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Characterization of a Novel Thermostable Mn(II)-dependent 2,3-Dihydroxybiphenyl 1,2-Dioxygenase from a Polychlorinated Biphenyl- and Naphthalene-degrading Bacillus sp. JF8

2003; Elsevier BV; Volume: 278; Issue: 24 Linguagem: Inglês

10.1074/jbc.m210240200

1083-351X

Takashi Hatta, Gouri Mukerjee-Dhar, Jir̆ı́ Damborský, Hohzoh Kiyohara, Kazuhide Kimbara,

Oxidative Organic Chemistry Reactions

A novel thermostable Mn(II)-dependent 2,3-dihydroxybiphenyl-1,2-dioxygenase (BphC_JF8) catalyzing the meta-cleavage of the hydroxylated biphenyl ring was purified from the thermophilic biphenyl and naphthalene degrader, Bacillus sp. JF8, and the gene was cloned. The native and recombinant BphC enzyme was purified to homogeneity. The enzyme has a molecular mass of 125 ± 10 kDa and was composed of four identical subunits (35 kDa). BphC_JF8 has a temperature optimum of 85 °C and a pH optimum of 7.5. It exhibited a half-life of 30 min at 80 °C and 81 min at 75 °C, making it the most thermostable extradiol dioxygenase studied. Inductively coupled plasma mass spectrometry analysis confirmed the presence of 4.0–4.8 manganese atoms per enzyme molecule. The EPR spectrum of BphC_JF8 exhibited g = 2.02 and g = 4.06 signals having the 6-fold hyperfine splitting characteristic of Mn(II). The enzyme can oxidize a wide range of substrates, and the substrate preference was in the order 2,3-dihydroxybiphenyl > 3-methylcatechol > catechol > 4-methylcatechol > 4-chlorocatechol. The enzyme is resistant to denaturation by various chelators and inhibitors (EDTA, 1,10-phenanthroline, H2O2, 3-chlorocatechol) and did not exhibit substrate inhibition even at 3 mm 2,3-dihydroxybiphenyl. A decrease in Km accompanied an increase in temperature, and the Km value of 0.095 μm for 2,3-dihydroxybiphenyl (at 60 °C) is among the lowest reported. The kinetic properties and thermal stability of the native and recombinant enzyme were identical. The primary structure of BphC_JF8 exhibits less than 25% sequence identity to other 2,3-dihydroxybiphenyl 1,2-dioxygenases. The metal ligands and active site residues of extradiol dioxygenases are conserved, although several amino acid residues found exclusively in enzymes that preferentially cleave bicyclic substrates are missing in BphC_JF8. A three-dimensional homology model of BphC_JF8 provided a basis for understanding the substrate specificity, quaternary structure, and stability of the enzyme. A novel thermostable Mn(II)-dependent 2,3-dihydroxybiphenyl-1,2-dioxygenase (BphC_JF8) catalyzing the meta-cleavage of the hydroxylated biphenyl ring was purified from the thermophilic biphenyl and naphthalene degrader, Bacillus sp. JF8, and the gene was cloned. The native and recombinant BphC enzyme was purified to homogeneity. The enzyme has a molecular mass of 125 ± 10 kDa and was composed of four identical subunits (35 kDa). BphC_JF8 has a temperature optimum of 85 °C and a pH optimum of 7.5. It exhibited a half-life of 30 min at 80 °C and 81 min at 75 °C, making it the most thermostable extradiol dioxygenase studied. Inductively coupled plasma mass spectrometry analysis confirmed the presence of 4.0–4.8 manganese atoms per enzyme molecule. The EPR spectrum of BphC_JF8 exhibited g = 2.02 and g = 4.06 signals having the 6-fold hyperfine splitting characteristic of Mn(II). The enzyme can oxidize a wide range of substrates, and the substrate preference was in the order 2,3-dihydroxybiphenyl > 3-methylcatechol > catechol > 4-methylcatechol > 4-chlorocatechol. The enzyme is resistant to denaturation by various chelators and inhibitors (EDTA, 1,10-phenanthroline, H2O2, 3-chlorocatechol) and did not exhibit substrate inhibition even at 3 mm 2,3-dihydroxybiphenyl. A decrease in Km accompanied an increase in temperature, and the Km value of 0.095 μm for 2,3-dihydroxybiphenyl (at 60 °C) is among the lowest reported. The kinetic properties and thermal stability of the native and recombinant enzyme were identical. The primary structure of BphC_JF8 exhibits less than 25% sequence identity to other 2,3-dihydroxybiphenyl 1,2-dioxygenases. The metal ligands and active site residues of extradiol dioxygenases are conserved, although several amino acid residues found exclusively in enzymes that preferentially cleave bicyclic substrates are missing in BphC_JF8. A three-dimensional homology model of BphC_JF8 provided a basis for understanding the substrate specificity, quaternary structure, and stability of the enzyme. The catabolic versatility exhibited by microorganism plays an essential role in the carbon cycle, and this depends to a large extent on the use of oxygenases. In the degradation of aromatic compounds, oxygenases play a significant role both by hydroxylating the aromatic ring and by catalyzing the ring fission reaction. Nearly all bacterial pathways for the degradation of aromatic compounds transform initial substrates into intermediates that carry two or more hydroxyl groups on the aromatic ring, which are then substrates for the ring cleavage dioxygenases. Cleavage is generally catalyzed by metalloenzymes of one of the two functional classes: intradiol dioxygenases, which cleave ortho to the hydroxyl substituents, or extradiol dioxygenases, which cleave meta to the hydroxyl substituents.Harayama and Rekik (1Harayama S. Rekik M. J. Biol. Chem. 1989; 264: 15328-15333Abstract Full Text PDF PubMed Google Scholar) proposed that extradiol dioxygenases could be divided into two families, those exhibiting a preference for bicyclic substrates and those with a preference for monocyclic substrates. Since then, several extradiol dioxygenases have been sequenced and characterized, and the evolutionary relationship among them has been investigated. The three-dimensional structures of three Type I extradiol dioxygenases, two of which cleave bicyclic compounds (2Han S. Eltis L.D. Timmis K.T.N. Muchmore S.W. Bolin J.T. Science. 1995; 270: 976-980Crossref PubMed Scopus (309) Google Scholar, 3Senda T. Sugiyama K. Narita H. Yamamoto H. Kimbara K. Fukuda M. Sato M. Yano K. Mitsui Y. J. Mol. Biol. 1996; 255: 735-752Crossref PubMed Scopus (197) Google Scholar) and one of which cleaves monocyclic compounds (4Kita K. Kita S. Fujisawa I. Inaka K. Ishida T. Horiike K. Nozaki M. Miki K. Structure. 1999; 7: 25-34Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar), have been reported. Whereas a majority of the bacterial extradiol dioxygenases that have been characterized contain Fe(II) as a catalytic metal center, there are only three known bacterial 3,4-dihydroxyphenylacetate 2,3-dioxygenase that utilize metals other than Fe(II); the enzyme from Bacillus brevis (5Que Jr., L. Widom J. Crawford R.L. J. Biol. Chem. 1981; 256: 10941-10944Abstract Full Text PDF PubMed Google Scholar) and Arthrobacter globiformis CM-2 (6Boldt Y.R. Sadowsky M.J. Ellis L.B.M. Que Jr., L. Wackett L.P. J. Bacteriol. 1995; 177: 1225-1232Crossref PubMed Google Scholar) are manganese-dependent, whereas that from Klebsiella pneumoniae exhibits magnesium dependence (7Gibello A. Ferrer E. Martin M. Garrido-Pertierra A. Biochem. J. 1994; 301: 145-150Crossref PubMed Scopus (43) Google Scholar).The degradation of biphenyl by bacteria has been well characterized at the genetic and biochemical level (8Taira K. Hirose J. Hayashida S. Furukawa K. J. Biol. Chem. 1992; 267: 4844-4853Abstract Full Text PDF PubMed Google Scholar, 9Masai E. Yamada A. Healy J.M. Hatta T. Fukuda M. Yano K. Appl. Environ. Microbiol. 1995; 61: 2079-2085Crossref PubMed Google Scholar, 10Hofer B. Eltis L.D. Dowling D.N. Timmis K.N. Gene (Amst.). 1993; 130: 47-55Crossref PubMed Scopus (154) Google Scholar). The major pathway for biphenyl degradation is a four-step process initiated by the insertion of two atoms of oxygen at carbon positions 2 and 3 of the aromatic ring by biphenyl dioxygenase, the product of bphA genes. The resulting 2,3-dihydrodiol is dehydrogenated by a dihydrodiol dehydrogenase, the product of the bphB gene, to 2,3-dihydroxybiphenyl. This is cleaved at the meta position by the extradiol dioxygenase, 2,3-dihydroxybiphenyl 1,2-dioxygenase (BphC), the product of the bphC gene (Fig. 1). Then a hydrolase encoded by the bphD gene hydrolyzes the 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid into benzoate and 2-hydroxypenta-2,4-dienoate.This pathway has been well studied for its potential to cometabolize polychlorinated biphenyls (PCB), 1The abbreviations used are: PCB, polychlorinated biphenyl(s); X-gal, 5-bromo-4-chloro-3-indolyl-β-d-thiogalactopyranoside; ICP-MS, inductively coupled plasma mass spectrometry; mT, millitesla.1The abbreviations used are: PCB, polychlorinated biphenyl(s); X-gal, 5-bromo-4-chloro-3-indolyl-β-d-thiogalactopyranoside; ICP-MS, inductively coupled plasma mass spectrometry; mT, millitesla. a family of recalcitrant, toxic environmental pollutants. Considerable differences have been found in congener selectivity pattern and range of activity among various PCB-degrading bacteria (11Bedard D.L. Haberl M.H. Microb. Ecol. 1990; 20: 87-102Crossref PubMed Scopus (138) Google Scholar, 12Gibson D.T. Cruden D.L. Haddock J.D. Zylstra G.J. Brand J.M. J. Bacteriol. 1993; 175: 4561-4564Crossref PubMed Google Scholar). Although it is the initial oxygenase (BphA) that is crucially responsible for recognition and binding of the substrate (13Erickson B.D. Mondello F.J. Appl. Environ. Microbiol. 1993; 59: 3858-3862Crossref PubMed Google Scholar), the ability of the bph pathway is also limited in parts by the BphC enzyme, which is incapable of transforming certain chlorinated dihydroxy biphenyls (14Furukawa K. Tomizuka N. Kamibayashi A. Appl. Environ. Microbiol. 1979; 38: 301-310Crossref PubMed Google Scholar, 15Seeger M. Timmis K.N. Hofer B. Appl. Environ. Microbiol. 1995; 61: 2654-2655Crossref PubMed Google Scholar) and is inhibited by 3-chlorocatechol (16Sondossi M. Sylvestre M. Ahmad D. Appl. Environ. Microbiol. 1992; 58: 485-495Crossref PubMed Google Scholar, 17Arensdorf J.J. Focht D.D. Appl. Environ. Microbiol. 1994; 60: 2884-2889Crossref PubMed Google Scholar, 18Astuiras J.A. Timmis K.N. J. Bacteriol. 1993; 175: 4631-4640Crossref PubMed Google Scholar). Extradiol dioxygenases are also susceptible to mechanism-based inactivation by their aromatic substrates. Therefore, the potential of this pathway for the remediation of PCB-contaminated soils may not be fully realized until more stable forms of the enzymes are available. Thermophilic bacteria produce enzyme variants with vastly improved stability. To be able to rationally engineer such properties into mesophilic enzymes, a study of the determinants of the stability is an important task for basic and applied research. Although thermophiles degrading aromatic compounds such as BTEX (benzene, toluene, ethylbenzene, xylene isomers) and phenol/cresol have been isolated (19Chen C. Taylor R. Biotechnol. Bioeng. 1995; 48: 614-624Crossref PubMed Scopus (54) Google Scholar, 20Gurugeyalakshmi G. Oriel P. Appl. Environ. Microbiol. 1989; 55: 500-502Crossref PubMed Google Scholar, 21Mutzel A. Reinscheid U.M. Antranikian G. Muller R. Appl. Microbiol. Biotechnol. 1996; 46: 593-596Crossref Scopus (52) Google Scholar), the aromatic pathways in these organisms are not well studied, and there have been few reports on the characterization of the genes/proteins involved (22Dong F.-M. Wang L.-L. Wang C.-M. Cheng J.-P. He Z.-Q. Sheng Z.-J. Shen R.-Q. Appl. Environ. Microbiol. 1992; 58: 2531-2535Crossref PubMed Google Scholar, 23Natarajan M.R. Lu Z. Oriel P. Biodegradation. 1994; 5: 77-82Crossref PubMed Scopus (18) Google Scholar).We have recently isolated a thermophilic bacterium Bacillus sp. JF8, which besides utilizing biphenyl and naphthalene as the sole source of carbon and energy, can transform several PCB congeners (24Shimura M. Mukerjee-Dhar G. Kimbara K. Nagato H. Kiyohara H. Hatta T. FEMS Microbiol. Lett. 1999; 178: 87-93Crossref PubMed Google Scholar). From our analysis of the chlorobenzoic acids produced during mineralization of selected PCB congeners by strain JF8, we had concluded that the less chlorinated ring was oxidized, indicating similarity to the mesophilic PCB-degrading pathway. Probably, the upper biphenyl/PCB metabolic pathway in the thermophilic strain JF8 is identical to the metabolic pathway in mesophilic biphenyl/PCB degraders. Strain JF8N, a spontaneous mutant that lost the ability to utilize biphenyl as a carbon source while retaining the ability to utilize naphthalene, had indicated the presence of multiple dioxygenases in Bacillus sp. JF8. Here we report on the cloning and characterization of the extradiol dioxygenase, involved in the meta-cleavage of the biphenyl ring. To the best of our knowledge, this is the first report of a Mn(II)-dependent, thermostable 2,3-dihydroxybiphenyl 1,2-dioxygenase.EXPERIMENTAL PROCEDURESBacterial Strains, Media, and Cloning of bphCBacillus sp. strain JF8 was grown on Castenholz D medium (24Shimura M. Mukerjee-Dhar G. Kimbara K. Nagato H. Kiyohara H. Hatta T. FEMS Microbiol. Lett. 1999; 178: 87-93Crossref PubMed Google Scholar). Biphenyl was added at a concentration of 2 g/liter. For solid medium (1.5% agar), biphenyl was provided as vapor. Escherichia coli MV1190 was used as a host strain for DNA manipulation. Luria broth (pH 7.5) containing 10 g/liter tryptone, 5 g/liter yeast extract, and 5 g/liter NaCl was used as a complex medium. Ampicillin was added to the medium at a concentration of 100 μg/ml. Isopropyl-β-d-thiogalactopyranoside and 5-bromo-4-chloro-3-indolyl-β-d-thiogalactopyranoside (X-gal) were used at concentrations of 100 and 40 μg/ml, respectively. Bacillus sp. JF8 was incubated at 60 °C, whereas Escherichia coli was incubated at 37 °C.DNA ManipulationTotal DNA of strain JF8 was isolated by a modification of the procedure described by Marmur (25Johnson J.L. Gerhardt P. Murray R.G.E. Wood W.A. Kreig N.R. Similarity Analysis of DNAs. Methods for General and Molecular Bacteriology, American Society for Microbiology, Washington, D. C.1994Google Scholar), and plasmid DNA was isolated by the alkaline lysis method (26Birnboim H. Doly J. Nucleic Acids Res. 1979; 7: 1513-1523Crossref PubMed Scopus (9847) Google Scholar). The total DNA of JF8 was digested by different restriction enzymes and DNA manipulations carried out as described by Sambrook et al. (27Sambrook J. Fritsch E. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). The recovered DNA fragments were ligated into appropriate vectors and transformed into E. coli cells by the CaCl2 procedure (28Mukerjee-Dhar G. Hatta T. Shimura M. Kimbara K. Arch. Microbiol. 1998; 169: 61-70Crossref PubMed Scopus (11) Google Scholar). Transformants were selected on solid Luria broth plates supplemented with ampicillin, isopropyl-β-d-thiogalactopyranoside, and X-gal by spraying with 2,3-dihydroxybiphenyl solution. Southern hybridization was performed under stringent conditions using Hybond N+ nylon membrane filter (Amersham Biosciences). DNA sequencing was carried out by the dideoxy chain termination method of Sanger et al. (29Sanger F. Nicklen S. Coulson V. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (52363) Google Scholar). The bphC gene of Rhodococcus sp. RHA1 (9Masai E. Yamada A. Healy J.M. Hatta T. Fukuda M. Yano K. Appl. Environ. Microbiol. 1995; 61: 2079-2085Crossref PubMed Google Scholar) was introduced into the multicopy tac promoter vector, pTTQ18 (Amersham Biosciences), to give pTT122X. The MPC_mt2 expression plasmid, pIX121, was similarly constructed by inserting the xylE gene (4Kita K. Kita S. Fujisawa I. Inaka K. Ishida T. Horiike K. Nozaki M. Miki K. Structure. 1999; 7: 25-34Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar) encoded by the TOL plasmid pWWO into pTTQ18.Purification and Characterization of the Native and Recombinant BphC ProteinThe plasmid pQW1, constructed by inserting the bphC gene from strain JF8 into pTTQ18, was introduced into E. coli MV1190. Cells were grown in 1.5 liters of Luria broth containing 250 μg/ml of ampicillin to an A600 = 0.4. The bphC gene was induced by the addition of 1 mm isopropyl-β-d-thiogalactopyranoside. After 4 h of induction, cells were collected by centrifuging at 4000 × g for 10 min. For the native BphC protein, cells of strain JF8 were grown overnight on Castenholz D medium or on Luria broth with biphenyl. The cells were washed twice with 25 mm phosphate buffer (pH 7.5) and resuspended in the same buffer. The cells were disrupted by a French pressure cell (Aminco Corp.) and centrifuged at 12,000 × g for 30 min and then at 105,000 × g for 60 min. The supernatant was used as a cell-free extract for assaying 2,3-dihydroxybiphenyl 1,2-dioxygenase activity, which was determined by measuring the formation of the meta-cleavage reaction product at 434 nm with a Beckman DU7500 spectrophotometer equipped with a thermocontrolled cuvette holder, and the pH of the Tris-buffer was set at the assay temperature. Enzymatic activity was assayed at 60 °C in 50 mm Tris-HCl buffer (pH 7.5) containing 330 μm 2,3-dihydroxybiphenyl. One unit of enzyme activity was defined as the amount of enzyme that converts 1 μmol of substrate/min. The molar extinction coefficient of the product under assay conditions was taken to be 13,200 cm–1m–1. The relative meta-cleavage activities were determined from the extinction coefficients of the ring fission products formed from the following substrates: catechol (λmax, 375 nm; e, 33,000 cm–1m–1), 3-methylcatechol (λmax, 388 nm; e, 32,000 cm–1m–1), 4-methylcatechol (λmax, 382 nm; e, 17,000 cm–1m–1), and 4-chlorocatechol (λmax, 379 nm; e, 40,000 cm–1m–1). For purification of the enzyme, all manipulations were carried out at 10 °C in 25 mm potassium phosphate buffer (pH 7.5) containing 1 mm β-mercaptoethanol (Buffer A) unless otherwise mentioned.DEAE-Toyopearl Chromatography—The crude extract was loaded onto a DEAE-Toyopearl column (5.0 × 20 cm) previously equilibrated with Buffer A. Proteins were eluted with a linear gradient of KCl from 0 to 0.25 m in a total volume of 2500 ml of Buffer A. Active fractions, eluted around 0.2 m KCl, were collected.Phenyl-Sepharose Column Chromatography—The collected fractions were dialyzed against Buffer A containing 1.2 m ammonium sulfate. The resulting protein solution was loaded onto phenyl-Sepharose HP 2.6/10 column (Amersham Biosciences) equilibrated with Buffer A containing 1.2 m ammonium sulfate. The enzyme was eluted with an 800-ml gradient of 1.2 to 0.0 m ammonium sulfate. The enzyme was eluted around 0.1 m ammonium sulfate.MonoQ Column Chromatography—The active fractions eluted from phenyl-Sepharose column were pooled and dialyzed against Buffer A. The resulting solution was applied to a MonoQ HR 16/10 column (Amersham Biosciences) equilibrated with Buffer A. After the column was washed with 60 ml of Buffer A, the enzyme was eluted with 400 ml of a linear gradient from 0.0 to 0.5 m KCl. The enzyme was eluted around 0.3 m KCl.Protein concentration was estimated by the method of Bradford (30Bradford M.M. Ann. Biochem. 1976; 72: 248-254Crossref Scopus (213510) Google Scholar) using bovine serum albumin as a standard. The purity and size of the enzyme proteins were estimated by SDS-PAGE according to the method of Laemmli (31Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (206057) Google Scholar). Protein staining of the gel was performed with Coomassie Brilliant Blue R-250.Determination of Molecular MassThe relative molecular mass of the native and recombinant enzyme was estimated by gel filtration on a Superdex 200HR 10/30 column (Amersham Biosciences) calibrated with ferritin (440 kDa), catalase (232 kDa), bovine serum albumin (67 kDa), and chymotrypsin (25 kDa). The relative subunit molecular mass was determined by SDS-PAGE. The Amersham Biosciences low molecular mass calibration kit was used.N-terminal Sequence AnalysisPurified recombinant and native BphC_JF8 were subjected to N-terminal amino acid sequencing by the Edman degradation process using a model 477A protein sequencer (Applied Biosystems) in accordance with the manufacturer's procedure.Kinetic MeasurementsMichaelis-Menten kinetics of the reaction was verified by plotting reaction rates against substrate concentration. The Km and Vmax values were determined by nonlinear regression analysis of the plots and graphically from Lineweaver-Burk plotting of the initial cleavage rate. The substrate concentrations were in the range 0.05–0.25 μm 2,3-dihydroxybiphenyl at 60 °C and 0.1–0.5 μm at 25 °C. An activity assay of the enzyme was performed as mentioned above. For temperature stability and pH optimum, a temperature range of 30–90 °C and pH range of 6.0–9.0 (Tris-HCl and potassium phosphate buffer) was utilized, and the residual activities of the native and recombinant enzyme were determined. The buffer pH values were adjusted at the experimental temperature. The influence of metal cations, chelators, and inhibitors on enzyme activity was tested by incubating with samples of the purified enzyme (0.1 mg/ml) dialyzed against Tris-HCl buffer (pH 7.5) for different time intervals at 25 °C. The activation energy (Ea) was estimated using the Arrhenius equation for the temperature range of 30–75 °C. The value of Ea was determined from the slope of the straight line that resulted when the logarithm of the reaction constant, k, was plotted against 1/T.Determination of Metal ContentMetal content was determined by inductively coupled plasma mass spectrometry (ICP-MS) using a SEIKO SPQ6500 spectrometer (Seiko Instruments Inc.). Samples for ICP-MS were prepared using acid-washed glassware. Samples and standards were prepared in 0.1% HNO3. Separate standard curves were routinely prepared for iron and manganese, and samples were measured in quadruplicates.EPR Sample Preparation and Spectroscopic MethodSamples of 0.3 ml were inserted in 4.0-mm inner diameter quartz tubes and frozen by slow immersion in liquid nitrogen. X-band EPR spectra were measured and analyzed using JEOL JES-CT470 (JEOL, Tokyo, Japan) equipped with a JEOL JES-G470 liquid helium variable temperature system. The EPR spectrometer setting was as follows: microwave power, 1 milliwatt; modulation amplitude, 1 mT at 100 kHz. Spectra were obtained with a microwave frequency of 8.93 GHz at 10 K as a single 4-min scan from 50 to 550 mT.Phylogenetic AnalysisA homology search was performed with BLAST 2.0 (gapped BLAST) (32Altschul S.F. Madden T.L. Schaffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (59182) Google Scholar). Amino acid sequences retrieved from the protein databases were aligned using ClustalW version 1.9 (33Thompson J.D. Higgins D.G. Gibson T.J. Nucleic Acids Res. 1994; 11: 4673-4680Crossref Scopus (55217) Google Scholar). A phylogenetic tree was constructed by the neighbor-joining method using the Blosum62 distance matrix.Molecular ModelingSecondary structure predictions were done by building a consensus from the predicted results of PSIPRED (34Jones D.T. J. Mol. Biol. 1999; 292: 195-202Crossref PubMed Scopus (4388) Google Scholar), JPRED (35Cuff J.A. Clamp M.E. Siddigni A.S. Finlay M. Barton G.J. Bioinformatics. 1998; 14: 892-893Crossref PubMed Scopus (916) Google Scholar), PHD (36Rost B. Methods Enzymol. 1996; 266: 525-539Crossref PubMed Google Scholar), and SSpro (37Baldi P. Brunak S. Frasconi P. Pollastri G. Soda G. Bioinformatics. 1999; 15: 937-946Crossref PubMed Scopus (363) Google Scholar) methods. The BphC_JF8 protein was modeled in the tetrameric state using the crystal structure of catechol 2,3-dioxygenase (MPC; Protein Data Bank code 1MPY) as the template (4Kita K. Kita S. Fujisawa I. Inaka K. Ishida T. Horiike K. Nozaki M. Miki K. Structure. 1999; 7: 25-34Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). A preliminary model of the BphC_JF8 was constructed using automated modeling servers SWISS-MODEL (38Guex N. Peitsch M.C. Electrophoresis. 1997; 18: 2714-2723Crossref PubMed Scopus (9474) Google Scholar) and FAMS (39Ogata K. Umeyama H. Protein Eng. 1997; 10: 353-359Crossref PubMed Scopus (21) Google Scholar). The final model was derived with the program package MODELLER version 6.0 (40Sali A. Blundell T.L. J. Mol. Biol. 1993; 234: 779-815Crossref PubMed Scopus (10391) Google Scholar) and optimized by “refine1” molecular dynamic simulation as implemented in the same package. The stereochemical quality of the final model was assessed by the program PROCHECK version 3.0 (41Laskowski R.A. McArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystals. 1993; 26: 283-291Crossref Google Scholar). The presence of salt bridges was inferred when Asp or Glu side chain carbonyl oxygen atoms were found to be within a 4.0-Å distance from the nitrogen atoms in Arg and Lys side chains.RESULTSSequence Analysis of the Cloned meta-Cleavage Gene—A 4-kb HindIII fragment was isolated from the transformant (pBHC1), which could convert 2,3-dihydroxybiphenyl into the yellow meta-cleavage product. Restriction analysis and sub-cloning resulted in the identification of a 1.5-kb HindIII-SacI fragment (pBH1) that encoded the extradiol dioxygenase activity. Sequencing the 1.5-kb fragment revealed a 945-bp open reading frame with a G + C content of 48%. The C-terminal region of an open reading frame upstream of the meta-cleavage gene exhibited homology to known dihydrodiol dehydrogenase genes (bphB), implying that the meta-cleavage gene is not isolated but is most probably part of an operon. The 16 S rDNA sequence (97.8% identity to Bacillus stearothermophilus) and various chemotaxonomic markers indicate that strain JF8 is related to B. stearothermophilus (24Shimura M. Mukerjee-Dhar G. Kimbara K. Nagato H. Kiyohara H. Hatta T. FEMS Microbiol. Lett. 1999; 178: 87-93Crossref PubMed Google Scholar). Therefore, the codon usage of the bphC gene was compared with that of B. stearothermophilus and was found to be similar with one exception. The codon CCC, which is very rarely used in B. stearothermophilus, was found to be most frequently used to code for proline in BphC_JF8.Purification of the Biphenyl-induced Extradiol Dioxygenase and Its N-terminal Sequencing—Initially, the inducible extradiol dioxygenase from biphenyl-grown cells of Bacillus sp. JF8 was purified. The purification scheme for the native enzyme is given in Table I, and the enzyme was purified 32-fold with an overall yield of 54%. The SDS-PAGE of the purified enzyme showed one distinct band (Fig. 2). The N-terminal sequence of the native enzyme was determined by Edman degradation to be TAEIAKFGHIALITPNLEKSVWFFRdIVGLEEVdRQGdTI. This agrees with the DNA sequence data, except for the initial Met, of the cloned open reading frame, identifying the extradiol dioxygenase gene as a bphC. The bphC gene of JF8 was expressed from pQW1 in E. coli, and a three-step protocol (similar to one shown in Table I) was used to purify the recombinant extradiol dioxygenase, giving a 19-fold purification with a total recovery of 46%. The determined N-terminal sequence of the recombinant enzyme was identical to that of the native enzyme.Table IPurification of the native 2,3-dihydroxybiphenyl 1,2-dioxygenase of strain JF8Purification stepVolumeTotal proteinTotal activityaOne unit is defined as the amount of protein that converts 1 μmol of 2,3-dihydroxybiphenyl/min.Specific activityYieldmlmgunitsunits/mg%Crude extract15016802840.169100DEAE-Toyopearl 650M2821872411.2985Phenyl-Sepharose80321705.3160Mono Q40281535.4754a One unit is defined as the amount of protein that converts 1 μmol of 2,3-dihydroxybiphenyl/min. Open table in a new tab Fig. 2SDS-PAGE of purified 2,3-dihydroxybiphenyl 1,2-dioxygenase.Lane 1, molecular mass standards; lane 2, 2.5 μg of 2,3-dihydroxybiphenyl 1,2-dioxygenase recombinant protein purified from E. coli; lane 3, 1.0 μg of 2,3-dihydroxybiphenyl 1,2-dioxygenase purified from Bacillus sp. JF8.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Metal Analysis of BphC_JF8 —The recombinant BphC_JF8 grown on Luria broth had very low specific activity (0.68 units/mg) compared with the native enzyme (5.47 units/mg), and we tried to activate the recombinant enzyme in the presence of Fe(II) and Mn(II). Activation in the presence of 1 mm Mn(II) at 25 and 60 °C is shown in Fig. 3. Activation at 60 °C was faster and resulted in a higher specific activity as compared with 25 °C. When the enzyme was incubated with 1 mm Fe(II) and ascorbate in argon gas, the enzyme was activated 20-fold. However, the activity diminished rapidly and came down to the original level in 20 s (results not shown).Fig. 3Activation of purified, recombinant BphC_JF8. Enzymes were dialyzed against 50 mm Tris-HCl buffer (pH 7.5) and incubated in the presence of 1 mm Mn2+ at 25 and 60 °C.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Metal analysis using ICP-MS showed that the native Bph-C_JF8 contained between 4.0 and 4.8 manganese atoms per enzyme molecule, depending on the batch. The iron content was found to be consistently low at 0.05 iron atoms/enzyme molecule. The oxidation state of the manganese in BphC_JF8 was determined using EPR spectroscopy (Fig. 4A). The typical 6-fold signal centered at g = 2.02 clearly showed the presence of Mn(II). The hyperfine coupling constant, A, of 9.3 mT compares well with that observed for other Mn(II)-dependent enzymes (42Whiting A.K. Boldt Y.R. Hendrich M.P. Wackett L.P. Que Jr., L. Biochemistry. 1996; 35: 160-170Crossref PubMed Scopus (117) Google Scholar, 43Requena L. Bornemann S. Biochem. J. 1999; 343: 185-190Crossref PubMed Google Scholar, 44Tanner A. Bowater L. Fairhurst S.A. Bornemann S. J. Biol. Chem. 2001; 47: 43627-43634Abstract Full Text Full Text PDF Scopus (113) Google Scholar). An unusual feature of the BphC_JF8 spectrum was t