-GCGCCATGGGTGTTCAGCGACTGGGTT-3′), which introduces an NcoI site (underlined) at the beginning of the bphC1 gene and modifies the second codon (Ser→Gly), and C1rev (5′-CCGCTGCAGGCCCTTACTCTCCC-3′), which introduces a PstI site (underlined) after the stop codon of the bphC1 gene. The bphC3 gene was amplified with two other primers: C3for (5′-GCCCCATGGCCGTCACACCGCGCCTTG-3′), which introduces an NcoI site (underlined) at the start of the bphC3 gene and modifies the second codon (Thr→Ala), and C3rev (5′-CGGCTGCAGATCAGGGGGTCAG-3′), which introduces a PstI site (underlined) after the stop codon of the bphC3 gene. PCRs were performed by using Pfu DNA polymerase (Stratagene, La Jolla, Calif.) according to the manufacturer's instructions and an oligonucleotide hybridization step at 48°C. The resulting DNA fragments were digested with NcoI and PstI and cloned in pLEHP20 (20), yielding the expression plasmids pHMC1-1 and pHMC3-1 for bphC1 and bphC3, respectively. The genes were sequenced on an ABI 373 Strech (Applied Biosystems, Foster City, Calif.) apparatus with BigDye terminators to confirm that they contained no errors. Both genes were subcloned using XbaI and PstI into the pVLT31 vector (18), yielding plasmids pHMC1-2 and pHMC3-2, containing bphC1 and bphC3, respectively. DHBD_P6-I and DHBD_P6-III were overexpressed in P. putida strain KT2442 freshly transformed with pHMC1-2 and pHMC3-2, respectively. The cultures were grown to an optical density at 600 nm of approximately 0.5, at which point 0.25 mM isopropyl-1-thio-β-d-galactopyranoside was added to induce expression. The cultures were incubated for an additional 20 h before harvesting.

Protein purification.

All buffers were prepared by using water purified on a Barnstead NANOpure UV apparatus to a resistivity of greater than 17 MΩ×cm. After disruption of the cell preparations, DHBD_P6-I and DHBD_P6-III were manipulated under an inert atmosphere in an Mbraun (Stratham, N. H.) Labmaster glove box maintained at 2 ppm of O₂ or less. Chromatography was performed as previously described (43) on an AKTA Explorer 100 (Amersham Biosciences, Montreal, Quebec, Canada) configured to maintain an anaerobic atmosphere during purification. Buffers were sparged with N₂ and equilibrated in the glove box for at least 24 h prior to the addition of t-butanol, dithiothreitol (DTT), and ferrous ammonium sulfate when appropriate.

Cell pellets from 4 and 2 liters of culture were resuspended in 10 mM Tris, pH 7.5, for DHBD_P6-I and DHBD_P6-III, respectively. The cells were disrupted by two successive passages through a French press operated at 12,500 lb/in² and 4°C. The cell debris was removed by ultracentrifugation in gas-tight tubes at 180,000 × g for 60 min. The clear supernatant was carefully removed and referred to as the raw extract.

The raw extract containing DHBD_P6-I (approximately 17 ml) was divided into four equal portions, each of which was loaded onto an HR 10/10 Mono Q anion exchange column (Amersham Biosciences) equilibrated with buffer A (10 mM Tris [pH 7.5], 10% t-butanol, 2 mM DTT, 0.25 mM ferrous ammonium sulfate). The column was operated at a flow rate of 3.0 ml/min. The enzyme activity was eluted with a 180-ml linear gradient of 20 to 50% buffer B (buffer A containing 1.0 M NaCl). Fractions of 5 ml were collected. DHBD_P6-I eluted at approximately 400 mM NaCl, and purity was verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (9). Fractions containing activity from the four runs were combined and concentrated in an Amicon stirred cell concentrator (Millipore, Ltd., Nepean, Ontario, Canada) to 25 to 30 mg/ml and flash-frozen as beads in liquid nitrogen.

The raw extract containing DHBD_P6-III (approximately 19 ml) was divided into five equal portions, each of which was loaded onto an HR 10/10 Mono Q anion exchange column equilibrated with buffer C (10 mM Tris [pH 7.5], 1 mM DTT, 0.15 mM ferrous ammonium sulfate). The column was operated at a flow rate of 2.5 ml/min. Proteins that were not bound tightly to the resin were eluted with a 30-ml wash of 10% buffer D (buffer C containing 1.0 M NaCl). The enzyme activity was eluted using a 120-ml linear gradient of 10 to 40% buffer D. Fractions of 5 ml were collected. DHBD_P6-III eluted at approximately 190 mM NaCl. Fractions containing activity from the five runs were combined and concentrated in an Amicon stirred cell concentrator until the volume reached approximately 10 ml. To this solution, an equal volume of buffer E (buffer C containing 0.82 M ammonium sulfate) was added, and the protein was divided into five equal portions, each of which was loaded on a phenyl-Sepharose column (1 by 10 cm; Amersham Biosciences) equilibrated with 50% buffer E. The column was operated at a flow rate of 1.0 ml/min. Proteins that were not bound tightly to the resin were eluted with a 25-ml wash of 50% buffer E. The enzyme activity was eluted with a 60-ml linear gradient of 50 to 0% buffer E. Fractions of 2.5 ml were collected. DHBD_P6-III eluted at approximately 5% buffer E, and purity was verified by SDS-PAGE (9). Fractions containing activity from the five runs were combined, and several steps of concentration and dilution with buffer C were performed in an Amicon stirred cell concentrator until no ammonium sulfate remained. The final protein concentration reached 20 to 25 mg/ml, and the solution was flash-frozen as beads in liquid nitrogen. Preparations of purified DHBD_P6-III and DHBD_P6-I were stored at −80°C for several months without loss of activity.

Handling of DHBD samples.

Aliquots of DHBD were anaerobically thawed immediately prior to use and exchanged into 20 mM 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid (HEPPS)-80 mM NaCl (ionic strength [I] = 0.1), pH 8.0, by gel filtration chromatography (43) unless otherwise stated. Samples of DHBD were further diluted with the same buffer as required.

SDS-PAGE was performed in a Bio-Rad MiniPROTEAN II apparatus, and the gels were stained with Coomassie blue according to established procedures (9). Protein concentrations were determined by the Bradford method (14). Iron concentrations were determined colorimetrically by using Ferene S (24).

Kinetic measurements and analysis of steady-state data.

Enzymatic activity was routinely measured by following the consumption of dioxygen with a Clark-type polarographic O₂ electrode (model 5301; Yellow Springs Instruments, Yellow Springs, Ohio) as previously described (43). All experiments were performed with potassium phosphate buffer, pH 7.0 (I = 0.1), at 25.0 ± 0.1°C unless otherwise stated. The standard activity assay was performed with 80 μM DHB. Concentrations of active DHBD in the assay were defined by the iron content of the injected purified enzyme solution and were used in calculating the specificity, catalytic, and inactivation constants. One unit of enzymatic activity was defined as the quantity of enzyme required to consume 1 μmol of O₂ per min. The O₂ electrode was calibrated with standard concentrations of DHBD (either DHBD_LB400 [43], DHBD_P6-I, or DHBD_P6-III) and DHB.

For specificity experiments in which the concentration of the DHBs was varied in air-saturated buffer, the initial velocities obtained were fit to an equation describing a mechanism involving substrate inhibition (based on Fig. 2) or to the Michaelis-Menten equation (16), as appropriate. In each case, the substrate concentration and reaction velocities were monitored after the initiation of the reaction (10 to 20 s) and compared to the calculated values to reject assays involving greater than 15% inactivation of the enzyme (43).

The steady-state cleavage of 2′,6′-diCl DHB by DHBD could not be directly followed with the oxygen electrodes due to the very slow rate of catalysis. However, it could be observed spectrophotometrically by following the appearance of product at 391 nm in potassium phosphate buffer, pH 7.0 (I = 0.1); ɛ = 36.5 mM⁻¹ cm⁻¹. By using the spectrophotometric assay, the apparent catalytic constant kcatapp for 2′,6′-diCl DHB could be determined for both enzymes. The KmAapp of DHBD_P6-III for 2′,6′-diCl DHB was also determined by using this assay. The KmAapp of DHBD_P6-I for 2′,6′-diCl DHB was determined with DHB as a reporter substrate in the oxygen electrode assay. The concentration of DHB was varied from 3 to 85 μM (i.e., at concentrations below those at which substrate inhibition is observed), and the concentration of 2′,6′-diCl DHB was varied from 0.02 to 1.3 μM. An equation identical in form to that for competitive inhibition was fit to the data (16). In this equation, the KmAapp of 2′,6′-diCl DHB replaces the competitive inhibition constant K_ic.

For the O₂ specificity experiments, the concentration of monochlorinated DHB was fixed such that the enzyme was saturated (>10 × KmAapp) without substrate inhibition occurring. The assay buffer was equilibrated with gas mixtures containing between 2.5 and 100% O₂, as appropriate. Kinetic parameters were calculated from the initial velocities by using the Michaelis-Menten equation. All fitting was performed using the least-squares and dynamic weighting options of LEONORA (16).

Influence of copper on activity.

The influence of Cu(II) on the enzyme activity was tested by incubating DHBD_P6-I (0.24 mg/ml) and DHBD_P6-III (0.4 mg/ml) anaerobically for 2 min at 0°C in 20 mM HEPPS-80 mM NaCl, pH 8.0, containing various concentrations of CuCl₂ · 2H₂O prior to initiation of the reaction in the standard assay.

Stability in the presence of O₂ and mechanism-based inactivation studies.

The stability of the free enzyme in the presence of O₂ was studied by incubating DHBD in the oxygraph cuvette under standard assay conditions and monitoring A_t, the activity remaining after different time intervals, by adding 150 μM DHB to the cuvette. The apparent first-order rate constant of inactivation j1app was determined as previously described by using equation 1 (44), in which A_max is the activity observed in the absence of preincubation.

(1)

Partition ratios for all substrates except 2′,6′-diCl DHB were determined by an oxygraph assay according to standard procedures (44). For 2′,6′-diCl DHB, the partition ratio was determined spectrophotometrically by using the assay described in the previous section. The amount of DHBD added to the reaction cuvette was such that the enzyme was completely inactivated before 15% of either the catecholic substrate or O₂ was consumed in the reaction mixture. The partition ratio was calculated by dividing the amount of product formed by the amount of active DHBD added to the assay.

The apparent rate constant of inactivation during catalytic turnover in air-saturated buffer, j3app, was calculated from the partition ratio determined by using the oxygraph assay under saturating substrate conditions ([S] ≫ K_m) according to established procedures (equation 2; [44]) except for 2′,6′-diCl DHB. Under such conditions, the concentration of free enzyme, [E], is negligible and the partition ratio is equal to the ratio of the catalytic constant kcatapp to the inactivation constant j3app (i.e., Σj_i = j₃).

(2)

The rate constant of inactivation, j3app, for 2′,6′-diCl DHB was evaluated from the rate constant of saturation j_s by using equation 3 (44). The latter was determined from progress curves obtained spectrophotometrically at 391 nm from reactions performed at saturating substrate concentrations [S] (100 to 200 μM) by using equation 4 (44).

(3)

(4)

In equation 3, Kmapp is the apparent K_m of 2′,6′-diCl DHB in air-saturated buffer. In equation 4, P_i and P_∞ are the concentrations of product at the start and end of the assay, respectively, and P_t is the concentration of product formed after different time intervals.

RESULTS

Purification.

DHBD_P6-I and DHBD_P6-III were purified anaerobically to apparent homogeneity. Relevant details of the purifications are shown in Table 1. The enzymes were estimated to be greater than 99% pure by SDS-PAGE (data not shown). Total yields of 117 mg of DHBD_P6-I and 84 mg of DHBD_P6-III were obtained from 4 and 2 liters of cell culture, respectively. Ninefold and fivefold increases in specific activity were observed upon purification for DHBD_P6-I and DHBD_P6-III, respectively, and the iron content of the purified enzymes was typically higher than 85%.

TABLE 1.

Purification details of DHBD_P6-I and DHBD_P6-III expressed in P. putida strain KT2442^a

Isozyme and purification step	Total protein (mg)	Total act (U)	Sp act (U/mg)	Yield (%)
DHBD_P6-I
Raw extract	1,450	19,260	14.1	100
Mono Q	117	14,320	122.6	74
DHBD_P6-III
Raw extract	834	6,570	7.9	100
Mono Q	245	5,880	24.0	89
Phenyl-Sepharose	84	3,010	36.0	46

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Activity units are defined in Materials and Methods.

Coupling of the reaction and stability in the presence of O₂.

The coupling of DHB and O₂ utilization in DHBD_P6-I and DHBD_P6-III was investigated using an O₂ electrode that had been calibrated with DHB and DHBD_LB400, which constitutes a well-coupled system (43). For each P6 isozyme, the amount of O₂ consumed corresponded to the amount of DHB added to the reaction mixture, demonstrating that the utilization of DHB and O₂ was tightly coupled in both enzymes.

The stability of each enzyme in the presence of O₂ was evaluated by determining j1app, the pseudo-first-order rate constant of inactivation in air-saturated buffer. The respective j1app values of DHBD_P6-I and DHBD_P6-III were (0.7 ± 0.1) × 10⁻³ and (4.4 ± 0.5) × 10⁻³ s⁻¹, which correspond to half-lives of 16.5 ± 2 and 2.6 ± 0.3 min, respectively.

Influence of copper on DHB cleavage activity.

Incubation of DHBD_P6-I (0.24 mg/ml) in the presence of 10, 25, 50, 100, 200, and 500 μM copper chloride resulted in 24, 58, 76, 90, 94, and 95% inhibition of the enzyme's DHB cleavage activity, respectively. Similar incubation of DHBD_P6-III (0.4 mg/ml) inhibited the cleavage of DHB by 17, 35, 44, 63, 67, and 85%, respectively. Copper was therefore not included in any of the kinetic assays.

Steady-state kinetic analyses.

Steady-state kinetic analyses in which the DHB and O₂ concentrations were varied demonstrated that DHBD_P6-I and DHBD_P6-III utilize a compulsory-order, ternary-complex mechanism (Fig. 2 and data not shown). DHBD_P6-I, like DHBD_LB400, was subject to substrate inhibition. In contrast, DHBD_P6-III was not. Both P6 isozymes, like DHBD_LB400, were susceptible to inactivation during the steady-state turnover. Accordingly, the apparent specificities of DHBD_P6-I and DHBD_P6-III for chloro DHBs (kAapp = kcatapp/KmAapp) and their apparent inactivation (j3app) by these compounds were studied essentially as described previously for DHBD_LB400 (44).

The results of typical steady-state kinetic experiments performed by varying the concentration of monochlorinated DHBs in air-saturated buffer are shown in Fig. 3. Interestingly, DHBD_P6-I and DHBD_P6-III cleaved some chloro DHBs more specifically than DHB (Table 2). Thus, DHBD_P6-I cleaved 6-Cl DHB three times more specifically than DHB and cleaved 3′-Cl DHB, 4′-Cl DHB, and unchlorinated DHB with similar specificity. Overall, DHBD_P6-I cleaved monochlorinated DHBs in the following order of specificity: 6-Cl DHB > 3′-Cl DHB ∼ DHB ∼ 4′-Cl DHB > 2′-Cl DHB > 4-Cl DHB > 5-Cl DHB. Similarly, DHBD_P6-III cleaved a total of four monochlorinated DHBs more specifically than DHB, showing the highest apparent specificity for 4-Cl DHB in air-saturated buffer. DHBD_P6-III cleaved the DHBs in the following order of specificity: 4-Cl DHB > 5-Cl DHB ∼ 6-Cl DHB ∼ 3′-Cl DHB > DHB > 2′-Cl DHB ∼ 4′-Cl DHB. By comparison, DHBD_LB400 cleaved monochlorinated DHBs between 0.1 and 0.3 times that of unchlorinated DHB in the following order of specificity: DHB > 4′-Cl DHB > 6-Cl DHB > 3′-Cl DHB > 4-Cl DHB > 5-Cl DHB > 2′-Cl DHB.

FIG. 3. — Steady-state cleavage of 6-Cl DHB by DHBD_P6-I and 4-Cl DHB by DHBD_P6-III. The experiments were performed using air-saturated potassium phosphate buffer, pH 7.0 (I = 0.1), at 25°C. (A) DHBD_P6-I-catalyzed cleavage of 6-Cl DHB. The line represents a best fit of the substrate inhibition equation to the data. The fitted parameters are KmAapp = 1.9 ± 0.3 μM, KiAapp = 3.0 ± 0.9 mM, and Vmaxapp = 41.1 ± 1.2 μM/min. The inset shows the initial portion of the graph (0 to 30 μM 6-Cl DHB) in more detail. (B) DHBD_P6-III-catalyzed cleavage of 4-Cl DHB. The line represents a best fit of the Michaelis-Menten equation to the data. The fitted parameters are KmAapp = 1.8 ± 0.2 μM and v = 52.7 ± 1.2 μM/min. The inset shows the initial portion of the graph (0 to 30 μM 4-Cl DHB) in more detail.

TABLE 2.

Apparent steady-state kinetic parameters and inactivation parameters of DHBD_P6-I, DHBD_P6-III, and DHBD_LB400 for chlorinated DHBs^a

Isozyme and compound	K_mA^app (μM)	K_iA^app (mM)	k_cat^app (s⁻¹)	k_A^app (10⁶ M⁻¹ s⁻¹)	Partition ratio	j₃^app (10⁻³ s⁻¹)	j₃^app/K_mA^app (10³ M⁻¹ s⁻¹)
DHBD_P6-I
DHB	5.8 (1.0)	2.2 (0.7)	77.5 (2.7)	13.3 (1.9)	31,100 (3,300)	2.5 (0.4)	0.43 (0.14)
2′-Cl DHB	0.22 (0.06)		2.02 (0.04)	9 (2)	1,650 (80)	1.2 (0.1)	5.5 (2.0)
3′-Cl DHB	1.9 (0.3)	1.5 (0.3)	27.1 (0.8)	14.2 (1.8)	2,300 (200)	11.8 (1.3)	6.2 (1.7)
4′-Cl DHB	2.6 (0.3)	6.6 (3.4)	29.1 (0.7)	11.4 (1.0)	3,260 (130)	8.9 (0.6)	3.5 (0.6)
4-Cl DHB	22.0 (2.6)	2.6 (0.7)	109 (5)	4.9 (0.4)	27,800 (200)	3.9 (0.2)	0.18 (0.03)
5-Cl DHB	5.3 (0.6)		7.2 (0.2)	1.4 (0.1)	660 (35)	11.0 (0.8)	2.1 (0.4)
6-Cl DHB	1.9 (0.3)	3.0 (0.9)	72.5 (2.2)	39 (5)	4,085 (25)	17.7 (0.6)	9.4 (1.8)
2′6′-diCl DHB	0.144 (0.014)		0.088 (0.004)	0.61 (0.09)	49.9 (1.2)	1.7 (0.1)	11.7 (1.6)
DHBD_P6-III
DHB	16.7 (1.9)		17.0 (0.6)	1.0 (0.1)	895 (60)	19.0 (2.0)	1.1 (0.2)
2′-Cl DHB	10.4 (1.0)		5.3 (0.1)	0.51 (0.04)	1,990 (160)	2.7 (0.3)	0.25 (0.05)
3′-Cl DHB	10.4 (0.7)		14.2 (0.3)	1.36 (0.08)	870 (70)	16.3 (1.7)	1.6 (0.3)
4′-Cl DHB	27.0 (3.0)		12.7 (0.4)	0.47 (0.04)	410 (60)	31.2 (5.5)	1.2 (0.3)
4-Cl DHB	1.8 (0.2)		4.0 (0.1)	2.2 (0.2)	260 (40)	15.5 (3.0)	8.6 (2.6)
5-Cl DHB	4.3 (0.6)		6.6 (0.3)	1.5 (0.2)	97 (20)	68.0 (17.1)	15.8 (6.2)
6-Cl DHB	3.9 (0.8)		5.5 (0.3)	1.4 (0.2)	200 (10)	27.8 (3.1)	7.1 (2.2)
2′6′-diCl DHB	3.9 (0.4)		0.069 (0.002)	0.017 (0.001)	14.8 (0.3)	5.4 (0.2)	1.4 (0.1)
DHBD_LB400
DHB	12 (1)	2.7 (0.6)	251 (6)	21 (1)	84,900 (1,400)	3.0 (0.1)	0.25 (0.10)
2′-Cl DHB	2.3 (0.2)	2.7 (0.6)	5.0 (0.7)	2.1 (0.2)	1,390 (150)	3.6 (0.9)	1.6 (0.5)
3′-Cl DHB	58 (5)	0.40 (0.05)	305 (19)	5.3 (0.2)	42,000 (14,000)	7.3 (2.9)	0.13 (0.06)
4′-Cl DHB	68 (11)	0.38 (0.07)	430 (50)	6.4 (0.4)	40,600 (5,500)	10.6 (2.7)	0.16 (0.07)
4-Cl DHB	23 (1)		72 (1)	3.1 (0.1)	10,850 (400)	6.6 (0.3)	0.29 (0.06)
5-Cl DHB	26.4 (3.1)	2.5 (0.7)	63 (3)	2.4 (0.2)	12,000 (1,500)	5.2 (0.9)	0.20 (0.06)
6-Cl DHB	6.5 (0.6)	1.28 (0.15)	38.3 (0.9)	5.9 (0.4)	5,500 (470)	7.0 (0.8)	1.1 (0.2)
2′6′-diCl DHB	0.007 (0.001)		0.036 (0.001)	5.1 (0.9)	49.5 (1.6)	0.69 (0.01)	99 (16)

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Experiments with DHBD_P6-I and DHBD_P6-III were performed using air-saturated potassium phosphate buffer, pH 7.0 (I = 0.1), at 25°C. K_mA^app, K_iA^app, k_cat^app, k_A^app, and j₃^app represent the apparent K_m, inhibition constant, catalytic constant, specificity constant, and rate constant of inactivation during catalytic turnover, respectively, for DHB or chloro DHBs. All DHBD_LB400 data except K_iA^app were either taken or extrapolated from Dai et al. (17). Values in parentheses represent standard errors.

The apparent specificities of the two P6 isozymes for O₂ were studied at saturating concentrations of DHB and each of the six monochlorinated DHBs (Table 3). The apparent specificity constant of both DHBD_P6-I and DHBD_P6-III for O₂ (kBapp = kcatapp/KmBapp) was highest in the presence of DHB. In contrast, the k_B of DHBD_LB400 was highest in the presence of 3′-Cl DHB and 4′-Cl DHB (17). Moreover, chlorinated DHBs influenced the respective reactivities of DHBD_P6-I and DHBD_P6-III with O₂ in different ways. For example, the KmBapp of DHBD_P6-I was lowest in the presence of 4′-Cl DHB whereas that of DHBD_P6-III was highest in the presence of this same compound. Nevertheless, the lowest kBapp value of both isozymes was observed in the presence of 2′-Cl DHB, the most slowly cleaved substrate. However, the decrease in specificity versus that observed in the presence of DHB was more drastic for DHBD_P6-I (>60-fold) than for DHBD_P6-III (3.6-fold). In comparison, the k_B of DHBD_LB400 was 50-fold lower in the presence of 2′-Cl DHB than in the presence of DHB (17).

TABLE 3.

Apparent steady-state kinetic parameters of DHBD_P6-I and DHBD_P6-III for O₂ in the presence of monochlorinated DHBs^a

Isozyme and compound	Concn of (Cl-) DHB (μM)	K_mB^app (μM)	k_cat^app (s⁻¹)	k_B^app (10⁶ M⁻¹ s⁻¹)
DHBD_P6-I
DHB	275	253 (16)	121 (4)	0.48 (0.02)
2′-Cl DHB	35	>5,000	∼40	<0.008
3′-Cl DHB	150	240 (16)	32 (1)	0.134 (0.005)
4′-Cl DHB	150	167 (4)	32.1 (0.4)	0.193 (0.003)
4-Cl DHB	750	750 (120)	200 (16)	0.269 (0.022)
5-Cl DHB	250	334 (52)	10.7 (0.8)	0.032 (0.003)
6-Cl DHB	150	425 (28)	103 (4)	0.243 (0.008)
DHBD_P6-III
DHB	270	480 (80)	45.6 (3.5)	0.095 (0.009)
2′-Cl DHB	200	700 (150)	18.0 (1.8)	0.026 (0.003)
3′-Cl DHB	200	387 (50)	26.1 (1.3)	0.067 (0.006)
4′-Cl DHB	350	726 (82)	43.5 (2.8)	0.060 (0.003)
4-Cl DHB	85	116 (17)	5.0 (0.2)	0.044 (0.005)
5-Cl DHB	55	619 (100)	19.0 (1.4)	0.031 (0.003)
6-Cl DHB	70	440 (120)	15.7 (1.6)	0.036 (0.006)

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Experiments were performed with potassium phosphate buffer, pH 7.0 (I = 0.1), at 25°C with saturating, but noninhibiting, concentrations of (monochlorinated) DHBs. K_mB^app, k_cat^app, k_B^app represent the apparent K_m, catalytic constant, and specificity constant, respectively, for O₂ in the presence of DHM or chloro DHBs. Values in parentheses represent standard errors.

As with DHBD_LB400, the cleavage of 2′,6′-diCl DHB by DHBD_P6-I and DHBD_P6-III was too slow to follow directly with the oxygen electrodes. By using a spectrophotometric assay to follow product formation, the KmAapp of DHBD_P6-III for 2′,6′-diCl DHB was 3.9 ± 0.4 μM (Fig. 4). The KmAapp of DHBD_P6-I for 2′,6′-diCl DHB could not be determined by this assay due to the very low KmAapp of the enzyme for this compound. By using DHB as a reporter substrate in the oxygen electrode assay, 2′,6′-diCl DHB competitively inhibited the cleavage of DHB by DHBD_P6-I with a Kicapp of 0.144 ± 0.014 μM in air-saturated buffer (Fig. 4). By using the same assay, 2′,6′-diCl DHB competitively inhibited the cleavage of DHB by DHBD_P6-III with a Kicapp of 2.6 ± 0.2 μM, confirming the spectrophotometrically determined value. Under saturating substrate conditions, 2′,6′-diCl DHB was cleaved 900 and 250 times slower than DHB by DHBD_P6-I and DHBD_P6-III, respectively (Table 2).

FIG. 4. — Steady-state utilization of 2′,6′-diCl DHB by DHBD_P6-I and DHBD_P6-III. The experiments were performed using air-saturated potassium phosphate buffer, pH 7.0 (I = 0.1), at 25°C. (A) DHBD_P6-I-catalyzed cleavage of DHB in the presence of 2′,6′-diCl DHB. The rate of DHB cleavage was determined by using 3.1 μM (□), 5.4 μM (▪), 9.3 μM (▵), 13.1 μM (○), 16.9 μM (+), 54 μM (•), and 82.9 μM (×) DHB. Fits were obtained using an equation similar in form to that describing competitive inhibition as described in Materials and Methods. The fit of the equation to the data yielded the following parameters: KmAapp = 0.144 ± 0.014 μM, KmDHBapp = 3.4 ± 0.3 μM, and Vmaxapp = 60.4 ± 1.5 μM/min. (B) The DHBD_P6-III-catalyzed cleavage of 2′,6′-diCl DHB. The line represents a best fit of the Michaelis-Menten equation to the data. The fitted parameters are Kmaxapp = 3.9 ± 0.4 μM and Vmaxapp = 3.0 ± 0.1 μM/min. The inset shows the initial portion of the graph (0 to 50 μM 2′,6′-diCl DHB) in more detail.

The DHBD isozymes had different susceptibilities to inactivation (j3app/KmAapp) by the chlorinated substrates. Thus, DHBD_P6-I was inactivated by these compounds in the following order: 2′,6′-diCl DHB > 6-Cl DHB > 3′-Cl DHB > 2′-Cl DHB > 4′-Cl DHB > 5-Cl DHB > DHB > 4-Cl DHB. In contrast, DHBD_P6-III was inactivated by these compounds in the following order: 5-Cl DHB > 4-Cl DHB > 6-Cl DHB > 3′-Cl DHB > 2′,6′-diCl DHB > 4′-Cl DHB > DHB > 2′-Cl DHB. These patterns differ from that of DHBD_LB400 (2′,6′-diCl DHB > 2′-Cl DHB > 6-Cl DHB > 4-Cl DHB > DHB > 5-Cl DHB > 4′-Cl DHB > 3′-Cl DHB [17]). In the presence of saturating concentrations of individual substrates, DHBD_P6-I would be inactivated by these compounds in the following order: 6-Cl DHB > 3′-Cl DHB > 5-Cl DHB > 4′-Cl DHB > 4-Cl DHB > DHB > 2′,6′-diCl DHB > 2′-Cl DHB. In contrast, DHBD_P6-III would be inactivated by these compounds in the following order: 5-Cl DHB > 4′-Cl DHB > 6-Cl DHB > DHB > 3′-Cl DHB > 4-Cl DHB > 2′,6′-diCl DHB > 2′-Cl DHB. Again, these patterns differ from that of DHBD_LB400 (4′-Cl DHB > 3′-Cl DHB > 6-Cl DHB > 4-Cl DHB > 5-Cl DHB > 2′-Cl DHB > DHB > 2′,6′-diCl DHB [17]).

DISCUSSION

R. globerulus strain P6 contains three extradiol ring cleavage enzymes that have been identified as DHBDs. The properties of the highly active preparations of DHBD_P6-I and DHBD_P6-III described differed slightly from those previously reported for the DHBD_P6 isozymes. First, DHBD_P6-I and DHBD_P6-III in raw extracts of P. putida strain KT2442 displayed strong substrate inhibition for DHB (8). In contrast, these isozymes were subject to minor or no substrate inhibition by DHB in the present study. It is possible that the previous analyses did not adequately account for the rapid inactivation of the enzymes observed at high substrate concentrations. The substrate inhibition observed in DHBD_P6-I is similar in magnitude to that observed in DHBD_LB400 with most substrates and presumably is due to the binding of a second substrate molecule in the active site of the enzyme, as proposed for DHBD_LB400 (43).

A second difference in properties concerns the reactivity of the isozymes with copper. More particularly, a previous report established that 0.1 and 0.2 mM Cu(II) stimulated the DHB cleavage activity of DHBD_P6-II by 137 and 165%, respectively (6). In contrast, DHBD_P6-I and DHBD_P6-III were inhibited by Cu(II) at all of the concentrations tested in the present study. It is unclear whether this difference reflects differences in the enzymes or differences in experimental conditions. In a previous study, DTT was included in the incubation buffer. DTT is highly reactive with transition metals (31), and would likely generate small quantities of Cu(I) and otherwise influence the reactivity of the copper. Interestingly, the specificity of DHBD_P6-II for DHB estimated from the reported specific activity and KmAapp (6) is very similar to that of DHBD_P6-III reported here. The mechanism of activation of DHBD_P6-II by copper remains to be fully elucidated.

It is unclear whether all of the enzymes identified as DHBDs in R. globerulus strain P6 are involved in biphenyl or PCB degradation. Indeed, the apparent specificity of each of the P6 isozymes for DHB is significantly lower than that for DHBD_LB400. Thus, the respective apparent specificities of DHBD_P6-I and DHBD_P6-III for DHB were 1.6 and 21 times lower than that for DHBD_LB400 (43). It is unclear whether the low specificities of DHBD_P6-II and DHBD_P6-III in particular reflect their adaptation to the degradation of other compounds such as PCBs (see below). Interestingly, the apparent K_m for oxygen, the KmBapp values, of DHBD_P6-I and DHBD_P6-III in the presence of DHB are significantly lower than that of DHBD_LB400 so the differences in apparent specificity for DHB between the P6 isozymes and DHBD_LB400 would not be as great at lower O₂ concentrations.

The most striking result of the present study is that DHBD_P6-I and DHBD_P6-III cleaved certain chlorinated DHBs with higher apparent specificities than DHB. This is in clear contrast to DHBD_LB400, whose best substrate is DHB (17). Moreover, the respective specificities of the two P6 isozymes for chlorinated DHBs appear to be quite complementary. Thus, the worst substrate for DHBD_P6-III (4′-Cl DHB) was a good substrate for DHBD_P6-I and the best substrates for DHBD_P6-III (4-Cl DHB and 5-Cl DHB) were the worst substrates for DHBD_P6-I. It is likely that the differences in the specificities of the enzymes for polychlorinated DHBs would be even larger. Overall, these data suggest that the existence of multiple DHBD isozymes in a single bacterial strain improves the PCB-degrading capabilities of that strain and suggest that these enzymes may have been recruited or adapted to improve the PCB-degrading capabilities of R. globerulus strain P6.

The different reactivities of DHBD_P6-I, DHBD_P6-III, and DHBD_LB400 towards PCB metabolites is further illustrated by their respective reactivities towards 2′,6′-diCl DHB. All three isozymes turned over this compound very slowly, indicating that the chlorine substituents probably partially occlude the accessibility for O₂, as observed in DHBD_LB400 (17). However, the KmAapp of the isozymes for 2′,6′-diCl DHB differed by over 500-fold. Moreover, the KmAapp for 2′,6′-diCl DHB was 1,700 times lower than that for DHB in DHBD_LB400, 40 times lower than that for DHB in DHBD_P6-I, and only 4 times lower than that for DHB in DHBD_P6-III. Thus, the P6 isozymes, particularly DHBD_P6-III, are not as susceptible to competitive inhibition by 2′,6′-diCl DHB. As this inhibition would prevent the degradation of other chlorinated DHBs, the different KmAapp values of DHBD_P6-I and DHBD_P6-III for 2′,6′-diCl DHB further demonstrate how the existence of multiple DHBD isozymes in the same strain is advantageous to PCB degradation.

There appears to be no correlation between substrate specificity and susceptibility to inactivation in DHBD_P6-I and DHBD_P6-III: in general, the best substrates were not those that inactivated the enzymes more slowly. Similarly, both P6 isozymes were relatively resistant to inactivation by DHB although this was not the best substrate of either enzyme. This lack of correlation between specificity and susceptibility to inactivation might reflect the recent evolution of these enzymes for the degradation of PCB metabolites, as PCBs were only recently introduced into the environment in significant amounts. The tuning of the enzyme against inactivation versus optimization of specificity could be more difficult to evolve. Alternatively, R. globerulus strain P6 might possess a XylT-like ferredoxin (28, 34) to reduce oxidized iron in DHBD_P6-I and DHBD_P6-III. The existence of such a ferredoxin would prevent prolonged inactivation of the enzymes in vivo and might reduce the evolutionary pressure to minimize susceptibility to inactivation. It was recently shown that DHBD_LB400 in Burkholderia sp. strain was LB400 inactivated with 3-Cl catechol recovered in vivo in the absence of protein synthesis, indicating the presence of an agent capable of reducing the enzyme in this organism (44).

Commercial mixtures of PCBs typically contain up to 60 different congeners. The effective microbial degradation of these environmental pollutants thus requires enzymes of broad or complementary specificities. The present study of the specificities of DHBD_P6-I and DHBD_P6-III points to several similarities as well as significant structural or electronic differences in the respective active sites of DHBD_P6-I, DHBD_P6-III, and DHBD_LB400. Structural and spectroscopic studies of DHBD_P6-I and DHBD_P6-III may provide insights into the molecular basis of these differences. The poor cleavage of 2′,6′-diCl DHB by DHBD_P6-I and DHBD_P6-III further suggests that directed evolution of DHBD might be worthwhile to improve the cleavage of this recalcitrant metabolite and, ultimately, the microbial degradation of PCBs.

Acknowledgments

This work was supported by Strategic grants from the Natural Sciences and Engineering Research Council of Canada (NSERC). F.H.V. was the recipient of the Li Tze Fong Memorial and NSERC postgraduate scholarships.

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PERMALINK

Characterization of Extradiol Dioxygenases from a Polychlorinated Biphenyl-Degrading Strain That Possess Higher Specificities for Chlorinated Metabolites

Frédéric H Vaillancourt

María-Amparo Haro

Nathalie M Drouin

Zamil Karim

Halim Maaroufi

Lindsay D Eltis

Abstract

FIG. 1.

FIG. 2.