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

2001; Elsevier BV; Volume: 276; Issue: 32 Linguagem: Inglês

10.1074/jbc.m102025200

1083-351X

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

Microbial bioremediation and biosurfactants

Biphenyl dioxygenase (BphDox) inPseudomonas pseudoalcaligenes KF707 is a multicomponent enzyme consisting of an iron–sulfur protein (ISP) that is composed of α (BphA1) and β (BphA2) subunits, a ferredoxin (FDBphA3), and a ferredoxin reductase (FDRBphA4). A recombinant Escherichia colistrain expressing hybrid Dox that had replaced BphA1 with TodC1 (α 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 ISPTodC1BphA2, FDBphA3, and FDRBphA4 exhibited oxygenation activities toward biphenyl, toluene, and TCE. Native polyacrylamide gel electrophoresis followed by the Ferguson plot analyses demonstrated that ISPTodC1BphA2 and ISPBphA1A2 were present as heterohexamers, whereas ISPTodC1C2 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. TheK m value of the hybrid Dox for TCE was 130 µm, which was lower than 250 µm for TolDox. These results suggest that the α subunit of ISP is crucial for the determination of substrate specificity and that the change in the α subunit conformation of ISP from α2β2 to α3β3 results in the acquisition of higher affinity to TCE, which may lead to high TCE degradation activity. Biphenyl dioxygenase (BphDox) inPseudomonas pseudoalcaligenes KF707 is a multicomponent enzyme consisting of an iron–sulfur protein (ISP) that is composed of α (BphA1) and β (BphA2) subunits, a ferredoxin (FDBphA3), and a ferredoxin reductase (FDRBphA4). A recombinant Escherichia colistrain expressing hybrid Dox that had replaced BphA1 with TodC1 (α 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 ISPTodC1BphA2, FDBphA3, and FDRBphA4 exhibited oxygenation activities toward biphenyl, toluene, and TCE. Native polyacrylamide gel electrophoresis followed by the Ferguson plot analyses demonstrated that ISPTodC1BphA2 and ISPBphA1A2 were present as heterohexamers, whereas ISPTodC1C2 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. TheK m value of the hybrid Dox for TCE was 130 µm, which was lower than 250 µm for TolDox. These results suggest that the α subunit of ISP is crucial for the determination of substrate specificity and that the change in the α subunit conformation of ISP from α2β2 to α3β3 results in the acquisition of higher affinity to TCE, which may lead to high TCE degradation activity. biphenyl dioxygenase polychlorinated biphenyl iron–sulfur protein ferredoxin ferredoxin reductase toluene dioxygenase trichloroethylene polymerase chain reaction thioredoxin gene 4-morpholinepropanesulfonic acid 50 mm MOPS buffer, pH 7.0, containing 5% ethanol and 5% glycerol dichloroethylene 10 mm potassium phosphate buffer, pH 7.0 polyacrylamide gel electrophoresis Biphenyl dioxygenase (BphDox)1 inPseudomonas 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, wherebphA1 encodes an α subunit (BphA1) of the terminal dioxygenase (an iron–sulfur protein, ISPBph),bphA2 encodes the β subunit (BphA2) of ISPBph, bphA3 encodes ferredoxin (BphA3, FDBphA3), and bphA4 encodes ferredoxin reductase (BphA4, FDRBphA4) (1Taira K. Hirose J. Hayashida S. Furukawa K. J. Biol. Chem. 1992; 267: 4844-4853Abstract Full Text PDF PubMed Google Scholar). 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 (2Gibson D.T. Parales R.E. Curr. Opin. Biotechnol. 2000; 11: 236-243Crossref PubMed Scopus (513) Google Scholar). FD and FDR act as an electron transfer system from NADH to reduce ISP. Recent studies provided evidence that the α subunit of ISPBph plays a primary role in the determination of the substrate specificity for PCB (3Furukawa K. Hirose J. Suyama A. Zaiki T. Hayashida S. J. Bacteriol. 1993; 175: 5224-5232Crossref PubMed Google Scholar, 4Erickson B.D. Mondello F.J. Appl. Environ. Microbiol. 1993; 59: 3858-3862Crossref PubMed Google Scholar, 5Mondello F.J. Turcich M.P. Lobos J.H. Erickson B.D. Appl. Environ. Microbiol. 1997; 63: 3096-3103Crossref PubMed Google Scholar, 6Kimura N. Nishi A. Goto M. Furukawa K. J. Bacteriol. 1997; 179: 3936-3943Crossref PubMed Google Scholar, 7Kumamaru T. Suenaga H. Mitsuoka M. Watanabe T. Furukawa K. Nat. Biotechnol. 1998; 16: 663-666Crossref PubMed Scopus (192) Google Scholar, 8Suenaga H. Goto M. Furukawa K. J. Biol. Chem. 2001; 276: 22500-22506Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Modified BphDox constructed by subunit exchange (3Furukawa K. Hirose J. Suyama A. Zaiki T. Hayashida S. J. Bacteriol. 1993; 175: 5224-5232Crossref PubMed Google Scholar), site-directed mutagenesis (4Erickson B.D. Mondello F.J. Appl. Environ. Microbiol. 1993; 59: 3858-3862Crossref PubMed Google Scholar, 5Mondello F.J. Turcich M.P. Lobos J.H. Erickson B.D. Appl. Environ. Microbiol. 1997; 63: 3096-3103Crossref PubMed Google Scholar, 6Kimura N. Nishi A. Goto M. Furukawa K. J. Bacteriol. 1997; 179: 3936-3943Crossref PubMed Google Scholar), DNA shuffling (7Kumamaru T. Suenaga H. Mitsuoka M. Watanabe T. Furukawa K. Nat. Biotechnol. 1998; 16: 663-666Crossref PubMed Scopus (192) Google Scholar), and random-priming recombinations (8Suenaga H. Goto M. Furukawa K. J. Biol. Chem. 2001; 276: 22500-22506Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar) resulted in the dramatic alterations in the enzyme activities, the substrate selectivities, and the mode of oxygenation toward certain PCB congeners (9Furukawa K. Curr. Opin. Biotechnol. 2000; 11: 244-249Crossref PubMed Scopus (111) Google Scholar). 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 (10Gibson D.T. Cruden D.L. Haddock J.D. Zylstra G.J. Brand J.M. J. Bacteriol. 1993; 175: 4561-4564Crossref PubMed Google Scholar). Toluene dioxygenase (TolDox) in Pseudomonas putida F1 catalyzes the conversion of toluene to cis-toluene dihydrodiol (11Wackett L.P. Gibson D.T. Appl. Environ. Microbiol. 1988; 54: 1703-1708Crossref PubMed Google Scholar). Besides toluene, TolDox exhibits catalytic activity toward benzene, biphenyl, naphthalene, and trichloroethylene (TCE). The toluene catabolic tod operon is similar to the KF707bph gene cluster in terms of gene organization and the nucleotide sequence of the corresponding genes (1Taira K. Hirose J. Hayashida S. Furukawa K. J. Biol. Chem. 1992; 267: 4844-4853Abstract Full Text PDF PubMed Google Scholar, 12Zylstra G.J. Gibson D.T. J. Biol. Chem. 1989; 264: 14940-14946Abstract Full Text PDF PubMed Google Scholar). TolDox is a multicomponent enzyme consisting of α (TodC1) and β (TodC2) subunits of ISPTod, 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% (1Taira K. Hirose J. Hayashida S. Furukawa K. J. Biol. Chem. 1992; 267: 4844-4853Abstract Full Text PDF PubMed Google Scholar,12Zylstra G.J. Gibson D.T. J. Biol. Chem. 1989; 264: 14940-14946Abstract Full Text PDF PubMed Google Scholar). During the course of identifying the component responsible for the substrate specificity of BphDox and TolDox, hybrid bph-todgene clusters were constructed by replacing genes encoding the dioxygenase components from BphDox and TolDox. Among them, the recombinant Escherichia coli expressing the hybridtodC1-bphA2A3A4 genes exhibited substrate specificity similar to that of the original TolDox, but 3-fold higher activity toward TCE than TolDox (13Furukawa K. Hirose J. Hayashida S. Nakamura K. J. Bacteriol. 1994; 176: 2121-2123Crossref PubMed Google Scholar). Chloroethylenes such as TCE have been recognized to be significant environmental pollutants in the soil, groundwater, and atmosphere (14U. S. Environmental Protection Agency (1985) Document NPL-U3-6-3. US.Google Scholar). These compounds have been shown to persist over time in the environment and are suspected to be carcinogenic (15Infante P.F. Tsongas T.A. Environ. Sci. Res. 1982; 25: 301-327Google Scholar). In the recent past, it has been shown that TCE can be degraded with a variety of oxygenases such as methane monooxygenase (16Little C.D. Palumbo A.V. Herbes S.E. Lidstrom M.E. Tyndall R.L. Gilmer P.J. Appl. Environ. Microbiol. 1988; 54: 951-956Crossref PubMed Google Scholar), toluene monooxygenase (17Shields M.S. Montgomery S.O. Cuskey S.M. Chapman P.J. Pritchard P.H. Appl. Environ. Microbiol. 1991; 57: 1935-1941Crossref PubMed Google Scholar), ammonium monooxygenase (18Arciero D. Vannelli T. Logan M. Hooper A.B. Biochem. Biophys. Res. Commun. 1989; 159: 640-643Crossref PubMed Scopus (152) Google Scholar), phenol hydroxylase (19Nelson M.J. Montgomery S.O. Mahaffey W.R. Pritchard P.H. Appl. Environ. Microbiol. 1987; 53: 949-954Crossref PubMed Google Scholar), and TolDox (11Wackett L.P. Gibson D.T. Appl. Environ. Microbiol. 1988; 54: 1703-1708Crossref PubMed Google Scholar) from aerobic bacteria. The mechanism of TCE oxygenation by TolDox has been proposed by Li and Wackett (20Li S. Wackett L.P. Biochem. Biophys. Res. Commun. 1992; 185: 443-451Crossref PubMed Scopus (66) Google Scholar). 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. E. coli JM109 was used for the general propagation of the plasmids and for the expression of ISP. E. coliBL21(DE3) was used for the expression of bphA3 andbphA4. For the expression of bphA1A2 andtodC1BphA2, E. coli cells were grown at 37 °C for 12 h in Luria-Bertani medium containing 0.1 mmisopropyl-β-d-galactopyranoside and a concentration, 50 µg/ml, of the appropriate antibiotics. For expressions oftodC1C2, bphA3, and bphA4, E. coli cells were grown at 30 °C for 24 h in the same medium described above. pUC-A1A2 for the expression of ISPbphA1A2 was constructed by inserting the XhoI DNA fragment containing the bphA1A2 genes from pKTF18ΔORF3 (1Taira K. Hirose J. Hayashida S. Furukawa K. J. Biol. Chem. 1992; 267: 4844-4853Abstract Full Text PDF PubMed Google Scholar) into anSalI site of pUC119. pUC-C1A2, for the expression of the hybrid ISPTodC1BphA2, was constructed by removing thePstI DNA fragment containing the bphA3A4BC from pJHF10 (3Furukawa K. Hirose J. Suyama A. Zaiki T. Hayashida S. J. Bacteriol. 1993; 175: 5224-5232Crossref PubMed Google Scholar). pHSG-C1C2, for the expression of the ISPTodC1C2, was constructed as follows: ThetodC2 gene without the original Shine-Dalgarno sequence was amplified by PCR using pJHF-C1C2 (21Hirose J. Suyama A. Hayashida S. Furukawa K. Gene. 1994; 138: 27-33Crossref PubMed Scopus (71) Google Scholar) as the template DNA. An oligonucleotide corresponding to the 5′ sequence of thetodC2 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 andKpnI, was inserted into the corresponding sites of pBluescriptII SK+ (Stratagene) to generate pBlue-C2. ABamHI/EcoRI DNA fragment containing thetodC1 and the Shine-Dalgarno sequence of thebphA2 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 andKpnI sites. To express FDBphA3, 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 thebphA3 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 FDRBphA4, site-directed mutagenesis was carried out to introduce an NcoI site around the start codon of thebphA4 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 (21Hirose J. Suyama A. Hayashida S. Furukawa K. Gene. 1994; 138: 27-33Crossref PubMed Scopus (71) Google Scholar) with PpuMI and subsequent self-ligation. The mutagenized plasmid, pUC-A3A4N, was digested with NcoI and EcoRI, and thebphA4 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 (22Ashiuchi M. Yoshimura T. Kitamura T. Kawata Y. Nagai J. Gorlatov S. Esaki N. Soda K. J. Biochem. 1995; 117: 495-498Crossref PubMed Scopus (24) Google Scholar) was provided by Y. Kawata (Tottori University) and was used to produce the soluble FDRBphA4 fusion protein.Table IPlasmids used in this studyPlasmidsRelevant characteristics1-aApr, ampicilline resistance; Cmr, chloramphenicol resistance.Source or referencepUC-A1A2bphA1A2under the control of a lac promoter in pUC118, AprThis studypKTF18ΔORF3bphA1A2A3A4BC lacking orf3in pUC118, Apr1Taira K. Hirose J. Hayashida S. Furukawa K. J. Biol. Chem. 1992; 267: 4844-4853Abstract Full Text PDF PubMed Google ScholarpUC-C1A2todC1::bphA2 under the control of the lac promoter in pUC118, AprThis studypJHF10todC1::bphA2A3A4BCin pUC118, Apr3Furukawa K. Hirose J. Suyama A. Zaiki T. Hayashida S. J. Bacteriol. 1993; 175: 5224-5232Crossref PubMed Google ScholarpJHF-C1C2todC1C2 in pUC119, Apr21Hirose J. Suyama A. Hayashida S. Furukawa K. Gene. 1994; 138: 27-33Crossref PubMed Scopus (71) Google ScholarpBluescriptIISK+AprStratagenepBlue-C2todC2 in pBluescriptII SK+, AprThis studypBlue-C1C2todC1C2 in pBluescriptII SK+, AprThis studypHSG396CmrTakara ShuzopHSG-C1C2todC1C2 under the control of thelac promoter in pHSG396, CmrThis studypET32btrx-6Xhis under the control of the T7 promoter, AprNovagenpET32-A3trx-bphA3–6Xhis under the control of the T7 promoter in pET32b, AprThis studypJHF-A3A4BCbphA3A4BC in pUC119, Apr21Hirose J. Suyama A. Hayashida S. Furukawa K. Gene. 1994; 138: 27-33Crossref PubMed Scopus (71) Google ScholarpUC-A3A4bphA3A4 in pUC119, AprThis studypUC-A3A4NbphA3A4 with a Ncol site in pUC119, AprThis studypUC-A4trx::6Xhis::bphA4::6Xhisunder the control of the lac promoter in pUC119, AprThis studypKY206groELS, Cmr, origin from pACY10122Ashiuchi M. Yoshimura T. Kitamura T. Kawata Y. Nagai J. Gorlatov S. Esaki N. Soda K. J. Biochem. 1995; 117: 495-498Crossref PubMed Scopus (24) Google Scholar1-a Apr, ampicilline resistance; Cmr, chloramphenicol resistance. Open table in a new tab 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 ofTaq 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. ISP purification was carried out according to the method described by Haddock and Gibson (23Haddock J.D. Gibson D.T. J. Bacteriol. 1995; 177: 5834-5839Crossref PubMed Google Scholar) with some modifications. All the purification steps were carried out at 4 °C. Harvested recombinantE. 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 ISPTodC1BphA2 as well as the parental ISPBphA1A2 and ISPTodC1C2 were purified as follows. 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 mammonium 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. ISPBphA1A2 was eluted with 0.25 m KP. 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 mammonium 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 ISPTodC1C2 was the same as described above for ISPBphA1A2. 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 ISPTodC1BphA2 was the same as described for ISPBphA1A2. 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 0.3m NaCl (washing buffer). The FDBphA3 fusion protein was eluted with 50 mm KP containing 0.2m imidazole and 0.3 m NaCl. Purification of the FDRBphA4 fusion protein was done using the same method for FDBphA3 but changing the washing buffer to 50 mm KP containing 20 mm imidazole, 0.3m NaCl, and 0.5% Triton X-100. 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 itsA 434 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. Steady-state kinetic parameters for TCE, cis-DCE,trans-DCE, and 1,1-DCE were determined with purified ISP, FDBphA3, and FDRBphA4. Two milliliters of the reaction mixture containing 2.2∼3.2 µm ISP, 26∼38 µm FDBphA3, 2.2∼3.2 µmFDRBphA4, 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 inN,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 (13Furukawa K. Hirose J. Hayashida S. Nakamura K. J. Bacteriol. 1994; 176: 2121-2123Crossref PubMed Google Scholar), and those in the aqueous phase were calculated according to Henry's Law constants for the chloroethylenes (24Gossett J.M. Environ. Sci. Technol. 1987; 21: 202-208Crossref Scopus (707) Google Scholar). The values of V maxand K m were determined using the Hanes-Woolf (S/V − S plot) analysis. Purified ISP comprised of α and β 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 (25Gallagher, S. R. (1995) in Current Protocols in Protein Science (Coligan, J. E., Dunn, B. M., Ploegh, H. L., Speicher, D. W., and Wingfield, P. T., eds) Unit 10.3.5–10.3.11, John Wiley & Sons, New York.Google Scholar) to estimate the molecular mass. SDS-PAGE was carried out according to the method of Laemmli (26Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (212859) Google Scholar). The protein concentration was measured by a Bio-Rad protein assay using bovine serum albumin as the standard. The α and β 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. ISPBphA1A2 and ISPTodC1BphA2 were produced as the soluble and active forms in the recombinant E. coli at 37 °C. When ISPTodC1C2 was expressed in E. coli using a recombinant plasmid carrying the originaltodC1C2 genes, the level of expression of the β subunit was extremely low, as compared with that of the α subunit. Therefore, the Shine-Dalgarno sequence of todC2 was replaced by that ofbphA1, 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. FDBphA3 and FDRBphA4 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 FDBphA3 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 FDBphA3, the bphA4 gene was also expressed as a fusion protein with the N-terminal TRX and His-tag. However, the FDRBphA4 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 FDRBphA4fusion protein. In addition, coexpression of a chaperone, GroELS ofE. coli using pKY206, allowed the FDRBphA4fusion protein to efficiently solubilize. By using this coexpression system, the production of the FDRBphA4 fusion protein was improved to 4.5-fold compared with that without pKY206. The catalytically active components of ISPs, FDBphA3 and FDRBphA4, were purified as shown in Fig. 1. The yields of the purified ISPBphA1A2, ISPTodC1BphA2, ISPTodC1C2, FDBphA3, and FDRBphA4from a 1-liter culture were 11.0, 9.4, 8.6, 22.1, and 20.4 mg, respectively. The Dox activity was observed when all purified components of ISP, FDBphA3, and FDRBphA4 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 FDBphA3 was incubated with ISPTodC1BphA2 and FDRBphA4. Only 17%, 30%, and 70% of the maximum activity was observed when 3-, 6-, and 9-folds excess of FDBphA3were 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 FDBphA3, respectively. The specific activities of the purified ISP were determined in the presence of excess amounts of FDbphA3 by high performance liquid chromatography analysis and were evaluated from the rate of substrate depletion (TableII). 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.Table IISpecific activities of various dioxygenases toward original substratesEnzymesActivity toward biphenylActivity toward toluenenmol/min/nmol ISPBphDox183.3 ± 1538.5 ± 3.5TodC1-BphA2 hybrid Dox5.3 ± 1.3270.3 ± 6.7TolDox4.1 ± 0.6182.7 ± 0.9 Open table in a new tab 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 (α3β3) or heterotetramer (α2β2) conformations (27Maison J.R. Cammack R. Annu. Rev. Microbiol. 1992; 46: 277-305Crossref PubMed Scopus (382) Google Scholar). The α and β subunits of the parental ISPBphA1A2, ISPTodC1C2, and hybrid ISPTodC1BphA2 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 α and β 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 ISPTodC1BphA2 as well as the parental ISPBphA1A2 and ISPTodC1C2 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 ISPBphA1A2, ISPTodC1C2, and ISPTodC1BphA2 were estimated to be 209, 160, and 229 kDa, respectively (Fig.2). Because the theoretical molecular mass of ISPBphA1A2 is 219 kDa for α3β3 and 146 kDa for α2β2, the parental ISPBphA1A2is present as a heterohexamer. The theoretical molecular mass of ISPTodC1C2 is 222 kDa for α3β3and 148 kDa for α2β2, therefore, ISPTodC1C2 is present as a heterotetramer, which is in agreement with the result reported by Subramanian et al. (28Subramanian V. Liu T.N. Yeh W.K. Gibson D.T. Biochem. Biophys. Res. Commun. 1979; 91: 1131-1139Crossref PubMed Scopus (62) Google Scholar). The ISPTodC1BphA2 hybrid has a similar conformation to ISPBphA1A2, and the estimated molecular mass of 229 kDa is in good agreement with the theoretical 226.5 kDa of α3β3 conformation. 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 α2β2 heterotetramer and α3β3 heterohexamer, respectively, the catalytic center activity (k cat) of the hybrid Dox is calculated to be 1.4 min−1 site−1 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 theK 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 thetrans-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.Table IIIKinetic parameters of dioxygenases for various chloroethylensSubstratesCl ClCl ClH ClCl H\ /\ /\ /\ /C = CC = CC = CC = C/ \/ \/ \/ \Cl HH HCl HCl HTCEcis-DCEtrans-DCE1,1-DCEHybrid dioxygenase (TodC1-BphA2)k 0(min−1)4.1 ± 0.16.8 ± 0.2N.D.3-aN.D. indicates not determined.1.1k cat (min−1site−1)1.42.3 ± 0.1N.D.0.4K m(µm)130 ± 5370 ± 30N.D.227k cat/K m (min−1m−1)10,514 ± 2566,088 ± 494N.D.1,659Toluene dioxygenase (TodC1-TodC2)k 0 (min−1)4.2 ± 0.47.4 ± 2.42.1 ± 0.11.3k cat (min−1 site−1)2.1 ± 0.23.7 ± 1.21.1 ± 0.10.7K m (µm)250 ± 33824 ± 236754 ± 32383k cat/K m(m−1 min−1)8,462 ± 1,1174,488 ± 1,4561,422 ± 1291,6993-a N.D. indicates not determined. Open table in a new tab It is known that TolDox is irreversibly inactivated by coupling to TCE oxygenation (20Li S. Wackett L.P. Biochem. Biophys. Res. Commun. 1992; 185: 443-451Crossref PubMed Scopus (66) Google Scholar). 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, ISPTodC1BphA2, FDBphA3, or FDBphA4was, respectively, added to the inactivated reaction mixture. The addition of ISPTodC1BphA2 permitted the immediate restoration of the enzymatic activity of the TCE oxygenation (Fig. 3). On the other hand, the addition of FDBphA3 or FDBphA4 failed to restore the activity. These results indicate that the ISPTodC1BphA2 component is susceptible and involved in the inactivation of the hybrid Dox during TCE degradation. 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 (4Erickson B.D. Mondello F.J. Appl. Environ. Microbiol. 1993; 59: 3858-3862Crossref PubMed Google Scholar, 5Mondello F.J. Turcich M.P. Lobos J.H. Erickson B.D. Appl. Environ. Microbiol. 1997; 63: 3096-3103Crossref PubMed Google Scholar, 6Kimura N. Nishi A. Goto M. Furukawa K. J. Bacteriol. 1997; 179: 3936-3943Crossref PubMed Google Scholar, 7Kumamaru T. Suenaga H. Mitsuoka M. Watanabe T. Furukawa K. Nat. Biotechnol. 1998; 16: 663-666Crossref PubMed Scopus (192) Google Scholar, 8Suenaga H. Goto M. Furukawa K. J. Biol. Chem. 2001; 276: 22500-22506Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 9Furukawa K. Curr. Opin. Biotechnol. 2000; 11: 244-249Crossref PubMed Scopus (111) Google Scholar, 29Bruhlmann F. Chen W. Biotech. Bioeng. 1999; 63: 544-551Crossref PubMed Scopus (91) Google Scholar). We previously reported that recombinant bacteria producing hybrid TodC1-BphA2A3A4 dioxygenase constructed by subunit exchanges, exhibited the expanded capability of converting various aromatic compounds (21Hirose J. Suyama A. Hayashida S. Furukawa K. Gene. 1994; 138: 27-33Crossref PubMed Scopus (71) Google Scholar, 30Suyama A. Iwakiri R. Kimura N. Nishi A. Nakamura K. Furukawa K. J. Bacteriol. 1996; 178: 4039-4046Crossref PubMed Google Scholar) and the enhanced degradation activity toward TCE (13Furukawa K. Hirose J. Hayashida S. Nakamura K. J. Bacteriol. 1994; 176: 2121-2123Crossref PubMed Google Scholar, 30Suyama A. Iwakiri R. Kimura N. Nishi A. Nakamura K. Furukawa K. J. Bacteriol. 1996; 178: 4039-4046Crossref PubMed Google Scholar). To reconstitute ISP, we first mixed in vitro α and β 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. (31Hurtubise Y. Barriault D. Sylvestre M. J. Biol. Chem. 1996; 271: 8152-8156Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). They reported that His-tagged α and β 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, FDBphA3, and FDRBphA4 are highly functional, indicating that FDBphA3 and FDRBphA4originating 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 α 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 FDBphA3 were incubated with ISP and FDRBphA4. The recombinant FDBphA3 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 FDBphA3 may be associated with the instability of this protein. The FD of BphDox from C. testosteroni was also reported to be labile (32Hurtubise Y. Barriault D. Powlowski J. Sylvestre M. J. Bacteriol. 1995; 177: 6610-6618Crossref PubMed Google Scholar). The substrate specificities of the purified hybrid Dox were similar to those of TolDox (Table II), indicating the α 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 α3β3. Changing the subunit conformation from α2β2to α3β3 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 ISPTodC1BphA2 may be slightly relaxed to accommodate various chloroethylenes. Although the k 0 values of the hybrid Dox are comparable to those of TolDox, thek 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 ISPTodC1BphA2 is slightly changed compared with that in ISPTodC1C2 (33Jiang H. Parales R.E. Lynch N.A. Gibson D.T. J. Bacteriol. 1996; 178: 3133-3139Crossref PubMed Google Scholar). As previously reported (13Furukawa K. Hirose J. Hayashida S. Nakamura K. J. Bacteriol. 1994; 176: 2121-2123Crossref PubMed Google Scholar), 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 theKm 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 (34Newman L.M. Wackett L.P. J. Bacteriol. 1997; 179: 90-96Crossref PubMed Google Scholar) and dioxygenases (20Li S. Wackett L.P. Biochem. Biophys. Res. Commun. 1992; 185: 443-451Crossref PubMed Scopus (66) Google Scholar, 35Lee K. J. Bacteriol. 1999; 181: 2719-2725Crossref PubMed Google Scholar). 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 (35Lee K. J. Bacteriol. 1999; 181: 2719-2725Crossref PubMed Google Scholar). Li and Wackett (20Li S. Wackett L.P. Biochem. Biophys. Res. Commun. 1992; 185: 443-451Crossref PubMed Scopus (66) Google Scholar) reported that TolDox is inactivatedvia 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 α and β subunits occurred on ISPTodC1BphA2 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 ISPTodC1BphA2were 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 ISPTodC1BphA2 (data not shown). Therefore, it is likely that the hybrid ISPTodC1BphA2 is inactivated by a fashion similar to the ISP of TolDox (20Li S. Wackett L.P. Biochem. Biophys. Res. Commun. 1992; 185: 443-451Crossref PubMed Scopus (66) Google Scholar).