Functional analyses of Bph-Tod hybrid dioxygenase, which exhibits high degradation activity toward trichloroethylene.

Biphenyl dioxygenase (BphDox) in Pseudomonas pseudoalcaligenes KF707 is a multicomponent enzyme consisting of an iron-sulfur protein (ISP) that is composed of alpha (BphA1) and beta (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 (alpha 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 (k(0)) of the hybrid Dox for TCE was 4.1 min(-1), which is comparable to that of TolDox. The K(m) value of the hybrid Dox for TCE was 130 microm, which was lower than 250 microm for TolDox. These results suggest that the alpha subunit of ISP is crucial for the determination of substrate specificity and that the change in the alpha subunit conformation of ISP from alpha(2)beta(2) to alpha(3)beta(3) results in the acquisition of higher affinity to TCE, which may lead to high TCE degradation activity.

ISP Bph , bphA3 encodes ferredoxin (BphA3, FD BphA3 ), and bphA4 encodes ferredoxin reductase (BphA4, FDR BphA4 ) (1). ISP requires mononuclear iron (Fe 2ϩ ), 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)(4)(5)(6)(7)(8). Modified BphDox constructed by subunit exchange (3), sitedirected mutagenesis (4 -6), DNA shuffling (7), and randompriming 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 ironbound dioxygenated intermediate on the enzyme surface, and the intermediate compound is rearranged to form formate and HCl using H 2 O.
We now report the purification of the TodC1-BphA2 hybrid Dox and the kinetic analyses toward chloroethylenes.

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 g/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 pKTF18⌬ORF3 (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 5Ј sequence of the todC2 gene, 5Ј-AAGAATTCAACATGATGATTCAGCCAACA-3Ј (primer #1), was used as a forward primer, where the EcoRI site is underlined. An oligonucleotide corresponding to the 3Ј sequence of the todC2 gene, 5Ј-AAAGGTACCCTAGAAGAAGAAACTGAGG-3Ј (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 ϩ (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 5Ј sequence of the bphA3 gene, 5Ј-ATATCCATGGTTATGAAATTTAC-CAGAGTTTGTGAT-3Ј (primer #3), was used as the forward primer, where the NcoI site is underlined. An oligonucleotide corresponding to the 3Ј sequence of the bphA3 gene, 5Ј-TTTCTCGAGTGGCGCCAGAT-ACCCGGC-3Ј (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, 5Ј-GCGATGGTGTCGAC-CATGGCGCCAG-3Ј, 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 gro-ELS 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 l that contained the PCR buffer (Promega, Madison, WI), 100 M dNTPs, 1 M 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 ϫ 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. 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 l 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 A 434 value.
Enzymatic reactions were also detected and quantified by high performance liquid chromatography. The reaction mixture (800-l total volume) contained 50 mM MOPS buffer, pH 7.0, 1 mM NADH, 0.4 mM FeSO 4 , 1 mM biphenyl or 3 mM toluene as the substrates, and 40 -840 g 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 l of the reaction mixture was removed, then added to 300 l of methanol in a microtube. After centrifugation for 10 min, 40 l 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 2.2ϳ3.2 M ISP, 26ϳ38 M FD BphA3 , 2.2ϳ3.2 M FDR BphA4 , 20 M 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 Ϫ 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. 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.
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 1.5fold higher than that of BphDox and TolDox, respectively.
Subunit Conformation of Rieske Oxygenase-Rieske nonheme iron oxygenase (ISP) systems that are involved in the initial dioxygenation of aromatic compounds and which comprise heteromeric subunits are generally present as heterohexamer (␣ 3 ␤ 3 ) or heterotetramer (␣ 2 ␤ 2 ) 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

Functional Analyses of Hybrid Dioxygenase
( 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 ␣ 3 ␤ 3 and 146 kDa for ␣ 2 ␤ 2 , the parental ISP BphA1A2 is present as a heterohexamer. The theoretical molecular mass of ISP TodC1C2 is 222 kDa for ␣ 3 ␤ 3 and 148 kDa for ␣ 2 ␤ 2 , 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 ␣ 3 ␤ 3 conformation. 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 (k 0 ) of the hybrid Dox for TCE is comparable to that of TolDox. Because conformation of TolDox and hybrid Dox are ␣ 2 ␤ 2 heterotetramer and ␣ 3 ␤ 3 heterohexamer, respectively, the catalytic center activity (k cat ) of the hybrid Dox is calculated to be 1.4 min Ϫ1 site Ϫ1 and 33% lower than that of TolDox. The K m value for TCE of the hybrid Dox is 130 M, which is smaller than that of TolDox (250 M). 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.   2. Ferguson plots for estimation of molecular masses of ISP BphA1A2 , ISP TodC1C2 , and ISP TodC1BphA2 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 (R f ) of the standard proteins were determined at four different gel concentrations (%T) and plotted as logR f versus %T. The slope of Kr was determined using linear regression of the %T Ϫ logR f plot. The log (molecular mass of the standards) was then plotted versus ϪlogKr. The resultant equation, log (molecular mass of the standards) ϭ Ϫ1.9321 logKr Ϫ 7.0181 (r ϭ 0.99479), was used to estimate the molecular mass of the ISP.

DISCUSSION
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 max-imum 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 ␣ 3 ␤ 3 . Changing the subunit conformation from ␣ 2 ␤ 2 to ␣ 3 ␤ 3 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 M (Table III). Those of the parental TolDox ranged from 250 to 824 M (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 k 0 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 M. 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).