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J. Biol. Chem., Vol. 276, Issue 32, 29833-29838, August 10, 2001

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Functional Analyses of Bph-Tod Hybrid Dioxygenase, Which Exhibits High Degradation Activity toward Trichloroethylene^*

Tomohiro Maeda, Yukihiro Takahashi, Hikaru Suenaga, Akiko Suyama, Masatoshi Goto, and Kensuke Furukawa

From the Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, Fukuoka 812-8581, Japan

Received for publication, March 6, 2001, and in revised form, May 31, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Biphenyl dioxygenase (BphDox) in Pseudomonas pseudoalcaligenes KF707 is a multicomponent enzyme consisting of an iron-sulfur protein (ISP) that is composed of  (BphA1) and  (BphA2) subunits, a ferredoxin (FD_BphA3), and a ferredoxin reductase (FDR_BphA4). A recombinant Escherichia coli strain expressing hybrid Dox that had replaced BphA1 with TodC1 ( subunit of toluene dioxygenase (TolDox) of Pseudomonas putida) exhibited high activity toward trichloroethylene (TCE) (Furukawa, K., Hirose, J., Hayashida, S., and Nakamura, K. (1994) J. Bacteriol. 176, 2121-2123). In this study, ISP, FD, and FDR were purified and characterized. Reconstitution of the dioxygenase components consisting of purified ISP_TodC1BphA2, FD_BphA3, and FDR_BphA4 exhibited oxygenation activities toward biphenyl, toluene, and TCE. Native polyacrylamide gel electrophoresis followed by the Ferguson plot analyses demonstrated that ISP_TodC1BphA2 and ISP_BphA1A2 were present as heterohexamers, whereas ISP_TodC1C2 was present as a heterotetramer. The molecular activity (k₀) of the hybrid Dox for TCE was 4.1 min¹, which is comparable to that of TolDox. The K_m value of the hybrid Dox for TCE was 130 µM, which was lower than 250 µM for TolDox. These results suggest that the  subunit of ISP is crucial for the determination of substrate specificity and that the change in the  subunit conformation of ISP from ₂₂ to ₃₃ results in the acquisition of higher affinity to TCE, which may lead to high TCE degradation activity.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Biphenyl dioxygenase (BphDox)¹ in Pseudomonas pseudoalcaligenes KF707 catalyzes the introduction of two atoms of molecular oxygen into biphenyl and some polychlorinated biphenyls (PCB). BphDox is a multicomponent enzyme encoded by the four genes, bphA1A2A3A4, where bphA1 encodes an  subunit (BphA1) of the terminal dioxygenase (an iron-sulfur protein, ISP_Bph), bphA2 encodes the  subunit (BphA2) of ISP_Bph, bphA3 encodes ferredoxin (BphA3, FD_BphA3), and bphA4 encodes ferredoxin reductase (BphA4, FDR_BphA4) (1). ISP requires mononuclear iron (Fe²⁺), which is likely to be an active center for catalysis and contains a Rieske [2Fe-2S] cluster for electron transfer. Recently a classification has been proposed for these types of ISP as a family of Rieske non-heme iron oxygenases (2). FD and FDR act as an electron transfer system from NADH to reduce ISP. Recent studies provided evidence that the  subunit of ISP_Bph plays a primary role in the determination of the substrate specificity for PCB (3-8). Modified BphDox constructed by subunit exchange (3), site-directed mutagenesis (4-6), DNA shuffling (7), and random-priming recombinations (8) resulted in the dramatic alterations in the enzyme activities, the substrate selectivities, and the mode of oxygenation toward certain PCB congeners (9). It is also true that some BphDox from various bacteria share structural similarities to those of KF707, however, their substrate specificities toward PCB are different (10).

Toluene dioxygenase (TolDox) in Pseudomonas putida F1 catalyzes the conversion of toluene to cis-toluene dihydrodiol (11). Besides toluene, TolDox exhibits catalytic activity toward benzene, biphenyl, naphthalene, and trichloroethylene (TCE). The toluene catabolic tod operon is similar to the KF707 bph gene cluster in terms of gene organization and the nucleotide sequence of the corresponding genes (1, 12). TolDox is a multicomponent enzyme consisting of  (TodC1) and  (TodC2) subunits of ISP_Tod, FD (TodB), and FDR (TodA). The identities of the amino acid sequences between the corresponding components of BphDox and TolDox are in the range of 53 to 65% (1, 12).

During the course of identifying the component responsible for the substrate specificity of BphDox and TolDox, hybrid bph-tod gene clusters were constructed by replacing genes encoding the dioxygenase components from BphDox and TolDox. Among them, the recombinant Escherichia coli expressing the hybrid todC1-bphA2A3A4 genes exhibited substrate specificity similar to that of the original TolDox, but 3-fold higher activity toward TCE than TolDox (13). Chloroethylenes such as TCE have been recognized to be significant environmental pollutants in the soil, groundwater, and atmosphere (14). These compounds have been shown to persist over time in the environment and are suspected to be carcinogenic (15). In the recent past, it has been shown that TCE can be degraded with a variety of oxygenases such as methane monooxygenase (16), toluene monooxygenase (17), ammonium monooxygenase (18), phenol hydroxylase (19), and TolDox (11) from aerobic bacteria. The mechanism of TCE oxygenation by TolDox has been proposed by Li and Wackett (20). TCE is converted to an iron-bound dioxygenated intermediate on the enzyme surface, and the intermediate compound is rearranged to form formate and HCl using H₂O.

We now report the purification of the TodC1-BphA2 hybrid Dox and the kinetic analyses toward chloroethylenes.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Bacterial Strains and Culture Conditions

E. coli JM109 was used for the general propagation of the plasmids and for the expression of ISP. E. coli BL21(DE3) was used for the expression of bphA3 and bphA4. For the expression of bphA1A2 and todC1BphA2, E. coli cells were grown at 37 °C for 12 h in Luria-Bertani medium containing 0.1 mM isopropyl--D-galactopyranoside and a concentration, 50 µg/ml, of the appropriate antibiotics. For expressions of todC1C2, bphA3, and bphA4, E. coli cells were grown at 30 °C for 24 h in the same medium described above.

Plasmids for ISP Expression

pUC-A1A2 for the expression of ISP_bphA1A2 was constructed by inserting the XhoI DNA fragment containing the bphA1A2 genes from pKTF18ORF3 (1) into an SalI site of pUC119. pUC-C1A2, for the expression of the hybrid ISP_TodC1BphA2, was constructed by removing the PstI DNA fragment containing the bphA3A4BC from pJHF10 (3). pHSG-C1C2, for the expression of the ISP_TodC1C2, was constructed as follows: The todC2 gene without the original Shine-Dalgarno sequence was amplified by PCR using pJHF-C1C2 (21) as the template DNA. An oligonucleotide corresponding to the 5' sequence of the todC2 gene, 5'-AAGAATTCAACATGATGATTCAGCCAACA-3' (primer #1), was used as a forward primer, where the EcoRI site is underlined. An oligonucleotide corresponding to the 3' sequence of the todC2 gene, 5'-AAAGGTACCCTAGAAGAAGAAACTGAGG-3' (primer #2), was used as the reverse primer, where the KpnI site is underlined. The amplified DNA, which had been digested with EcoRI and KpnI, was inserted into the corresponding sites of pBluescriptII SK⁺ (Stratagene) to generate pBlue-C2. A BamHI/EcoRI DNA fragment containing the todC1 and the Shine-Dalgarno sequence of the bphA2 gene from pUC-C1A2 was inserted into the corresponding sites of pBlue-C2 to construct pBlue-C1C2. pHSG-C1C2 was finally constructed by replacing the pBluescriptII region of pBlue-C1C2 by pHSG396 (Toyobo, Kyoto, Japan) at the BamHI and KpnI sites.

Plasmids for FD_BphA3 and FDR_BphA4 Expression

To express FD_BphA3, the bphA3 gene was amplified by PCR using pJHF10 as the template DNA. An oligonucleotide corresponding to the 5' sequence of the bphA3 gene, 5'-ATATCCATGGTTATGAAATTTACCAGAGTTTGTGAT-3' (primer #3), was used as the forward primer, where the NcoI site is underlined. An oligonucleotide corresponding to the 3' sequence of the bphA3 gene, 5'-TTTCTCGAGTGGCGCCAGATACCCGGC-3' (primer #4), was used as the reverse primer, where the XhoI site is underlined. The amplified DNA fragment, which had been digested with NcoI and XhoI, was inserted downstream of the thioredoxin gene (TRX), the six-histidine tag (His-tag), and upstream of the His-tag of pET32b (Novagen), yielding pET32-A3 (see Table I below). To construct pUC-A4 for the expression of FDR_BphA4, site-directed mutagenesis was carried out to introduce an NcoI site around the start codon of the bphA4 gene. An oligonucleotide, 5'-GCGATGGTGTCGACCATGGCGCCAG-3', where the mutated nucleotide is indicated in boldface letters and the site newly introduced NcoI is underlined, was used as the mutagenic primer. As a template DNA for site-directed mutagenesis, pUC-A3A4 was constructed by digesting pJHFA3A4BC (21) with PpuMI and subsequent self-ligation. The mutagenized plasmid, pUC-A3A4N, was digested with NcoI and EcoRI, and the bphA4 fragment was inserted downstream of the TRX and His-tag of pET32b, generating pET32-A4. An XbaI/EcoRI DNA fragment containing the bphA4 gene from pET32-A4 was inserted into the corresponding site of pUC119 to generate pUC-A4. A plasmid, pKY206, expressing the groELS gene (22) was provided by Y. Kawata (Tottori University) and was used to produce the soluble FDR_BphA4 fusion protein.

PCR

PCR was performed in a total volume of 50 µl that contained the PCR buffer (Promega, Madison, WI), 100 µM dNTPs, 1 µM forward and reverse primers, 0.5 unit of Taq DNA polymerase (Promega), and 50 ng of a template DNA. PCR was carried out for 25 cycles under the following conditions: denaturation, 94 °C for 1 min; primer annealing, 55 °C for 1.5 min; and primer extension, 72 °C for 1 min.

Protein Purification

ISP purification was carried out according to the method described by Haddock and Gibson (23) with some modifications. All the purification steps were carried out at 4 °C. Harvested recombinant E. coli cells were suspended in 50 mM MOPS buffer, pH 7.0, containing 5% ethanol and 5% glycerol (MEG). Cells were disrupted by a French pressure cell (Ohtake Seisakusho, Tokyo) prior to centrifugation at 17,400 × g for 10 min. The resultant viscous liquid was treated with 3% streptomycin sulfate for 30 min and centrifuged. The resulting supernatant was used as a crude enzyme. The crude enzyme was applied to a Q-Sepharose FF column (Amersham Pharmacia Biotech) equilibrated with MEG. The hybrid ISP_TodC1BphA2 as well as the parental ISP_BphA1A2 and ISP_TodC1C2 were purified as follows.

ISP_BphA1A2-- Proteins were eluted in stepwise fashions with 0.1 M KCl and 0.2 M KCl in MEG. A fraction showing ISP activity was eluted with 0.2 M KCl. The active fraction that had added 2.0 M ammonium sulfate was applied to a Butyl-Toyopearl 650M column (Tohsho, Tokyo, Japan) equilibrated with 2.0 M ammonium sulfate in MEG, and the proteins were stepwise eluted with 2.0, 1.0, and 0.5 M ammonium sulfate in MEG. The fraction showing the ISP activity was eluted with 0.5 M ammonium sulfate and was dialyzed against 10 mM potassium phosphate buffer, pH 7.0 (KP). The dialysate was applied to a column of hydroxylapatite (Bio-Rad) equilibrated with KP, and the proteins were eluted in stepwise fashions with 0.01, 0.1, and 0.25 M KP. ISP_BphA1A2 was eluted with 0.25 M KP.

ISP_TodC1C2-- Proteins were eluted using a linear gradient from 0.2 to 0.4 M KCl in MEG. The fraction showing ISP activity was eluted with 0.3 M KCl. The fraction in the added 1.0 M ammonium sulfate was applied to a Butyl-Toyopearl 650M column equilibrated with 1.0 M ammonium sulfate in MEG, and the proteins were stepwise eluted with 1.0, 0.75, and 0.5 M ammonium sulfate in MEG. The fraction showing ISP activity was eluted with 0.5 M ammonium sulfate. The subsequent procedure for purification of the ISP_TodC1C2 was the same as described above for ISP_BphA1A2.

ISP_TodC1BphA2-- Proteins were eluted in stepwise fashions with 0.2 and 0.4 M KCl in MEG. A fraction showing ISP activity was eluted with 0.4 M KCl. The subsequent procedure for purification of the ISP_TodC1BphA2 was the same as described for ISP_BphA1A2.

FD_BphA3 Fusion Protein-- The crude enzyme was prepared by extracting with 50 mM KP containing 10 mM imidazole, pH 7.0, instead of that with MEG. The crude enzyme was applied to a nickel-nitrilotriacetic acid-agarose column (Qiagen) equilibrated with 50 mM KP containing 10 mM imidazole, pH 7.0. The agarose gel was washed with 50 mM KP containing 50 mM imidazole and 0.3 M NaCl (washing buffer). The FD_BphA3 fusion protein was eluted with 50 mM KP containing 0.2 M imidazole and 0.3 M NaCl.

FDR_BphA4 Fusion Protein-- Purification of the FDR_BphA4 fusion protein was done using the same method for FD_BphA3 but changing the washing buffer to 50 mM KP containing 20 mM imidazole, 0.3 M NaCl, and 0.5% Triton X-100.

Enzyme Assay

An assay for dioxygenase activity was spectrophotometrically done. A total test volume of 250 µl contained 50 mM MOPS buffer, pH 7.0, 0.4 mM ferrous ammonium sulfate, 0.4 mM NADH, 5 mM biphenyl, 1 mg of cell-free extracts containing dihydrodiol dehydrogenase, 2,3-dihydroxybiphenyl dioxygenase, and enzyme components. Incubation was done with shaking at 30 °C for the appropriate time. The product catalyzed by dioxygenase from biphenyl was further converted to a yellow compound, 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid by the sequential actions of dihydrodiol dehydrogenase and 2,3-dihydroxybiphenyl dioxygenase. The yellow compound formed was quantified by measuring its A₄₃₄ value.

Enzymatic reactions were also detected and quantified by high performance liquid chromatography. The reaction mixture (800-µl total volume) contained 50 mM MOPS buffer, pH 7.0, 1 mM NADH, 0.4 mM FeSO₄, 1 mM biphenyl or 3 mM toluene as the substrates, and 40-840 µg of each enzyme component. The reaction was initiated by adding the substrate, and the reaction mixtures were incubated at 37 °C. At appropriate intervals, 150 µl of the reaction mixture was removed, then added to 300 µl of methanol in a microtube. After centrifugation for 10 min, 40 µl of the supernatant was injected into an Ultrasphere ODS column (Beckman) that had been equilibrated with water:methanol:acetonitrile (40:30:30, v/v). The column was eluted for 6 min at 0.75 ml/min with the same solvent system, followed by elution with methanol:acetonitrile (45:55, v/v) for 20 min. The activity was evaluated from the substrate disappearance measurement by detection at 254 nm for biphenyl and at 260 nm for toluene.

Kinetic Analysis

Steady-state kinetic parameters for TCE, cis-DCE, trans-DCE, and 1,1-DCE were determined with purified ISP, FD_BphA3, and FDR_BphA4. Two milliliters of the reaction mixture containing 2.2~3.2 µM ISP, 26~38 µM FD_BphA3, 2.2~3.2 µM FDR_BphA4, 20 µM ferrous ammonium sulfate, and 50 mM MOPS buffer (pH 7.0) was added to a glass vial (20 ml), which was sealed with a rubber septum and an aluminum crimp seal. Chloroethylenes dissolved in N,N-dimethylformamide were added to the vial and preincubated at 30 °C for 30 min to allow equilibration of the chloroethylene between the gas and liquid phases. The reaction was initiated by the addition of 0.8 mM NADH, and incubation was carried out with shaking at 30 °C for 10 min. The amounts of the chloroethylenes in the gas phase were measured by gas chromatographic analysis according to a previously described method (13), and those in the aqueous phase were calculated according to Henry's Law constants for the chloroethylenes (24). The values of V_max and K_m were determined using the Hanes-Woolf (S/V  S plot) analysis.

General Analytical Procedure

Purified ISP comprised of  and  subunits was subjected to native PAGE. The mobilities of the ISP on 5%, 7.5%, 10%, and 12.5% acrylamide gels were treated with the Ferguson plot (25) to estimate the molecular mass. SDS-PAGE was carried out according to the method of Laemmli (26). The protein concentration was measured by a Bio-Rad protein assay using bovine serum albumin as the standard.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Expression of ISP, FD_BphA3, and FDR_BphA4-- The  and  subunits of ISP were successfully coexpressed in E. coli using pUC-A1A2, pUC-C1A2, and pHSG-C1C2 (Table I). In these expression systems, a lac promoter was applied to induce the ISP and was sufficient to produce soluble ISP. ISP_BphA1A2 and ISP_TodC1BphA2 were produced as the soluble and active forms in the recombinant E. coli at 37 °C. When ISP_TodC1C2 was expressed in E. coli using a recombinant plasmid carrying the original todC1C2 genes, the level of expression of the  subunit was extremely low, as compared with that of the  subunit. Therefore, the Shine-Dalgarno sequence of todC2 was replaced by that of bphA1, and the pHSG-C1C2 was finally constructed. The recombinant E. coli cells carrying pHSG-C1C2 produced TodC2 with amounts equivalent to TodC1. However, the proteins were produced as prominent inclusion bodies. A reduction of the cultivation temperature from 37 °C to 30 °C resulted in an elevated yield of the soluble ISP. FD_BphA3 and FDR_BphA4 were expressed as fusion proteins to facilitate protein purification and solubilization. Recombinant E. coli cells carrying pET32-A3 under the control of a strong T7 promoter produced the soluble FD_BphA3 fusion protein with the N-terminal TRX, His-tag, and C-terminal His-tag with a molecular mass of 36 kDa (Fig. 1). As in the case of FD_BphA3, the bphA4 gene was also expressed as a fusion protein with the N-terminal TRX and His-tag. However, the FDR_BphA4 fusion protein with a molecular mass of 63 kDa was produced in E. coli (pET32-A4) as prominent inclusion bodies. Replacement of the T7 promoter by the lac promoter resulted in increased amounts of the soluble FDR_BphA4 fusion protein. In addition, coexpression of a chaperone, GroELS of E. coli using pKY206, allowed the FDR_BphA4 fusion protein to efficiently solubilize. By using this coexpression system, the production of the FDR_BphA4 fusion protein was improved to 4.5-fold compared with that without pKY206.

View larger version (108K): [in this window] [in a new window]   Fig. 1.   SDS-PAGE of ISP, FD, and FDR fusion proteins. Proteins were separated on 10% polyacrylamide gel and stained with Coomassie Brilliant Blue. Lane 1, mass marker proteins; lane 2, ISPBphA1A2; lane 3, ISPTodC1BphA2; lane 4, ISPTodC1C2; lane 5, FD BphA3 fusion protein; lane 6, FDR BphA4 fusion protein.

The catalytically active components of ISPs, FD_BphA3 and FDR_BphA4, were purified as shown in Fig. 1. The yields of the purified ISP_BphA1A2, ISP_TodC1BphA2, ISP_TodC1C2, FD_BphA3, and FDR_BphA4 from a 1-liter culture were 11.0, 9.4, 8.6, 22.1, and 20.4 mg, respectively.

Reconstitution of Dox Components-- The Dox activity was observed when all purified components of ISP, FD_BphA3, and FDR_BphA4 were mixed, indicating that all components were successfully associated with one another in vitro. The maximum activity of the hybrid enzyme was observed when 12-fold excess of FD_BphA3 was incubated with ISP_TodC1BphA2 and FDR_BphA4. Only 17%, 30%, and 70% of the maximum activity was observed when 3-, 6-, and 9-folds excess of FD_BphA3 were added, respectively (data not shown). The maximum activities of the parental BphDox and TolDox were also observed in the presence of 15- and 12-fold excesses of FD_BphA3, respectively_. The specific activities of the purified ISP were determined in the presence of excess amounts of FD_bphA3 by high performance liquid chromatography analysis and were evaluated from the rate of substrate depletion (Table II). The specific activity of the hybrid Dox toward biphenyl was 5.3 nmol/min/nmol ISP, which was comparable to that of TolDox and was as low as 2.9% of that of BphDox. The specific activity of the hybrid Dox toward toluene was 270.3 nmol/min/nmol ISP, which was 7- and 1.5-fold higher than that of BphDox and TolDox, respectively.

Subunit Conformation of Rieske Oxygenase-- Rieske non-heme iron oxygenase (ISP) systems that are involved in the initial dioxygenation of aromatic compounds and which comprise heteromeric subunits are generally present as heterohexamer (₃₃) or heterotetramer (₂₂) conformations (27). The  and  subunits of the parental ISP_BphA1A2, ISP_TodC1C2, and hybrid ISP_TodC1BphA2 were copurified by column chromatographies, indicating that these two subunits were tightly associated with each other and formed the catalytically active ISP (Fig. 1). An analysis of the purified ISP on SDS-PAGE demonstrated that the  and  subunits were associated with a 1:1 stoichiometry. To investigate how the purified subunits are organized in ISP, nondenaturing PAGE was carried out with the purified ISP. The hybrid ISP_TodC1BphA2 as well as the parental ISP_BphA1A2 and ISP_TodC1C2 were separated on various concentrations of acrylamide gel, and the relative mobilities were determined with the various concentrations. Based on the Ferguson plot, the molecular masses of ISP_BphA1A2, ISP_TodC1C2, and ISP_TodC1BphA2 were estimated to be 209, 160, and 229 kDa, respectively (Fig. 2). Because the theoretical molecular mass of ISP_BphA1A2 is 219 kDa for ₃₃ and 146 kDa for ₂₂, the parental ISP_BphA1A2 is present as a heterohexamer. The theoretical molecular mass of ISP_TodC1C2 is 222 kDa for ₃₃ and 148 kDa for ₂₂, therefore, ISP_TodC1C2 is present as a heterotetramer, which is in agreement with the result reported by Subramanian et al. (28). The ISP_TodC1BphA2 hybrid has a similar conformation to ISP_BphA1A2, and the estimated molecular mass of 229 kDa is in good agreement with the theoretical 226.5 kDa of ₃₃ conformation.

View larger version (31K): [in this window] [in a new window]   Fig. 2.   Ferguson plots for estimation of molecular masses of ISPBphA1A2, ISPTodC1C2, and ISPTodC1BphA2 by native PAGE using various concentrations of acrylamide gels. Purified ISP consisting of the  and  subunits was subjected to native PAGE using chicken egg white albumin monomer (45 kDa), bovine serum albumin dimer (132 kDa), urease monomer (272 kDa), and urease dimer (544 kDa) as mass standard proteins. The mobilities of ISP on 5%, 7.5%, 10%, and 12.5% acrylamide gels were treated using the Ferguson plot as follows. The relative mobilities (Rf) of the standard proteins were determined at four different gel concentrations (%T) and plotted as logRf versus %T. The slope of Kr was determined using linear regression of the %T  logRf plot. The log (molecular mass of the standards) was then plotted versus logKr. The resultant equation, log (molecular mass of the standards) = 1.9321 logKr  7.0181 (r = 0.99479), was used to estimate the molecular mass of the ISP.

Kinetic Parameters of Hybrid Dox for Chloroethylenes-- Because the parental BphDox is inactive toward chloroethylenes, the steady-state kinetic parameters for TCE, cis-DCE, trans-DCE, and 1,1-DCE were determined with purified TolDox and hybrid Dox (Table III). The apparent molecular activity (k₀) of the hybrid Dox for TCE is comparable to that of TolDox. Because conformation of TolDox and hybrid Dox are ₂₂ heterotetramer and ₃₃ heterohexamer, respectively, the catalytic center activity (k_cat) of the hybrid Dox is calculated to be 1.4 min¹ site¹ and 33% lower than that of TolDox. The K_m value for TCE of the hybrid Dox is 130 µM, which is smaller than that of TolDox (250 µM). The resulting catalytic efficiency, k_cat/K_m value for TCE increased 24% for the hybrid Dox, as compared with that for TolDox. For the other chloroethylenes such as cis-DCE and 1,1-DCE, the k_cat values are higher in TolDox and the K_m values are smaller in the hybrid Dox. The results indicate that the hybrid Dox acquired higher affinities for a variety of chloroethylenes than the parental TolDox and that the catalytic efficiency is slightly higher in the hybrid Dox. For the trans-DCE, the hybrid Dox showed only a weak activity, and thus the kinetic parameter with a high regression coefficient could not be determined from the Hanes-Woolf plot.

Components Responsible for Enzyme Inactivation Coupled to TCE Degradation-- It is known that TolDox is irreversibly inactivated by coupling to TCE oxygenation (20). The hybrid Dox was also gradually inactivated during TCE oxygenation and was completely inactivated after 7.5 h of incubation (Fig. 3). To identify the components responsible for being inactivated, ISP_TodC1BphA2, FD_BphA3, or FD_BphA4 was, respectively, added to the inactivated reaction mixture. The addition of ISP_TodC1BphA2 permitted the immediate restoration of the enzymatic activity of the TCE oxygenation (Fig. 3). On the other hand, the addition of FD_BphA3 or FD_BphA4 failed to restore the activity. These results indicate that the ISP_TodC1BphA2 component is susceptible and involved in the inactivation of the hybrid Dox during TCE degradation.

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Components responsible for enzyme inactivation coupled to TCE degradation. A, the reaction mixture containing 3.5 µM ISPTodC1BphA2, 102 µM FDBphA3, 3.5 µM FDRBphA4, 20 µM ferrous ammonium sulfate, 6.4 mM NADH, and 1 mM TCE in 50 mM Mops, pH 7.0, was incubated with shaking at 30 °C for 7.5 h to allow complete inactivation of the enzyme. 1 mM TCE and 1 mM NADH were added to the reaction mixture and further incubated for 2.5 h to confirm inactivation of the enzyme. B, ISPTodC1BphA2, FDBphA3, and FDRBphA4 were then added, respectively, to the reaction mixture to recover the Dox activity. Symbols: A, residual amounts of TCE (open square); B, additions of none (open triangle), ISPTodC1BphA2 (closed square), FDBphA3 (closed circle), and FDRBphA4 (closed diamond).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

It is of great interest to construct microorganisms with enhanced and expanded degradation capabilities for environmental pollutants. Some attempts, including in vitro DNA shuffling, domain exchanges, and subunit exchanges have been achieved for structural remodeling of dioxygenases with minimum prior information about the enzymes (4-9, 29). We previously reported that recombinant bacteria producing hybrid TodC1-BphA2A3A4 dioxygenase constructed by subunit exchanges, exhibited the expanded capability of converting various aromatic compounds (21, 30) and the enhanced degradation activity toward TCE (13, 30).

To reconstitute ISP, we first mixed in vitro  and  subunits of BphDox, TolDox, and TodC1-BphA2 hybrid Dox that had been individually expressed in E. coli but failed to reconstitute ISP with high activity (data not shown). However, the ISP components of BphDox and hybrid Dox were fully active when both subunits were coexpressed in E. coli. This result is different from the previous work by Hurtubise et al. (31). They reported that His-tagged  and  subunits of ISP from Comamonas testosteroni were separately expressed in E. coli and that the mixture of these subunits exhibited high activity.

It was found that the reconstituted Dox with purified ISP, FD_BphA3, and FDR_BphA4 are highly functional, indicating that FD_BphA3 and FDR_BphA4 originating from BphDox interacted with a foreign ISP that originated from TolDox and hybrid Dox, where an electron from NADH is transferred to the Rieske [2Fe-2S] center of the  subunit. The reduced ISP activates molecular oxygen and introduces it to the substrates. The maximum activity of ISP was observed when more than 12-fold equivalents of FD_BphA3 were incubated with ISP and FDR_BphA4. The recombinant FD_BphA3 fusion protein may be less active than the native FD due to the additional fusion tags. It is also likely that relatively low activity of FD_BphA3 may be associated with the instability of this protein. The FD of BphDox from C. testosteroni was also reported to be labile (32).

The substrate specificities of the purified hybrid Dox were similar to those of TolDox (Table II), indicating the  subunit of ISP is critically responsible for recognition of the substrates and the catalytic activity. This result also implies that, in the hybrid Dox, the structure of the functional domains such as the mononuclear iron-binding residues and substrate binding pocket can be retained even in the subunit conformation of an ₃₃. Changing the subunit conformation from ₂₂ to ₃₃ may lead to a small change in the structure around the active site. The K_m values of the hybrid Dox for the chloroethylenes ranged from 130 to 370 µM (Table III). Those of the parental TolDox ranged from 250 to 824 µM (Table III). These results suggest that the hybrid Dox gains slightly higher affinity for substrates used in this study than TolDox. A binding pocket around the active center of ISP_TodC1BphA2 may be slightly relaxed to accommodate various chloroethylenes. Although the k₀ values of the hybrid Dox are comparable to those of TolDox, the k_cat values of the hybrid Dox are slightly lower than those of TolDox. It is likely that the conformation of the catalytic residues involved in the mononuclear iron binding in ISP_TodC1BphA2 is slightly changed compared with that in ISP_TodC1C2 (33). As previously reported (13), the recombinant resting cells expressing hybrid Dox degraded TCE 3-fold faster than those expressing TolDox. The TCE used in this experiment was as low as 76 µM. Under conditions below the K_m value, the hybrid Dox is able to better exert its higher activity than TolDox. Thus, the elevated activity of the recombinant bacteria expressing the hybrid Dox toward TCE reflects the elevated affinity of the hybrid Dox for TCE.

There are some reports on the irreversible inactivation of monooxygenases (34) and dioxygenases (20, 35). Lee reported that benzene dioxygenase was inactivated via the Fenton-type reaction that formed hydroxyl radicals from the uncoupled reaction of hydrogen peroxide with ferrous mononuclear iron at the catalytic center (35). Li and Wackett (20) reported that TolDox is inactivated via alkylation of the enzyme during TCE degradation. In this study, an irreversibly inactivated component of the hybrid Dox was determined to be ISP not FD or FDR (Fig. 3). Neither cleavage of the peptide bond nor dissociation of the  and  subunits occurred on ISP_TodC1BphA2 during TCE oxygenation as judged by the SDS-PAGE and native PAGE (data not shown). Some attempts to understand the mechanism for the TCE inactivation of ISP_TodC1BphA2 were carried out using chelating agents and catalase, indicating that the hydroxyl radical caused by the Fenton reaction was not implicated in the TCE inactivation of ISP_TodC1BphA2 (data not shown). Therefore, it is likely that the hybrid ISP_TodC1BphA2 is inactivated by a fashion similar to the ISP of TolDox (20).

	FOOTNOTES

^* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Laboratory of Applied Microbiology, Dept. of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, Fukuoka 812-8581, Japan. Tel./Fax: 81-92-642-2849; E-mail address: kfurukaw@agr.kyushu-u.ac.jp.

Published, JBC Papers in Press, June 4, 2001, DOI 10.1074/jbc.M102025200

	ABBREVIATIONS

The abbreviations used are: BphDox, biphenyl dioxygenase; PCB, polychlorinated biphenyl; ISP, iron-sulfur protein; FD, ferredoxin; FDR, ferredoxin reductase; TolDox, toluene dioxygenase; TCE, trichloroethylene; PCR, polymerase chain reaction; TRX, thioredoxin gene; MOPS, 4-morpholinepropanesulfonic acid; MEG, 50 mM MOPS buffer, pH 7.0, containing 5% ethanol and 5% glycerol; DCE, dichloroethylene; KP, 10 mM potassium phosphate buffer, pH 7.0; PAGE, polyacrylamide gel electrophoresis.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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