Phenol Hydroxylase from Bacillus thermoglucosidasius A7, a Two-protein Component Monooxygenase with a Dual Role for FAD
2003; Elsevier BV; Volume: 278; Issue: 48 Linguagem: Inglês
10.1074/jbc.m307397200
ISSN1083-351X
AutoresU. Kirchner, Adrie H. Westphal, Rudolf Müller, Willem J. H. van Berkel,
Tópico(s)Microbial bioremediation and biosurfactants
ResumoA novel phenol hydroxylase (PheA) that catalyzes the first step in the degradation of phenol in Bacillus thermoglucosidasius A7 is described. The two-protein system, encoded by the pheA1 and pheA2 genes, consists of an oxygenase (PheA1) and a flavin reductase (PheA2) and is optimally active at 55 °C. PheA1 and PheA2 were separately expressed in recombinant Escherichia coli BL21(DE3) pLysS cells and purified to apparent homogeneity. The pheA1 gene codes for a protein of 504 amino acids with a predicted mass of 57.2 kDa. PheA1 exists as a homodimer in solution and has no enzyme activity on its own. PheA1 catalyzes the efficient ortho-hydroxylation of phenol to catechol when supplemented with PheA2 and FAD/NADH. The hydroxylase activity is strictly FAD-dependent, and neither FMN nor riboflavin can replace FAD in this reaction. The pheA2 gene codes for a protein of 161 amino acids with a predicted mass of 17.7 kDa. PheA2 is also a homodimer, with each subunit containing a highly fluorescent FAD prosthetic group. PheA2 catalyzes the NADH-dependent reduction of free flavins according to a Ping Pong Bi Bi mechanism. PheA2 is structurally related to ferric reductase, an NAD(P)H-dependent reductase from the hyperthermophilic Archaea Archaeoglobus fulgidus that catalyzes the flavin-mediated reduction of iron complexes. However, PheA2 displays no ferric reductase activity and is the first member of a newly recognized family of short-chain flavin reductases that use FAD both as a substrate and as a prosthetic group. A novel phenol hydroxylase (PheA) that catalyzes the first step in the degradation of phenol in Bacillus thermoglucosidasius A7 is described. The two-protein system, encoded by the pheA1 and pheA2 genes, consists of an oxygenase (PheA1) and a flavin reductase (PheA2) and is optimally active at 55 °C. PheA1 and PheA2 were separately expressed in recombinant Escherichia coli BL21(DE3) pLysS cells and purified to apparent homogeneity. The pheA1 gene codes for a protein of 504 amino acids with a predicted mass of 57.2 kDa. PheA1 exists as a homodimer in solution and has no enzyme activity on its own. PheA1 catalyzes the efficient ortho-hydroxylation of phenol to catechol when supplemented with PheA2 and FAD/NADH. The hydroxylase activity is strictly FAD-dependent, and neither FMN nor riboflavin can replace FAD in this reaction. The pheA2 gene codes for a protein of 161 amino acids with a predicted mass of 17.7 kDa. PheA2 is also a homodimer, with each subunit containing a highly fluorescent FAD prosthetic group. PheA2 catalyzes the NADH-dependent reduction of free flavins according to a Ping Pong Bi Bi mechanism. PheA2 is structurally related to ferric reductase, an NAD(P)H-dependent reductase from the hyperthermophilic Archaea Archaeoglobus fulgidus that catalyzes the flavin-mediated reduction of iron complexes. However, PheA2 displays no ferric reductase activity and is the first member of a newly recognized family of short-chain flavin reductases that use FAD both as a substrate and as a prosthetic group. Phenolic compounds constitute one of the largest groups of natural products. They are predominantly found in plants, where they occur in a great variety of structures and functions. During the last century, the natural pool of phenolic compounds has been increased with products of industrial origin. Many of these synthetic compounds cause environmental pollution and human health problems as a result of their persistence, toxicity, and transformation into hazardous intermediates (1Timmis K.N. Pieper D.H. Trends Biotechnol. 1999; 17: 200-204Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar, 2Gupta S.S. Stadler M. Noser C.A. Ghosh A. Steinhoff B. Lenoir D. Horwitz C.P. Schramm K.W. Collins T.J. Science. 2002; 296: 326-328Crossref PubMed Scopus (357) Google Scholar). The aerobic mineralization of natural and xenobiotic phenolic compounds by mesophilic microorganisms has been intensively investigated, and numerous pathways are known (3Moiseeva O.V. Solyanikova I.P. Kaschabek S.R. Gröning J. Thiel M. Golovleva L.A. Schlömann M. J. Bacteriol. 2002; 184: 5282-5292Crossref PubMed Scopus (80) Google Scholar). Almost invariably, phenols are first converted into more reactive dihydroxylated intermediates and then subjected to intraor extradiol ring cleavage by molecular oxygen. The initial hydroxylation of the phenolic ring usually is catalyzed by single-component NAD(P)H-dependent flavoprotein monooxygenases (4Moonen M.J.H. Fraaije M.W. Rietjens I.M.C.M. Laane C. van Berkel W.J.H. Adv. Synth. Catal. 2002; 344: 1023-1035Crossref Scopus (53) Google Scholar). These enzymes share a typical dinucleotide-binding fold for complexation of the FAD cofactor while lacking a common NAD(P)-binding fold (5Eppink M.H.M. Overkamp K.M. Schreuder H.A. van Berkel W.J.H. J. Mol. Biol. 1999; 292: 87-96Crossref PubMed Scopus (52) Google Scholar, 6Wang J. Ortiz-Maldonado M. Entsch B. Massey V. Ballou D.P. Gatti D.L. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 608-613Crossref PubMed Scopus (94) Google Scholar). Because the regioselective hydroxylation of phenols is notoriously difficult to achieve by chemical methods, the mechanistic and structural features of single-component flavoprotein monooxygenases have received much attention (7Massey V. J. Biol. Chem. 1994; 269: 22459-22462Abstract Full Text PDF PubMed Google Scholar, 8Entsch B. van Berkel W.J.H. FASEB J. 1995; 9: 476-483Crossref PubMed Scopus (184) Google Scholar, 9Palfey B.A. Massey V. Sinnott M. 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Biochemistry. 1999; 38: 1153-1158Crossref PubMed Scopus (87) Google Scholar) and phenol hydroxylase from yeast (15Enroth C. Neujahr H. Schneider G. Lindqvist Y. Structure. 1998; 6: 605-617Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar), it was shown that the oxygenation reaction takes place in the inner part of the protein and that this aprotic environment is crucial for preventing uncoupling of flavin reduction from substrate hydroxylation. With 4-hydroxyphenylacetate 3-hydroxylase from Pseudomonas putida, the binding of a second protein component prevents the uncoupling of substrate hydroxylation (16Arunachalam U. Massey V. Vaidyanathan C.S. J. Biol. Chem. 1992; 267: 25848-25855Abstract Full Text PDF PubMed Google Scholar, 17Arunachalam U. Massey V. Miller S.M. J. Biol. Chem. 1994; 269: 150-155Abstract Full Text PDF PubMed Google Scholar, 18Arunachalam U. Massey V. J. Biol. Chem. 1994; 269: 11795-117801Abstract Full Text PDF PubMed Google Scholar). This enzyme is an unusual example of a two-component flavoprotein hydroxylase in which flavin reduction and substrate oxygenation take place in the same protein. Relatively little is known about the mineralization of phenolic compounds by thermophilic microorganisms (19Kim I.C. Oriel P.J. Appl. Environ. Microbiol. 1995; 61: 1252-1256Crossref PubMed Google Scholar). Several Bacillus species isolated from geographically distinct thermal sources degrade phenol at 65 °C via the meta-cleavage pathway (Fig. 1) (20Mutzel A. Reinscheid U.M. Antranikian G. Müller R. Appl. Microbiol. Biotechnol. 1996; 46: 593-596Crossref Scopus (52) Google Scholar, 21Reinscheid U.M. Bauer M.P. Müller R. Biodegradation. 1996; 7: 455-461Crossref Google Scholar, 22Duffner F.M. Reinscheid U.M. Bauer M.P. Mutzel A. Müller R. Syst. Appl. Microbiol. 1997; 20: 602-611Crossref Scopus (7) Google Scholar). The initial conversion of phenol to catechol in these microorganisms requires two protein components that are encoded by the pheA1 and pheA2 genes (23Duffner F.M. Müller R. FEMS Microbiol. Lett. 1998; 161: 37-45Crossref PubMed Google Scholar). Characterization of the Bacillus phenol hydroxylase system appeared to be severely hampered by the low yield and instability of the purified enzymes. This prompted us to clone the Bacillus pheA genes in an Escherichia coli expression system (24Duffner F.M. Kirchner U. Bauer M.P. Müller R. Gene (Amst.). 2000; 256: 215-221Crossref PubMed Scopus (85) Google Scholar). In this study, we describe the overexpression, purification, and characterization of the recombinant phenol hydroxylase of Bacillus thermoglucosidasius A7. We show that this two-protein enzyme belongs to a newly recognized family of flavin-dependent monooxygenases that carry out the reductive and oxidative half-reactions on separate polypeptide chains. Members of this family are involved in various biological processes, including the biosynthesis of antibiotics (25Kendrew S.G. Harding S.E. Hopwood D.A. Marsh E.N. J. Biol. Chem. 1995; 270: 17339-17343Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 26Thibaut D. Ratet N. Bisch D. Faucher D. Debussche L. Blanche F. J. Bacteriol. 1995; 177: 5199-5205Crossref PubMed Google Scholar, 27Parry R.J. Li W. J. Biol. Chem. 1997; 272: 23303-23311Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar), the desulfurization of fossil fuels (28Gray K.A. Pogrebinsky O.S. Mrachko G.T. Xi L. Monticello D.J. Squires C.H. Nat. Biotechnol. 1996; 14: 1705-1709Crossref PubMed Scopus (307) Google Scholar), the degradation of chelating agents (29Uetz T. Schneider R. Snozzi M. Egli T. J. Bacteriol. 1992; 174: 1179-1188Crossref PubMed Google Scholar), and the oxidation of aromatic compounds (30Gálan B. Díaz E. Prieto M.A. García J.L. J. Bacteriol. 2000; 182: 627-636Crossref PubMed Scopus (155) Google Scholar). In contrast to most other family members, the reductase component of phenol hydroxylase from B. thermoglucosidasius A7 harbors a tightly bound FAD. Evidence is provided that the FAD cofactor is directly involved in the reduction of free flavins. Chemicals, Bacterial Strains, and Plasmids—E. coli strain BL21(DE3) pLysS and plasmid pET3a were used for expression of the pheA genes (31Studier F.W. Rosenberg A.H. Dunn J.J. Dubendorff J.W. Methods Enzymol. 1990; 185: 60-89Crossref PubMed Scopus (6006) Google Scholar). Isopropyl-β-d-thiogalactopyranoside was purchased from Invitrogen. Phenyl-Sepharose, Q-Sepharose Fast Flow, HiLoad Q-Sepharose, Superdex 200 pg, Superdex 200 HR10/30, Superdex 75 HR10/30, and PhastGel precast isoelectric focusing gels were from Amersham Biosciences. Macro-prep ceramic hydroxylapatite (type I, particle size of 20 μm) was obtained from Bio-Rad. FAD, FMN, riboflavin, and 3-acetylpyridine-adenine dinucleotide (AcPyAD+) 1The abbreviations used are: AcPyAD+3-acetylpyridine-adenine dinucleotideHPLChigh performance liquid chromatographyTricineN-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycineFPLCfast protein liquid chromatographyFeRferric reductase. were purchased from Sigma. NADH, NADPH, NAD+, and glucose oxidase (grade II) were from Roche Applied Science. Phenolic compounds were obtained from Aldrich. All other chemicals were from Merck and were of the purest grade available. 3-acetylpyridine-adenine dinucleotide high performance liquid chromatography N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine fast protein liquid chromatography ferric reductase. Enzyme Purification—All purification steps were carried out at room temperature. PheA1 and PheA2 were purified from transformed E. coli cells harboring the pheA1 and pheA2 genes (24Duffner F.M. Kirchner U. Bauer M.P. Müller R. Gene (Amst.). 2000; 256: 215-221Crossref PubMed Scopus (85) Google Scholar), respectively. Recombinant cells were grown for 16 h in medium containing 10 g/liter Tryptone, 5 g/liter yeast, and 5 g/liter NaCl (pH 7.4) at 30 °C, followed by induction with 1 mm isopropyl-β-d-thiogalactopyranoside for 3 h. Purification of PheA1 (Oxygenase Component)—Washed recombinant E. coli cells (15 g) were suspended in 15 ml of 50 mm sodium phosphate (pH 7.0) containing 0.5 mm EDTA, 0.5 mm phenylmethylsulfonyl fluoride, and 0.5 mg of DNase and passed two times through a precooled French press operating at 10,000 p.s.i. Following centrifugation for 30 min at 15,000 × g to remove cellular debris, the supernatant was made 0.5% in protamine sulfate from a 2% stock solution. The protamine sulfate aggregates were removed by centrifugation for 15 min at 15,000 × g, and the resulting supernatant was applied to a Q-Sepharose Fast Flow column (1.6 × 10 cm) equilibrated with 50 mm sodium phosphate (pH 7.0) containing 0.5 mm EDTA (starting buffer). After washing with starting buffer, the bound protein was eluted with a linear gradient of 0–0.6 m NaCl in 100 ml of starting buffer. The PheA1 protein eluting between 0.3 and 0.5 m NaCl was concentrated by ultrafiltration (Amicon YM-30 membrane) and dialyzed against 5 mm sodium phosphate (pH 7.0). The PheA1 protein was then loaded onto a hydroxylapatite column (1.6 × 10 cm) pre-equilibrated with 5 mm sodium phosphate (pH 7.0). After washing, PheA1 was eluted with a linear gradient of 5–500 mm sodium phosphate (pH 7.0) in 100 ml of water. Pooled fractions (80–200 mm) were concentrated by ultrafiltration and stored at 5 mg/ml in 50 mm sodium phosphate (pH 7.0) at –70 °C. Purification of PheA2 (Reductase Component)—The preparation of cell extract from recombinant E. coli cells, the protamine sulfate precipitation, and the ion exchange chromatography step were carried out under the same conditions as described above. HiLoad Q-Sepharose fractions containing FAD reductase activity (eluting between 0.1 and 0.5 m NaCl) were pooled and concentrated by ultrafiltration (Amicon YM-30 membrane). After the addition of pulverized ammonium sulfate to a final concentration of 1.4 m, the protein solution was loaded onto a phenyl-Sepharose column (1.6 × 10 cm) equilibrated with 50 mm potassium phosphate (pH 7.0) containing 1.4 m ammonium sulfate. After washing with starting buffer, bound protein was eluted with a linear descending gradient of ammonium sulfate (1.4 to 0.4 m) in 200 ml of starting buffer. Fractions containing reductase activity (0.4–1.0 m) were pooled, concentrated by ultrafiltration, and loaded onto a Superdex 200 preparative gel filtration column (2.6 × 60 cm) equilibrated with 50 mm potassium phosphate (pH 7.0) containing 150 mm NaCl. Pure reductase fractions were pooled, concentrated, and stored in 50 mm potassium phosphate (pH 7.0) at –70 °C. Activity Determinations—NADH:flavin reductase activity was determined at 25 or 53 °C in 25 mm potassium phosphate and 150 mm KCl (pH 7.0). To determine the specificity of the reaction, FMN, riboflavin, and NADPH were tested as substrates. NADH:cytochrome c reductase activity was determined spectrophotometrically at 25 °C by recording the NADH-dependent reduction of cytochrome c at 550 nm (ϵ550 = 21.1 mm–1 cm–1). The assay mixture contained 0.2 mm cytochrome c and 0.2 mm NADH in 50 mm potassium phosphate (pH 7.5). NADH oxidase activity was determined spectrophotometrically at 25 °C by monitoring the decrease in absorption of NADH at 340 nm (ϵ340 = 6.22 mm–1 cm–1). The assay mixture contained 0.2 mm NADH in 50 mm potassium phosphate (pH 7.0). The NADH-dependent reduction of 2,6-dichlorophenolindophenol was measured at 25 °C by following the decrease in absorption of 2,6-dichlorophenolindophenol at 600 nm (ϵ600 = 21.0 mm–1 cm–1, pH 7.0). The assay mixture contained 25 mm potassium phosphate (pH 7.0), 150 mm KCl, 200 μm NADH, and varying concentrations of 2,6-dichlorophenolindophenol. The NADH-dependent reduction of AcPyAD+ (transhydrogenase activity) was measured at 25 °C by following the increase in absorption of reduced AcPyAD+ at 363 nm (ϵ363 = 5.6 mm–1 cm–1). The assay mixture contained 25 mm potassium phosphate (pH 7.0), 150 mm KCl, 200 μm NADH, and varying concentrations of AcPyAD+. Kinetic Analysis—NADH:flavin reductase activity was determined at 25, 40, and 53 °C in 25 mm potassium phosphate and 150 mm KCl (pH 7.0). For estimation of kinetic parameters, the NADH concentration was varied at a fixed flavin concentration and vice versa. Kinetic parameters were determined from saturation curves, fitted with the Michaelis-Menten equation, using a Levenberg-Marquardt algorithm. For estimation of the type of mechanism, the NADH:flavin reductase activity was determined as a function of FAD concentration at several constant levels of NADH and as a function of NADH at several constant levels of FAD. Reciprocal initial velocities were plotted against reciprocal substrate concentrations and fitted with a straight line determined by a linear regression program. Inhibition constants for NAD+ inhibition were determined according to Fromm (32Fromm H.J. Methods Enzymol. 1979; 63: 467-486Crossref PubMed Scopus (98) Google Scholar). Other Assays—Phenol monooxygenase activity was determined by measuring phenol consumption colorimetrically (33Gurujeyalakshmi G. Oriel P.J. Appl. Environ. Microbiol. 1989; 55: 500-502Crossref PubMed Google Scholar). The reaction mixture (6 ml) contained 0.1 mm phenol, 0.5 mm NADH, 10 μm FAD, and 1.0 nm PheA2 in 50 mm potassium phosphate (pH 7.0). After equilibration at 50 °C and the addition of the desired amount of PheA1 (10–200 nm), samples of 1 ml were taken for 10 min at 1-min intervals. The reaction was stopped by the addition of 12 μl of 2% 4-aminoantipyrine followed by 40 μl of 2 n ammonium hydroxide and 40 μl of 2% potassium ferricyanide, and the final volume was adjusted to 2 ml with water. After 15 min of incubation at room temperature, the absorbance at 510 nm was read and compared with those of phenol standards. 1 unit is defined as the amount of enzyme that catalyzes the conversion of 1 μmol of substrate/min under the assay conditions. Oxygen consumption was determined polarographically at 50 °C in a closed reaction vessel fitted with a Clark-type oxygen electrode. Reaction mixtures contained 50 mm sodium phosphate (pH 7.0), 0.1 mm phenol, 5–10 μm FAD, and 0.5 nm PheA2 in the absence or presence of 100 nm PheA1. NADH was added to a final concentration of 0.1 mm to initiate the reaction. When oxygen consumption ceased, 90 units of catalase were added to determine the degree of uncoupling of hydroxylation. Thermostability—Studies on the thermostability of PheA1 and PheA2 were performed by incubating the enzymes (46 μm PheA1 or 48 μm PheA2) in closed vials at different temperatures (50, 60, 70, and 85 °C) in 25 mm phosphate (pH 7.0) for 2 h. At timed intervals, aliquots were withdrawn from the incubation mixtures and assayed for residual phenol hydroxylase (PheA1) and NADH:flavin reductase (PheA2) activities. Analytical Methods—HPLC experiments were performed with an Applied Biosystems pump equipped with a Waters 996 photodiode array detector. Reaction products were separated with a 3.9 × 100-mm Lichrospher RP18 column running in methanol/water (50:50, v/v) containing 0.7% acetic acid. The flow rate was 1 ml/min. Protein was determined by the method of Bradford (34Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217544) Google Scholar) with bovine serum albumin as a standard. SDS-PAGE was carried out with 15% slab gels (35Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar) or with 16.5% Tricine gels (36Schägger H. von Jagow G. Anal. Biochem. 1987; 166: 368-379Crossref PubMed Scopus (10505) Google Scholar). The gels were scanned and analyzed with a computing densitometer system (Scanjet ADF, Hewlett-Packard Intelligent Scanning Technology). Analytical gel filtration was performed on an Amersham Biosciences Åkta FPLC system equipped with a Superdex 200 HR10/30 column running in 50 mm potassium phosphate (pH 7.0) containing 0.15 m NaCl. Isoelectric focusing was performed with a PhastSystem (Amersham Biosciences) using PhastGel 3–9 isoelectric focusing precast gels. Gels were stained with Coomassie Blue R-250. Spectral Analysis—Absorption spectra were recorded at 25 °C using a Hewlett-Packard 8453 diode array spectrophotometer or an Aminco DW-2000 double-beam spectrophotometer. For anaerobic reduction experiments, enzyme and substrate solutions were made anaerobic by alternated flushing with deoxygenated argon and evacuating. Anaerobic reduction of PheA2 was performed by adding aliquots of oxygen-free NADH or dithionite to the enzyme solution, followed by spectral recording. Photoreduction in the presence of EDTA and 5-deazaflavin as catalyst was performed essentially as described (37de Jong E. van Berkel W.J.H. van der Zwan R.P. de Bont J.A.M. Eur. J. Biochem. 1992; 208: 651-657Crossref PubMed Scopus (112) Google Scholar). Circular dichroism spectra were measured at 25 °C on a Jasco J-715 spectropolarimeter. Fluorescence emission spectra were recorded at 25 °C on an SLM-AMINCO SPF500C spectrofluorometer. Cofactor Analysis—The flavin prosthetic group of PheA2 was isolated by boiling an enzyme sample for 3 min. After removal of the protein precipitate, the nature of the extracted flavin was identified by fluorescence analysis (38Mayhew S.G. Wassink J.H. Methods Enzymol. 1980; 66: 217-220Crossref PubMed Scopus (13) Google Scholar) and reverse-phase HPLC (39Eppink M.H.M. Boeren S.A. Vervoort J. van Berkel W.J.H. J. Bacteriol. 1997; 179: 6680-6687Crossref PubMed Google Scholar). Cysteine Determination—The estimation of sulfhydryl groups of native or unfolded PheA2 was carried out by the procedure of Ellman (40Ellman G.L. Arch. Biochem. Biophys. 1959; 82: 70-77Crossref PubMed Scopus (21624) Google Scholar) employing the modifications of Habeeb (41Habeeb A.F.S.A. Methods Enzymol. 1972; 25: 457-464Crossref PubMed Scopus (878) Google Scholar). All thiol determinations were carried out on enzyme preparations that were freshly incubated with 1 mm dithiothreitol, 50 μm FAD, and 0.5 mm EDTA for 15 min and subsequently passed through a Bio-Gel P-6DG column equilibrated with 50 mm potassium phosphate (pH 7.0) containing 0.5 mm EDTA. The enzyme was diluted in 100 mm Tris sulfate (pH 8.0) immediately before assaying the time-dependent release of 5-thio-2-nitrobenzoate at 412 nm (final pH 7.9). Sequence Analysis—N-terminal protein sequence analysis was performed by Edman degradation with an Applied Biosystems Model 473A pulsed-liquid sequencer fitted with an on-line phenylthiohydantoin analytical system following the procedures suggested by the manufacturer. Protein sequence similarity searches were performed with PSI-BLAST (42Altschul S.F. Madden T.L. Schäffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (60233) Google Scholar) using the facilities offered by the National Center for Biotechnology Information (NCBI). Multiple sequence alignments were made with ClustalX (43Thompson J.D. Gibson T.J. Plewniak F. Jeanmougin F. Higgins D.G. Nucleic Acids Res. 1997; 25: 4876-4882Crossref PubMed Scopus (35620) Google Scholar) using the PAM350 matrix. Protein Homology Modeling—Model building of PheA2 was performed with MODELLER (44Sali A. Blundell T.L. J. Mol. Biol. 1993; 234: 779-815Crossref PubMed Scopus (10636) Google Scholar) using the CVFF force field (45Dauber-Osguthorpe P. Roberts V.A. Osguthorpe D.J. Wolff J. Genest M. Hagler A.T. Proteins. 1988; 4: 31-47Crossref PubMed Scopus (1951) Google Scholar). The structure of ferric reductase (FeR) from Archaeoglobus fulgidus with NADP+ bound (Protein Data Bank code 1IOS) (46Chiu H.-J. Johnson E. Schröder I. Rees D.C. Structure. 2001; 9: 311-319Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar) was used as a template. The model was verified after several rounds of energy minimization. The stereochemical quality of the homology model was verified by PROCHECK (47Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar), and the protein folding was assessed with PROSAII (48Sippl M.J. Proteins. 1993; 17: 355-362Crossref PubMed Scopus (1792) Google Scholar), which evaluates the compatibility of each residue with its environment independently. The FMN part of FAD was placed in the model structure at the same position and orientation as the FMN in the template structure. The AMP part of FAD was placed in the groove continuing from the binding pocket of FMN. NADP+ was oriented in the same position as found in the crystal structure of FeR. Overexpression of pheA Genes—Both protein components PheA1 and PheA2 from B. thermoglucosidasius A7 are required for phenol hydroxylase activity (24Duffner F.M. Kirchner U. Bauer M.P. Müller R. Gene (Amst.). 2000; 256: 215-221Crossref PubMed Scopus (85) Google Scholar). However, E. coli cells containing a plasmid with the genes for PheA1 and PheA2 in tandem did not express PheA2, as only a clear protein band for PheA1 (but not for PheA2) could be detected by SDS-PAGE analysis of cell extracts (24Duffner F.M. Kirchner U. Bauer M.P. Müller R. Gene (Amst.). 2000; 256: 215-221Crossref PubMed Scopus (85) Google Scholar). A likely explanation is that, in this construct, PheA2 expression must use its own B. thermoglucosidasius A7 ribosome-binding site, which might not be effective in E. coli. When PheA2 was expressed separately, expression was under the control of the highly efficient phage T7 promoter, and a clear protein band of PheA2 was visible upon SDS-PAGE analysis of cell extracts (24Duffner F.M. Kirchner U. Bauer M.P. Müller R. Gene (Amst.). 2000; 256: 215-221Crossref PubMed Scopus (85) Google Scholar). Therefore, PheA1 and PheA2 were separately purified from E. coli cells containing the appropriate plasmid with the gene for PheA1 or PheA2. Purification of PheA1—Extracts of E. coli cells containing the plasmid with only the pheA1 gene exhibited phenol hydroxylase activity. Because these cells do not contain PheA2, this indicates that a flavin reductase activity present in the E. coli host substitutes for PheA2 to give substrate conversion. A similar observation was made for 4-hydroxyphenylacetate 3-monooxygenase from E. coli W (30Gálan B. Díaz E. Prieto M.A. García J.L. J. Bacteriol. 2000; 182: 627-636Crossref PubMed Scopus (155) Google Scholar). During purification of PheA1, phenol hydroxylase activity was determined with and without complementation with PheA2. The phenol hydroxylase activity determined in the absence of PheA2 decreased after every purification step (Table I). The specific activity of purified PheA1 determined in the presence of PheA2 was 0.32 units/mg. SDS-PAGE analysis of the purified PheA1 protein revealed the presence of a single polypeptide chain corresponding to a molecular mass of ∼57 kDa (Fig. 2). This value is in good agreement with the molecular mass predicted from gene sequence analysis (24Duffner F.M. Kirchner U. Bauer M.P. Müller R. Gene (Amst.). 2000; 256: 215-221Crossref PubMed Scopus (85) Google Scholar).Table IPurification of the PheA1 oxygenase component of phenol hydroxylase from B. thermoglucosidasius A7 expressed in E. coli PH2StepProteinActivityaIn the absence of 1 mm PheA2.Specific activityaIn the absence of 1 mm PheA2.ActivitybIn the presence of 1 mm PheA2.Specific activitybIn the presence of 1 mm PheA2.Yieldmgunitsunits/mgunitsunits/mg%Cell extract764430.056460.060100Q-Sepharose3022.40.008160.05335Hydroxyapatite270.110.0048.60.32018a In the absence of 1 mm PheA2.b In the presence of 1 mm PheA2. Open table in a new tab Purified PheA1 did not contain any chromophore as judged by absorption spectral analysis and eluted from an analytical Superdex 200 HR10/30 column in a single symmetrical peak with an apparent molecular mass of 120 ± 5 kDa. This suggests that the PheA1 protein is a dimer composed of identical subunits. Isoelectric focusing of PheA1 revealed a main protein band with an isoelectric point of 5.2 ± 0.1. This value is considerably lower than the theoretical value of 6.29 calculated from the amino acid sequence. Purified PheA1 was stable for 2 h at 60 °C, as no significant decrease in phenol hydroxylase activity was observed in complementation experiments with PheA2. In contrast, at 70 °C, inactivation of PheA1 was complete after 10 min of incubation. The purified PheA1 protein was not very stable when stored at –70 °C because it formed aggregates after thawing. Therefore, PheA1 was stored as a protein precipitate in 80% ammonium sulfate at 4 °C. Purification of PheA2—The PheA2 protein expressed in E. coli cells containing the plasmid with the pheA2 gene was purified to apparent homogeneity using three chromatographic steps (Table II). The specific NADH:FAD reductase activity of the purified PheA2 protein at 53 °C was 800 units/mg. Analysis by SDS-PAGE revealed the presence of a single band corresponding to a polypeptide chain molecular mass of ∼18 kDa (Fig. 3). Again, this value is in good agreement with the molecular mass predicted from gene sequence analysis (17,660 Da) (24Duffner F.M. Kirchner U. Bauer M.P. Müller R. Gene (Amst.). 2000; 256: 215-221Crossref PubMed Scopus (85) Google Scholar). The purified PheA2 protein was bright yellow and eluted from an analytical Superdex 200 HR10/30 column in a single symmetrical peak with an apparent molecular mass of 35 ± 3 kDa, indicative of a homodimeric structure. When PheA
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