Biochemical and Molecular Characterization of a Ring Fission Dioxygenase with the Ability to Oxidize (Substituted) Salicylate(s) from Pseudaminobacter salicylatoxidans
2004; Elsevier BV; Volume: 279; Issue: 36 Linguagem: Inglês
10.1074/jbc.m313500200
ISSN1083-351X
AutoresJan-Peter Hintner, Thorsten Reemtsma, A. Stolz,
Tópico(s)Metal-Catalyzed Oxygenation Mechanisms
ResumoThe gene coding for a dioxygenase with the ability to cleave salicylate by a direct ring fission mechanism to 2-oxohepta-3,5-dienedioic acid was cloned from Pseudaminobacter salicylatoxidans strain BN12. The deduced amino acid sequence encoded a protein with a molecular mass of 41,176 Da, which showed 28 and 31% sequence identity, respectively, to a gentisate 1,2-dioxygenase from Pseudomonas alcaligenes NCIMB 9867 and a 1-hydroxy-2-naphthoate 1,2-dioxygenase from Nocardioides sp. KP7. The highest degree of sequence identity (58%) was found to a presumed gentisate 1,2-dioxygenase from Corynebacterium glutamicum. The enzyme from P. salicylatoxidans BN12 was heterologously expressed in Escherichia coli and purified as a His-tagged enzyme variant. The purified enzyme oxidized in addition to salicylate, gentisate, 5-aminosalicylate, and 1-hydroxy-2-naphthoate also 3-amino- and 3- and 4-hydroxysalicylate, 5-fluorosalicylate, 3-, 4-, and 5-chlorosalicylate, 3-, 4-, and 5-bromosalicylate, 3-, 4-, and 5-methylsalicylate, and 3,5-dichlorosalicylate. The reactions were analyzed by high pressure liquid chromatography/mass spectrometry, and the reaction products were tentatively identified. For comparison, the putative gentisate 1,2-dioxygenase from C. glutamicum was functionally expressed in E. coli and shown to convert gentisate but not salicylate or 1-hydroxy-2-naphthoate. The gene coding for a dioxygenase with the ability to cleave salicylate by a direct ring fission mechanism to 2-oxohepta-3,5-dienedioic acid was cloned from Pseudaminobacter salicylatoxidans strain BN12. The deduced amino acid sequence encoded a protein with a molecular mass of 41,176 Da, which showed 28 and 31% sequence identity, respectively, to a gentisate 1,2-dioxygenase from Pseudomonas alcaligenes NCIMB 9867 and a 1-hydroxy-2-naphthoate 1,2-dioxygenase from Nocardioides sp. KP7. The highest degree of sequence identity (58%) was found to a presumed gentisate 1,2-dioxygenase from Corynebacterium glutamicum. The enzyme from P. salicylatoxidans BN12 was heterologously expressed in Escherichia coli and purified as a His-tagged enzyme variant. The purified enzyme oxidized in addition to salicylate, gentisate, 5-aminosalicylate, and 1-hydroxy-2-naphthoate also 3-amino- and 3- and 4-hydroxysalicylate, 5-fluorosalicylate, 3-, 4-, and 5-chlorosalicylate, 3-, 4-, and 5-bromosalicylate, 3-, 4-, and 5-methylsalicylate, and 3,5-dichlorosalicylate. The reactions were analyzed by high pressure liquid chromatography/mass spectrometry, and the reaction products were tentatively identified. For comparison, the putative gentisate 1,2-dioxygenase from C. glutamicum was functionally expressed in E. coli and shown to convert gentisate but not salicylate or 1-hydroxy-2-naphthoate. The oxygenolytic cleavage of the aromatic nucleus by bacteria requires in most cases the presence of two hydroxy groups attached to the aromatic ring (1Que Jr., L. Ho R.Y.N. Chem. Rev. 1996; 96: 2607-2624Crossref PubMed Scopus (758) Google Scholar, 2Bugg T.D.H. Lin G. Chem. Comm. 2001; : 941-952Crossref Scopus (154) Google Scholar, 3Mishra V. Lal R. Srinivisan Crit. Rev. Microbiol. 2001; 27: 133-166Crossref PubMed Scopus (45) Google Scholar). Only a few examples have been described previously in which monohydroxylated aromatic compounds were cleaved by ring fission dioxygenases, and in most of these examples aminohydroxybenzene derivatives were observed as ring fission substrates. The ability of "traditional" ring fission dioxygenases to oxidize aminohydroxybenzene derivatives is mechanistically easily explained, because the amino group activates the aromatic nucleus in a similar way as the hydroxy group for an electrophilic attack of the ring-cleaving dioxygenases (4Davis J.K. He Z. Somerville C.C. Spain J.C. Arch. Microbiol. 1999; 172: 330-339Crossref PubMed Scopus (35) Google Scholar, 5Harpel M.R. Lipscomb J.D. J. Biol. Chem. 1990; 265: 6301-6311Abstract Full Text PDF PubMed Google Scholar, 6Harpel M.R. Lipscomb J.D. J. Biol. Chem. 1990; 265: 22187-22196Abstract Full Text PDF PubMed Google Scholar, 7Lendenmann U. Spain J.C. J. Bacteriol. 1996; 178: 6227-6232Crossref PubMed Google Scholar, 8Stolz A. Nörtemann B. Knackmuss H.-J. Biochem. J. 1992; 282: 675-680Crossref PubMed Scopus (30) Google Scholar, 9Takenaka S. Murakami S. Shinke R. Hatakeyama K. Yukawa H. Aoki K. J. Biol. Chem. 1997; 272: 14727-14732Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). There are also a few examples that describe the ring fission of monohydroxylated aromatic compounds that do not possess a second electron-donating substituent. This has been described for the oxidation of 5-chlorosalicylate by a "Bacillus" sp. and for the conversion of 1-hydroxy-2-naphthoate by several Gram-negative (e.g. Aeromonas sp.) and Gram-positive bacteria (Nocardioides sp.) From these two reactions only the oxidation of 1-hydroxy-2-naphthoate by Nocardioides sp. KP7 has been analyzed on an enzymatic and genetic level, and the ring fission product was isolated and characterized by various spectroscopic techniques (10Adachi K. Iwabuchi T. Sano H. Harayama S. J. Bacteriol. 1999; 181: 757-763Crossref PubMed Google Scholar, 11Crawford R.L. Olson P.E. Frick T.D. Appl. Environ. Microbiol. 1979; 38: 379-384Crossref PubMed Google Scholar, 12Iwabuchi T. Harayama S. J. Biol. Chem. 1998; 273: 8332-8336Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 13Kiyohara H. Nakao K. Agric. Biol. Chem. 1977; 41: 705-707Google Scholar, 14Kiyohara H. Nakao K. J. Gen. Microbiol. 1978; 105: 69-75Crossref Scopus (127) Google Scholar).We recently described a new ring fission dioxygenase from the naphthalenesulfonate-degrading strain Pseudaminobacter salicylatoxidans, which oxidized salicylate by a novel ring fission mechanism to 2-oxohepta-3,5-dienedioic acid (Scheme 1). The ring fission dioxygenase resembled gentisate 1,2-dioxygenases or 1-hydroxy-2-naphthoate dioxygenases, because of the ability of the enzyme to convert gentisate and 1-hydroxy-2-naphthoate, the size of the subunits, the structure of the holoenzyme, and the dependence of the enzyme on Fe2+ ions (15Hintner J.-P. Lechner C. Riegert U. Kuhm A.E. Storm T. Reemtsma T. Stolz A. J. Bacteriol. 2001; 183: 6936-6942Crossref PubMed Scopus (59) Google Scholar). In order to allow a more detailed analysis of the ability of ring fission dioxygenases to oxidatively cleave the aromatic ring of monohydroxylated benzene derivatives in the current study, the encoding gene was cloned and the substrate specificity of the enzyme analyzed in greater detail.MATERIALS AND METHODSBacterial Strains and Media—The isolation and characterization of P. salicylatoxidans strain BN12 DSM 6986T has been described previously (16Kämpfer P. Müller C. Mau M. Neef A. Auling G. Busse H.-J. Osborn A.M. Stolz A. Int. J. Syst. Bacteriol. 1999; 49: 887-897Crossref PubMed Scopus (60) Google Scholar, 17.Nörtemann, B. (1987) Bakterieller Abbau von Amino-und Hydroxynaphthalinsulfonsäuren, Ph.D. Thesis, University of Stuttgart, GermanyGoogle Scholar). Corynebacterium glutamicum ATCC 13032 was obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany. Escherichia coli JM109 and E. coli BL21(DE3) were used as host strains for recombinant DNA work.Mineral media were prepared as described by Dorn et al. (18Dorn E. Hellwig M. Reineke W. Knackmuss H.-J. Arch. Microbiol. 1974; 99: 61-70Crossref PubMed Scopus (372) Google Scholar) and were supplemented with 100 mg/liter of yeast extract. P. salicylatoxidans was routinely grown in this supplemented mineral medium with 5 mm 6-aminonaphthalene-2-sulfonate. For the cultivation of C. glutamicum, a complex medium proposed by the Deutsche Sammlung von Mikroorganismen und Zellkulturen was used. The recombinant E. coli strains were routinely cultured in Luria-Bertani medium supplemented with ampicillin (100 μg/ml).Heat-labile and autoxidable substrates were sterilized by membrane filtration (pore size, 0.2 μm; Sartorius, Göttingen, Germany); all other substrates were autoclaved at 121 °C.Oxygen Uptake Experiments—P. salicylatoxidans BN12 was grown in a mineral medium with 6-aminonaphthalene-2-sulfonate (5 mm) supplemented with 100 mg/liter yeast extract. The cells were harvested during the late exponential growth phase by centrifugation and resuspended in Tris-HCl buffer (20 mm, pH 8) to an optical density (A546 nm) of 14. The resting cells were incubated at 30 °C in an oxygen electrode (YSI 5350, YSI Inc., Yellow Springs, OH). The endogenous respiration was determined for 2 min, and then the respective salicylates were added (1 mm each). The oxygen uptake was recorded for 5 min, and the reaction rates were corrected for the endogenous respiration.Preparation of Cell Extracts—Cell suspensions in 20 mm Tris-HCl buffer (pH 8.0) were disrupted by using a French press (Aminco, Silver Springs, MD) at 80 MPa. Cell debris was removed by centrifugation at 100,000 × g for 30 min at 4 °C. Protein content of the cell extracts was determined by the method of Bradford (19Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (213377) Google Scholar) with bovine serum albumin as a standard.Protein Purification, Enzymatic Cleavage of the Protein, Isolation of Peptides, and Sequencing of Peptides and Amino Termini—The dioxygenase was purified from P. salicylatoxidans BN12 by fast protein liquid chromatography as described previously (15Hintner J.-P. Lechner C. Riegert U. Kuhm A.E. Storm T. Reemtsma T. Stolz A. J. Bacteriol. 2001; 183: 6936-6942Crossref PubMed Scopus (59) Google Scholar). The digestion of the purified dioxygenase by trypsin and the subsequent separation of tryptic digests by reversed-phase HPLC 1The abbreviations used are: HPLC, high pressure liquid chromatography; MS, mass spectrometry; LC-MS, liquid chromatographymass spectrometry. were performed by established procedures (20Stone K.L. LoPresti M.B. Crawford J.M. DeAngelis R. Williams K.R. Matsudaira P.T. A Practical Guide to Protein and Peptide Purification for Micro sequencing. Academic Press, Inc., San Diego1989: 31-47Google Scholar). The amino acid sequences were determined by automated Edman degradation by using an ABI 476 protein sequencer (Applied Biosystems, Foster City, CA).PAGE—SDS-PAGE was performed by the method of Laemmli (21Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (206024) Google Scholar), and the gels were routinely stained with Coomassie Blue.Enzyme Assays with Purified Enzyme Preparations—One unit of enzyme activity was defined as the amount of enzyme that converts 1 μmol of substrate/min. The conversion of 1-hydroxy-2-naphthoate, gentisate, 5-aminosalicylate, and salicylate were determined spectrophotometrically as described previously (15Hintner J.-P. Lechner C. Riegert U. Kuhm A.E. Storm T. Reemtsma T. Stolz A. J. Bacteriol. 2001; 183: 6936-6942Crossref PubMed Scopus (59) Google Scholar).The conversion of all other salicylates and benzoic acids was analyzed by HPLC. The reaction mixtures contained, in a total volume of 1 ml, 20 mm Tris-HCl (pH 8.0) and the respective substrates (0.1 mm each). The reactions were started by the addition of the purified enzyme (0.04–0.1 mg of protein). After 10–30 s, 50 μl of the reaction mixtures were removed and the reactions terminated by the addition of 5 μl of 1 m HCl or by boiling for 10 min (for the experiments with 3,5-dichloro-, 3,5-dibromo-, and 3,5-diiodosalicylate). The precipitated proteins were removed by centrifugation in an Eppendorf centrifuge (2 min at 14,000 rpm). The concentrations of the substrates were subsequently analyzed by HPLC.For the determination of the kinetic Vmax and Km values, the kinetic parameters were determined basically as described above but using a substrate concentration range from 0.01 to 5 mm.Analytical Methods—The turnover of the substituted salicylates was analyzed by reversed-phase HPLC (HPLC pumps model 510 equipped with a photo-diode array detector model 996 and Millenium Chromatography Manager 2.0, Waters, Milford, MA.). A reversed-phase column (125 × 4.0 mm, internal diameter), packed with 5-μm particles of Merck Lichrospher RP8 (end capped) was used. The separated compounds were detected photometrically at 210 nm and at the wavelength indicated in Table I by using a photodiode array detector.Table ISolvent systems, retention times, and detection wavelength used for HPLC analysisSolvent system (% methanol)Substrate(Main) productRtAbsorption maximumRtAbsorption maximum%minnmminnm3-Aminosalicylic acid103.0300NDaND, not detected.4-Aminosalicylic acid107.63001.72745-Aminosalicylic acid404.8317ND3-Hydroxysalicylic acid404.83172.33324-Hydroxysalicylic acid404.72951.62466-Hydroxysalicylic acid403.6308NRbNR, no reaction observed.3-Methylsalicylic acid606.33091.72104-Methylsalicylic acid605.13021.72105-Methylsalicylic acid605.13151.72055-Fluorosalicylic acid604.43131.62933-Chlorosalicylic acid605.33091.82174-Chlorosalicylic acid607.73021.72175-Chlorosalicylic acid606.93151.83003-Bromosalicylic acid606.13091.82234-Bromosalicylic acid608.93021.72225-Bromosalicylic acid608.03151.83005-Iodosalicylic acid609.4320NR5-Nitrosalicylic acid603.6308NR5-Sulfosalicylic acid104.1302NR3,5-Dichlorosalicylic acid6010.83191.62103,5-Dibromosalicylic acid6014.33191.72003,5-Diiodosalicylic acid6021.5328NRSalicylic acid1522.13003.3283a ND, not detected.b NR, no reaction observed. Open table in a new tab Liquid Chromatography-Mass Spectrometry—Product identification was performed by liquid chromatography-mass spectrometry (HP1100, Agilent) coupled to a triple quadrupole mass spectrometer (Quattro LC, Micromass, Manchester, UK) using electrospray ionization in the negative ion mode. Substrate solutions before and after addition of the enzyme and 20 min after addition of the enzyme were injected (20 μl) into the HPLC system without any pretreatment. Analytes were separated by ion-pair chromatography on a Luna C18 (2Bugg T.D.H. Lin G. Chem. Comm. 2001; : 941-952Crossref Scopus (154) Google Scholar) 3-μm column, 15 cm × 3 mm inner diameter at 40 °C. Eluent A was H2O/MeOH (80:20 v/v), and eluent B was H2O/MeOH (5:95 v/v) with 1 mm tributylamine and 1 mm acetic acid each. Gradient elution started with 20% (v/v) eluent B at 0 min, 90% eluent B at 11 min, isocratic to 15 min, and 16 min 20% (v/v) eluent B, 21 min, 20% (v/v) eluent B. A diode array detector and the MS were coupled in a series. The mass spectrometric interface was operated at a cone voltage of 18 V and a capillary voltage of 2.9 kV. Probe temperature was 220 °C, and source block temperature was 120 °C. Product ion spectra were recorded at collision energies of 10 and 15 eV with a scan rate of 0.5 s.PCR—Oligonucleotides were custom-synthesized according to the known or deduced sequences of the amino-terminal amino acid sequence and various internal peptides of the ring fission dioxygenase. PCR mixtures (25–50 μl) for the amplification of genomic DNA contained 2 mm of each primer, 10–100 ng of genomic template DNA, 0.1 mm of each deoxynucleotide triphosphates, 1.5 mm MgCl2, 0.5 units of Taq (Eppendorf, Hamburg, Germany) or Pwo (Peqlab, Erlangen, Germany) DNA polymerase and the corresponding reaction buffers.For the amplification reaction with the primers deduced from the amino terminus and the peptide P28, the following PCR program was used: an initial denaturation (94 °C, 3 min) was followed by 35 cycles consisting of an annealing temperature of 38.7 °C (30 s), a polymerization step (72 °C, 2 min), and denaturation (94 °C, 30 s). The last polymerization step was extended to 15 min.For the determination of the complete sequence of the gene encoding the ring fission dioxygenase, a partial inverse PCR was performed (22Pang K.M. Knecht D.A. BioTechniques. 1997; 22: 1046-1048Crossref PubMed Scopus (39) Google Scholar). The template was prepared by digesting the chromosomal DNA of strain BN12 with PsuI (for the 3′-sequence) or NcoI (for the 5′-sequence), each of which possessed one restriction site within the 0.7-kb sequence obtained from the initial PCR experiment. The fragments obtained were religated using T4 DNA ligase. Thus intramolecular ligation of these DNA fragments resulted in circular DNA molecules, which were then used as a template for the following PCR. Primers for the PCR experiments were deduced from the sequence of the incomplete gene present on the 0.7-kb PCR product previously obtained and were facing in both directions outward from the known DNA sequence. The following nucleotide primers were used for the amplification of the DNA sequence 3′ of the known part of the dioxygenase gene: Paul-fwd, 5′-ATTGGGGCCTATCGCTGG-3′, and Paul-rev, 5′-CTGATCGGTGTCGTTGTGG-3′. For the determination of the nucleotide sequence encoding the amino terminus of the protein, the primers NcoI-fwd 5′-CGCATGTCTCGTGGC-3′and NcoI-rev 5′-CTTCGGCTGCATTGC-3′ were used. For the amplification reactions the following PCR programs were used: an initial denaturation (94 °C, 3 min) was followed by 35 cycles consisting of an annealing temperature of 55 (for the 5′-sequence) or 60 °C (for the 3′-sequence) (30 s each), a polymerization step (72 °C, 2.5 min), and denaturation (94 °C, 30 s). The last polymerization step was extended to 15 min. The PCR products were initially cloned into the T-tailed EcoRV-site of pBluescript II KS(+) (23Marchuk D. Drumm M. Saulino A. Collins F.S. Nucleic Acids Res. 1991; 19: 1154Crossref PubMed Scopus (1129) Google Scholar).Expression of the Dioxygenase in E. coli—For expression in E. coli, the dioxygenase gene was inserted in the plasmid vector pET28a (Novagen, Madison, WI) under the control of the T7 promoter. The DNA segment encompassing the dioxygenase gene was amplified by PCR (using a Pwo DNA-polymerase; Peqlab). The upstream primer (5′-GGAGGTCCATATGCAGAACG-3′) incorporated an NdeI site (underlined), and the downstream primer (5′-CGGGATCCTCACTTCTGCCCCTCG-3′) incorporated a BamHI site (underlined). The amplified product was cloned into the EcoRV site of pBluescript II KS(+). The resulting plasmid was cleaved with NdeI and BamHI and the DNA fragment with the dioxygenase gene ligated into pET28a, which was also previously cut with NdeI and BamHI. The resulting plasmid (pJPH100exN) was subsequently transformed into E. coli BL21(DE3). The expression of the dioxygenase gene was induced by isopropyl-1-thio-β-d-galactopyranoside as suggested by the supplier of the pET system.Purification of the His-tagged Enzyme—Cell extracts of E. coli JM109(pJPH100exN) were prepared in Tris-HCl buffer (20 mm, pH 8) as described above. The nickel-nitrilotriacetic acid-agarose (Qiagen, Hilden, Germany) was suspended in Tris-HCl (20 mm, pH 8) and transferred (2 ml) to an empty 10-ml polypropylene column. The filled column was equilibrated with a buffer system (pH 8) consisting of Tris-HCl (20 mm), NaCl (300 mm), and imidazole (50 mm). The cell extracts (about 100 mg of protein) were applied to the column, and the protein was eluted with subsequent steps of the Tris-HCl/NaCl buffer (2–4 ml each) with increasing imidazole concentrations (50, 60, 150, and 500 mm). The fractions with dioxygenase activity eluted at an imidazole concentration of 150 mm. The imidazole was removed from the active fractions on a "HiTrap desalting column" (Amersham Biosciences) using Tris-HCl (20 mm, pH 8) plus 100 mm NaCl as eluent buffer.Cloning of the Presumed Gentisate 1,2-Dioxygenase from C. glutamicum—The gene was amplified from the genomic DNA of C. glutamicum by using the oligonucleotide primers GDO-Cglu-fwd 5′-CACATATGGGCGCCCCAGG-3′ and GDO-Cglu-rev 5′-CAGGATCCCTAGATTCCTTCCGGAG-3′, which simultaneously introduced NdeI and BamHI cleavage sites (underlined). The following PCR program was used: an initial denaturation (94 °C, 3 min) was followed by 30 cycles consisting of annealing at 54.5 °C (for 30 s), polymerization at 72 °C (for 90 s), and denaturation at 94 °C (for 30 s). The last polymerization step was extended to 5 min. The PCR product was initially cloned into the T-tailed EcoRV site of pBluescript II KS(+). Finally, the gene was isolated from positive clones by using the restriction enzymes NdeI and BamHI and cloned into the expression vector pJOE2702 under the control of an l-rhamnose-inducible promoter (24Volff J.N. Eichenseer C. Viell P. Piendl W. Altenbuchner J. Mol. Microbiol. 1996; 21: 1037-1047Crossref PubMed Scopus (69) Google Scholar, 25Stumpp T. Wilms B. Altenbuchner J. Biospektrum. 2000; 6: 33-36Google Scholar).DNA Sequencing and Nucleotide Sequence Analysis—The DNA sequence was determined by dideoxy chain termination with double-stranded DNA of clones and overlapping subclones in an automated DNA sequencing system (ALFexpress-Sequencer, Amersham Biosciences) with fluorescently labeled primers or nucleotides.Sequence analysis, data base searches, and comparisons were done using the NCBI facilities. The alignment of the ring fission dioxygenases was obtained with the program Clustal using the default parameters.Chemicals—The chemicals used were obtained from Aldrich, Fluka (Neu-Ulm, Germany), Merck, Serva (Heidelberg, Germany), and Sigma. 6-Aminonaphthalene-2-sulfonate was kindly provided by Bayer AG (Leverkusen, Germany). The complex media were purchased from Difco and Oxoid (Wesel). The suppliers of the substituted salicylates have been described previously (26Rubio M.A. Engesser K.-H. Knackmuss H.-J. Arch. Microbiol. 1986; 145: 116-122Crossref PubMed Scopus (21) Google Scholar). The reagents for molecular biology were supplied by Invitrogen, Peqlab (Erlangen, Germany), MBI Fermentas (St. Leon-Rot, Germany), New England Biolabs (Schwalbach, Germany), and Roche Applied Science.RESULTSOxidation of Different Salicylates by Resting Cells of P. salicylatoxidans BN12—It was demonstrated previously that the enzyme from P. salicylatoxidans BN12 oxidized salicylate, gentisate, 5-aminosalicylate, and 1-hydroxy-2-naphthoate by an 1,2-dioxygenolytic cleavage (15Hintner J.-P. Lechner C. Riegert U. Kuhm A.E. Storm T. Reemtsma T. Stolz A. J. Bacteriol. 2001; 183: 6936-6942Crossref PubMed Scopus (59) Google Scholar). This ring fission mechanism principally allows the cleavage of various substituted salicylates. Therefore, resting cells of P. salicylatoxidans BN12 were incubated with a wide range of substituted salicylates, and the substrate-dependent oxygen uptake rates were determined. These experiments suggested that the bacteria were also able to oxidize a wide range of methyl-, chloro-, or bromo-substituted salicylates. Most surprisingly, the cells oxidized 5-fluorosalicylate with its exceptionally electronegative fluorine substituent with higher rates than salicylate (Fig. 1).Fig. 1Relative oxygen uptake of resting cells from P. salicylatoxidans BN12 after addition of different salicylates. The reaction rates are expressed as percentages of that taken for 1-hydroxy-2-naphthoate (0.15 units/mg of protein = 100%).View Large Image Figure ViewerDownload (PPT)Cloning of the Gene Encoding the Salicylate 1,2-Dioxygenase Activity—The ring fission dioxygenase was purified from cell extracts of P. salicylatoxidans BN12 as described previously (15Hintner J.-P. Lechner C. Riegert U. Kuhm A.E. Storm T. Reemtsma T. Stolz A. J. Bacteriol. 2001; 183: 6936-6942Crossref PubMed Scopus (59) Google Scholar). The purified dioxygenase was digested with trypsin, and several peptides were isolated by HPLC. The amino acid sequences of the amino terminus and five internal fragments were determined and used for the design of oligonucleotide primers for PCR experiments (Table II). Using genomic DNA of P. salicylatoxidans BN12 as template and primers derived from the amino-terminal sequence and of the peptide P28 (Table II), a DNA fragment with a size of about 0.7 kb was amplified. The amplified fragment was sequenced, and the sequence obtained was used to complete the sequence of the gene by using partial inverse PCR (see "Material and Methods"). Thus a continuous stretch of DNA of 1249 bp was obtained. The gene for the salicylate dioxygenase was unequivocally identified in this sequence by the presence of the amino-terminal region and the internal peptides determined before by Edman degradation (Fig. 2).Table IISequences of the amino terminus, tryptic peptides, and deduced oligonucleotidesProtein or peptideAmino acid sequenceDeduced oligonucleotide sequencesAmino terminusMQNEKLDHESVTQAMQPKDTPELRALYKS5′-CA(AG)GCNATGCA(AG)CC-3′P28WEFTDR5′-(AG)TCNGT(AG)AA(TC)TCCCA-3′P32WSTLLRP36ALTEQLLLEDEGQPATVAPGHAAIRP52ALGLANPGLGGNAYISPTM Open table in a new tab Fig. 2DNA sequence and amino acid sequence of the salicylate dioxygenase from P. salicylatoxidans BN12. The amino-terminal and the internal amino acid sequences, which were determined by chemical Edman degradation, are underlined. The stop codon is indicated by a dash.View Large Image Figure ViewerDownload (PPT)Sequence Analysis—The size of the gene encoding the ring fission dioxygenase was 1107 bp, and it demonstrated a GC content of 62.6% which is in good agreement with the total GC content of the organism (63.9 mol % (16Kämpfer P. Müller C. Mau M. Neef A. Auling G. Busse H.-J. Osborn A.M. Stolz A. Int. J. Syst. Bacteriol. 1999; 49: 887-897Crossref PubMed Scopus (60) Google Scholar)). The gene encoded a protein consisting of 368 amino acids which corresponded to a molecular mass of 41,176 Da. This value agreed with the molecular mass of the dioxygenase subunits (45 kDa) determined earlier by SDS-gel electrophoresis after purification of the enzyme from P. salicylatoxidans BN12 (15Hintner J.-P. Lechner C. Riegert U. Kuhm A.E. Storm T. Reemtsma T. Stolz A. J. Bacteriol. 2001; 183: 6936-6942Crossref PubMed Scopus (59) Google Scholar). The deduced complete amino acid sequence showed significant sequence similarities with different gentisate 1,2-dioxygenases and 1-hydroxy-2-naphthoate 1,2-dioxygenases from different bacteria (Fig. 3). The highest degree of sequence identity (31%) to proteins with a known function was observed with a gentisate 1,2-dioxygenase from Pseudomonas alcaligenes NCIMB 9867 (27Feng Y. Khoo H.E. Poh C.L. Appl. Environ. Microbiol. 1999; 65: 946-950Crossref PubMed Google Scholar). Yet a much higher degree of sequence identity (58%) was found to a presumed gentisate 1,2-dioxygenase that had been identified in C. glutamicum in the course of a genome project.Fig. 3Conversion of various substituted salicylates by cell extracts from E. coli JM109 (pJPH100exN). The reaction mixtures contained in 1 ml of 20 mm Tris-HCl (pH 8.0), 0.1 mm of the respective salicylate and 1–140 μg of protein. The spectra were recorded every minute against a reference cuvette, which contained the same amount of protein in Tris-HCl buffer but in which the respective substrates were omitted.View Large Image Figure ViewerDownload (PPT)Expression of the Dioxygenase Gene in E. coli—The dioxygenase gene was amplified by PCR from the genomic DNA of strain BN12 by using a set of primers that created new NdeI and BamHI restriction sites. The amplified fragment was then ligated into plasmid pBluescript II SK+ (previously cut with EcoRV) and finally into the expression vector pET28a, which resulted in the addition of an amino-terminal His tag in the synthesized proteins. The resulting expression plasmid (pJPH100exN) was used to transform E. coli BL21(DE3), and the dioxygenase gene was induced by the addition of isopropyl-1-thio-β-d-galactopyranoside. A comparison of SDS-PAGE gels from induced and uninduced cells of E. coli BL21(DE3)(pJPH100exN) demonstrated the induction of an additional protein with a molecular mass of about 45 kDa. The dioxygenase activity of these cell extracts with salicylate as substrate was 0.43 units mg–1 of protein. This was about 25-fold higher compared with the activity found in the wild-type strain of P. salicylatoxidans BN12. The His-tagged dioxygenase was purified by affinity chromatography using nickel-nitrilotriacetic acid-agarose (see "Materials and Methods"). This resulted in an enzyme preparation that was more than 95% homogenous, according to SDS-PAGE, and that converted salicylate as substrate with a specific activity of about 2 units/mg protein.Spectrophotometric Analysis of the Substrate Conversion by the Purified Dioxygenase—The oxygen uptake experiments with resting cells of P. salicylatoxidans (see above) suggested a broad substrate specificity of the salicylate 1,2-dioxygenase activity. It was demonstrated previously that the conversion of salicylate, 5-amino-, and 5-hydroxysalicylate and also 1-hydroxy-2-naphthoate could be analyzed spectrophotometrically because of the pronounced UV-visible spectra of the products formed (15Hintner J.-P. Lechner C. Riegert U. Kuhm A.E. Storm T. Reemtsma T. Stolz A. J. Bacteriol. 2001; 183: 6936-6942Crossref PubMed Scopus (59) Google Scholar). Therefore, the reactions of other salicylates were also analyzed by UV-visible spectroscopy. Thus it was found that cell extracts of the recombinant E. coli strain converted 3-, 4-, and 5-substituted amino-, hydroxy-, chloro-, bromo-, and methylsalicylates (Fig. 3). The conversion of all isomers of the chloro- and bromosalicylates (and also of 5-fluorosalicylate which was the only available fluorosalicylate) resulted in the formation of products with absorption maxima in the range of λmax = 290–302 nm. The turnover of the methylsalicylates also resulted in the initial formation of new absorbance maxima in the range from 293 to 300 nm. The changes in the spectra observed during the oxidatio
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