Phenol Hydroxylase from Bacillus thermoglucosidasius A7, aTwo-protein Component Monooxygenase with a Dual Role for FAD*

Received for publication, July 10, 2003, and in revised form, September 9, 2003Published, JBC Papers in Press, September 10, 2003, DOI 10.1074/jbc.M307397200

Ulrike Kirchner‡, Adrie H. Westphal§, Rudolf Muller‡, and Willem J. H. van Berkel§¶

From the ‡Department of Technical Biochemistry, Biotechnology II, Technical University Hamburg-Harburg,Denickestrasse 15, D-21071 Hamburg, Germany and the §Laboratory of Biochemistry, Wageningen University,Dreijenlaan 3, NL-6703HA Wageningen, The Netherlands

A novel phenol hydroxylase (PheA) that catalyzes thefirst step in the degradation of phenol in Bacillus ther-moglucosidasius A7 is described. The two-protein sys-tem, encoded by the pheA1 and pheA2 genes, consists ofan oxygenase (PheA1) and a flavin reductase (PheA2)and is optimally active at 55 °C. PheA1 and PheA2 wereseparately expressed in recombinant Escherichia coliBL21(DE3) pLysS cells and purified to apparent homo-geneity. The pheA1 gene codes for a protein of 504 aminoacids with a predicted mass of 57.2 kDa. PheA1 exists asa homodimer in solution and has no enzyme activity onits own. PheA1 catalyzes the efficient ortho-hydroxyla-tion of phenol to catechol when supplemented withPheA2 and FAD/NADH. The hydroxylase activity isstrictly FAD-dependent, and neither FMN nor riboflavincan replace FAD in this reaction. The pheA2 gene codesfor a protein of 161 amino acids with a predicted mass of17.7 kDa. PheA2 is also a homodimer, with each subunitcontaining a highly fluorescent FAD prosthetic group.PheA2 catalyzes the NADH-dependent reduction of freeflavins according to a Ping Pong Bi Bi mechanism.PheA2 is structurally related to ferric reductase, anNAD(P)H-dependent reductase from the hyperthermo-philic Archaea Archaeoglobus fulgidus that catalyzesthe flavin-mediated reduction of iron complexes. How-ever, PheA2 displays no ferric reductase activity and isthe first member of a newly recognized family of short-chain flavin reductases that use FAD both as a substrateand as a prosthetic group.

Phenolic compounds constitute one of the largest groups ofnatural 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 com-pounds has been increased with products of industrial origin.Many of these synthetic compounds cause environmental pol-lution and human health problems as a result of their persist-ence, toxicity, and transformation into hazardous intermedi-ates (1, 2).

The aerobic mineralization of natural and xenobiotic pheno-lic compounds by mesophilic microorganisms has been inten-sively investigated, and numerous pathways are known (3).Almost invariably, phenols are first converted into more reac-

tive dihydroxylated intermediates and then subjected to intra-or extradiol ring cleavage by molecular oxygen. The initialhydroxylation of the phenolic ring usually is catalyzed by sin-gle-component NAD(P)H-dependent flavoprotein monooxygen-ases (4). These enzymes share a typical dinucleotide-bindingfold for complexation of the FAD cofactor while lacking a com-mon NAD(P)-binding fold (5, 6). Because the regioselectivehydroxylation of phenols is notoriously difficult to achieve bychemical methods, the mechanistic and structural features ofsingle-component flavoprotein monooxygenases have receivedmuch attention (7–11). The reduced forms of these enzymesreact with molecular oxygen to yield a transiently stable flavinC4a-hydroperoxide species that is involved in substrate oxy-genation. For Pseudomonas p-hydroxybenzoate hydroxylase(12–14) and phenol hydroxylase from yeast (15), it was shownthat the oxygenation reaction takes place in the inner part ofthe protein and that this aprotic environment is crucial forpreventing uncoupling of flavin reduction from substrate hy-droxylation. With 4-hydroxyphenylacetate 3-hydroxylase fromPseudomonas putida, the binding of a second protein compo-nent prevents the uncoupling of substrate hydroxylation (16–18). This enzyme is an unusual example of a two-componentflavoprotein hydroxylase in which flavin reduction and sub-strate oxygenation take place in the same protein.

Relatively little is known about the mineralization of pheno-lic compounds by thermophilic microorganisms (19). SeveralBacillus species isolated from geographically distinct thermalsources degrade phenol at 65 °C via the meta-cleavage pathway(Fig. 1) (20–22). The initial conversion of phenol to catechol inthese microorganisms requires two protein components thatare encoded by the pheA1 and pheA2 genes (23). Characteriza-tion of the Bacillus phenol hydroxylase system appeared to beseverely hampered by the low yield and instability of the puri-fied enzymes. This prompted us to clone the Bacillus pheAgenes in an Escherichia coli expression system (24).

In this study, we describe the overexpression, purification,and characterization of the recombinant phenol hydroxylase ofBacillus thermoglucosidasius A7. We show that this two-pro-tein enzyme belongs to a newly recognized family of flavin-de-pendent monooxygenases that carry out the reductive and ox-idative half-reactions on separate polypeptide chains. Membersof this family are involved in various biological processes, in-cluding the biosynthesis of antibiotics (25–27), the desulfuriza-tion of fossil fuels (28), the degradation of chelating agents (29),and the oxidation of aromatic compounds (30). In contrast tomost other family members, the reductase component of phenolhydroxylase from B. thermoglucosidasius A7 harbors a tightlybound FAD. Evidence is provided that the FAD cofactor isdirectly involved in the reduction of free flavins.

* This work was supported by a grant (to U. K.) from the DutchGraduate School of Environmental Chemistry and Toxicology. The costsof publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby marked “advertise-ment” in accordance with 18 U.S.C. Section 1734 solely to indicate thisfact.

¶ To whom correspondence should be addressed. Tel.: 31-317-482861;Fax: 31-317-484801; E-mail: willem.vanberkel@wur.nl.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 278, No. 48, Issue of November 28, pp. 47545–47553, 2003© 2003 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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EXPERIMENTAL PROCEDURES

Chemicals, Bacterial Strains, and Plasmids—E. coli strainBL21(DE3) pLysS and plasmid pET3a were used for expression of thepheA genes (31). Isopropyl-�-D-thiogalactopyranoside was purchasedfrom Invitrogen. Phenyl-Sepharose, Q-Sepharose Fast Flow, HiLoadQ-Sepharose, Superdex 200 pg, Superdex 200 HR10/30, Superdex 75HR10/30, and PhastGel precast isoelectric focusing gels were fromAmersham Biosciences. Macro-prep ceramic hydroxylapatite (type I,particle size of 20 �m) was obtained from Bio-Rad. FAD, FMN, ribofla-vin, and 3-acetylpyridine-adenine dinucleotide (AcPyAD�)1 were pur-chased from Sigma. NADH, NADPH, NAD�, and glucose oxidase (gradeII) were from Roche Applied Science. Phenolic compounds were ob-tained from Aldrich. All other chemicals were from Merck and were ofthe purest grade available.

Enzyme Purification—All purification steps were carried out at roomtemperature. PheA1 and PheA2 were purified from transformed E. colicells harboring the pheA1 and pheA2 genes (24), respectively. Recom-binant cells were grown for 16 h in medium containing 10 g/literTryptone, 5 g/liter yeast, and 5 g/liter NaCl (pH 7.4) at 30 °C, followedby induction with 1 mM isopropyl-�-D-thiogalactopyranoside for 3 h.

Purification of PheA1 (Oxygenase Component)—Washed recombi-nant E. coli cells (15 g) were suspended in 15 ml of 50 mM sodiumphosphate (pH 7.0) containing 0.5 mM EDTA, 0.5 mM phenylmethylsul-fonyl fluoride, and 0.5 mg of DNase and passed two times through aprecooled French press operating at 10,000 p.s.i. Following centrifuga-tion for 30 min at 15,000 � g to remove cellular debris, the supernatantwas made 0.5% in protamine sulfate from a 2% stock solution. Theprotamine sulfate aggregates were removed by centrifugation for 15min at 15,000 � g, and the resulting supernatant was applied to aQ-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 witha linear gradient of 0–0.6 M NaCl in 100 ml of starting buffer. ThePheA1 protein eluting between 0.3 and 0.5 M NaCl was concentrated byultrafiltration (Amicon YM-30 membrane) and dialyzed against 5 mM

sodium phosphate (pH 7.0). The PheA1 protein was then loaded onto ahydroxylapatite column (1.6 � 10 cm) pre-equilibrated with 5 mM

sodium phosphate (pH 7.0). After washing, PheA1 was eluted with alinear gradient of 5–500 mM sodium phosphate (pH 7.0) in 100 ml ofwater. Pooled fractions (80–200 mM) were concentrated by ultrafiltra-tion and stored at 5 mg/ml in 50 mM sodium phosphate (pH 7.0) at�70 °C.

Purification of PheA2 (Reductase Component)—The preparation ofcell extract from recombinant E. coli cells, the protamine sulfate pre-cipitation, and the ion exchange chromatography step were carried outunder the same conditions as described above. HiLoad Q-Sepharosefractions containing FAD reductase activity (eluting between 0.1 and0.5 M NaCl) were pooled and concentrated by ultrafiltration (AmiconYM-30 membrane). After the addition of pulverized ammonium sulfateto a final concentration of 1.4 M, the protein solution was loaded onto aphenyl-Sepharose column (1.6 � 10 cm) equilibrated with 50 mM po-tassium phosphate (pH 7.0) containing 1.4 M ammonium sulfate. Afterwashing with starting buffer, bound protein was eluted with a lineardescending gradient of ammonium sulfate (1.4 to 0.4 M) in 200 ml ofstarting buffer. Fractions containing reductase activity (0.4–1.0 M) werepooled, concentrated by ultrafiltration, and loaded onto a Superdex 200preparative gel filtration column (2.6 � 60 cm) equilibrated with 50 mM

potassium phosphate (pH 7.0) containing 150 mM NaCl. Pure reductasefractions were pooled, concentrated, and stored in 50 mM potassiumphosphate (pH 7.0) at �70 °C.

Activity Determinations—NADH:flavin reductase activity was deter-

mined 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 reductaseactivity was determined spectrophotometrically at 25 °C by recordingthe NADH-dependent reduction of cytochrome c at 550 nm (�550 � 21.1mM�1 cm�1). The assay mixture contained 0.2 mM cytochrome c and 0.2mM NADH in 50 mM potassium phosphate (pH 7.5). NADH oxidaseactivity was determined spectrophotometrically at 25 °C by monitoringthe decrease in absorption of NADH at 340 nm (�340 � 6.22 mM�1 cm�1).The assay mixture contained 0.2 mM NADH in 50 mM potassium phos-phate (pH 7.0). The NADH-dependent reduction of 2,6-dichloropheno-lindophenol was measured at 25 °C by following the decrease in absorp-tion of 2,6-dichlorophenolindophenol at 600 nm (�600 � 21.0 mM�1 cm�1,pH 7.0). The assay mixture contained 25 mM potassium phosphate(pH 7.0), 150 mM KCl, 200 �M NADH, and varying concentrations of2,6-dichlorophenolindophenol. The NADH-dependent reduction ofAcPyAD� (transhydrogenase activity) was measured at 25 °C by follow-ing the increase in absorption of reduced AcPyAD� at 363 nm (�363 � 5.6mM�1 cm�1). The assay mixture contained 25 mM potassium phosphate(pH 7.0), 150 mM KCl, 200 �M NADH, and varying concentrationsof AcPyAD�.

Kinetic Analysis—NADH:flavin reductase activity was determinedat 25, 40, and 53 °C in 25 mM potassium phosphate and 150 mM KCl (pH7.0). For estimation of kinetic parameters, the NADH concentrationwas varied at a fixed flavin concentration and vice versa. Kinetic pa-rameters were determined from saturation curves, fitted with theMichaelis-Menten equation, using a Levenberg-Marquardt algorithm.For estimation of the type of mechanism, the NADH:flavin reductaseactivity was determined as a function of FAD concentration at severalconstant levels of NADH and as a function of NADH at several constantlevels of FAD. Reciprocal initial velocities were plotted against recipro-cal substrate concentrations and fitted with a straight line determinedby a linear regression program. Inhibition constants for NAD� inhibi-tion were determined according to Fromm (32).

Other Assays—Phenol monooxygenase activity was determined bymeasuring phenol consumption colorimetrically (33). The reaction mix-ture (6 ml) contained 0.1 mM phenol, 0.5 mM NADH, 10 �M FAD, and 1.0nM PheA2 in 50 mM potassium phosphate (pH 7.0). After equilibrationat 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 reactionwas stopped by the addition of 12 �l of 2% 4-aminoantipyrine followedby 40 �l of 2 N ammonium hydroxide and 40 �l of 2% potassiumferricyanide, and the final volume was adjusted to 2 ml with water.After 15 min of incubation at room temperature, the absorbance at 510nm was read and compared with those of phenol standards. 1 unit isdefined as the amount of enzyme that catalyzes the conversion of 1�mol of substrate/min under the assay conditions. Oxygen consumptionwas determined polarographically at 50 °C in a closed reaction vesselfitted with a Clark-type oxygen electrode. Reaction mixtures contained50 mM sodium phosphate (pH 7.0), 0.1 mM phenol, 5–10 �M FAD, and0.5 nM PheA2 in the absence or presence of 100 nM PheA1. NADH wasadded to a final concentration of 0.1 mM to initiate the reaction. Whenoxygen consumption ceased, 90 units of catalase were added to deter-mine the degree of uncoupling of hydroxylation.

Thermostability—Studies on the thermostability of PheA1 andPheA2 were performed by incubating the enzymes (46 �M PheA1 or 48�M PheA2) in closed vials at different temperatures (50, 60, 70, and85 °C) in 25 mM phosphate (pH 7.0) for 2 h. At timed intervals, aliquotswere withdrawn from the incubation mixtures and assayed for residualphenol hydroxylase (PheA1) and NADH:flavin reductase (PheA2)activities.

Analytical Methods—HPLC experiments were performed with anApplied Biosystems pump equipped with a Waters 996 photodiodearray detector. Reaction products were separated with a 3.9 � 100-mmLichrospher RP18 column running in methanol/water (50:50, v/v) con-taining 0.7% acetic acid. The flow rate was 1 ml/min. Protein was

1 The abbreviations used are: AcPyAD�, 3-acetylpyridine-adeninedinucleotide; HPLC, high performance liquid chromatography; Tricine,N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; FPLC, fast proteinliquid chromatography; FeR, ferric reductase.

FIG. 1. Degradation pathway of phenol in B. thermoglucosidasius A7. Step 1, phenol hydroxylase; step 2, catechol 2,3-dioxygenase; step3, 2-hydroxymuconic-semialdehyde hydrolase; step 4, 2-hydroxypenta-2,4-dienoate hydratase.

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determined by the method of Bradford (34) with bovine serum albuminas a standard. SDS-PAGE was carried out with 15% slab gels (35) orwith 16.5% Tricine gels (36). The gels were scanned and analyzed witha computing densitometer system (Scanjet ADF, Hewlett-Packard In-telligent Scanning Technology). Analytical gel filtration was performedon an Amersham Biosciences Åkta FPLC system equipped with a Su-perdex 200 HR10/30 column running in 50 mM potassium phosphate(pH 7.0) containing 0.15 M NaCl. Isoelectric focusing was performedwith a PhastSystem (Amersham Biosciences) using PhastGel 3–9 iso-electric focusing precast gels. Gels were stained with Coomassie BlueR-250.

Spectral Analysis—Absorption spectra were recorded at 25 °C usinga Hewlett-Packard 8453 diode array spectrophotometer or an AmincoDW-2000 double-beam spectrophotometer. For anaerobic reduction ex-periments, enzyme and substrate solutions were made anaerobic byalternated flushing with deoxygenated argon and evacuating. Anaero-bic reduction of PheA2 was performed by adding aliquots of oxygen-freeNADH or dithionite to the enzyme solution, followed by spectral record-ing. Photoreduction in the presence of EDTA and 5-deazaflavin ascatalyst was performed essentially as described (37). Circular dichroismspectra 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 isolatedby boiling an enzyme sample for 3 min. After removal of the proteinprecipitate, the nature of the extracted flavin was identified by fluores-cence analysis (38) and reverse-phase HPLC (39).

Cysteine Determination—The estimation of sulfhydryl groups of na-tive or unfolded PheA2 was carried out by the procedure of Ellman (40)employing the modifications of Habeeb (41). All thiol determinationswere carried out on enzyme preparations that were freshly incubatedwith 1 mM dithiothreitol, 50 �M FAD, and 0.5 mM EDTA for 15 min andsubsequently passed through a Bio-Gel P-6DG column equilibratedwith 50 mM potassium phosphate (pH 7.0) containing 0.5 mM EDTA.The enzyme was diluted in 100 mM Tris sulfate (pH 8.0) immediatelybefore assaying the time-dependent release of 5-thio-2-nitrobenzoate at412 nm (final pH 7.9).

Sequence Analysis—N-terminal protein sequence analysis was per-formed by Edman degradation with an Applied Biosystems Model 473Apulsed-liquid sequencer fitted with an on-line phenylthiohydantoin an-alytical system following the procedures suggested by the manufac-turer. Protein sequence similarity searches were performed with PSI-BLAST (42) using the facilities offered by the National Center forBiotechnology Information (NCBI). Multiple sequence alignments weremade with ClustalX (43) using the PAM350 matrix.

Protein Homology Modeling—Model building of PheA2 was per-formed with MODELLER (44) using the CVFF force field (45). Thestructure of ferric reductase (FeR) from Archaeoglobus fulgidus withNADP� bound (Protein Data Bank code 1IOS) (46) was used as atemplate. The model was verified after several rounds of energy mini-mization. The stereochemical quality of the homology model was veri-fied by PROCHECK (47), and the protein folding was assessed withPROSAII (48), which evaluates the compatibility of each residue withits environment independently. The FMN part of FAD was placed in themodel structure at the same position and orientation as the FMN in thetemplate structure. The AMP part of FAD was placed in the groovecontinuing from the binding pocket of FMN. NADP� was oriented in thesame position as found in the crystal structure of FeR.

RESULTS

Overexpression of pheA Genes—Both protein componentsPheA1 and PheA2 from B. thermoglucosidasius A7 are re-quired for phenol hydroxylase activity (24). However, E. coli

cells containing a plasmid with the genes for PheA1 and PheA2in tandem did not express PheA2, as only a clear protein bandfor PheA1 (but not for PheA2) could be detected by SDS-PAGEanalysis of cell extracts (24). A likely explanation is that, in thisconstruct, PheA2 expression must use its own B. thermogluco-sidasius A7 ribosome-binding site, which might not be effectivein E. coli. When PheA2 was expressed separately, expressionwas under the control of the highly efficient phage T7 pro-moter, and a clear protein band of PheA2 was visible uponSDS-PAGE analysis of cell extracts (24). Therefore, PheA1 andPheA2 were separately purified from E. coli cells containingthe appropriate plasmid with the gene for PheA1 or PheA2.

Purification of PheA1—Extracts of E. coli cells containingthe plasmid with only the pheA1 gene exhibited phenol hydrox-ylase activity. Because these cells do not contain PheA2, thisindicates that a flavin reductase activity present in the E. colihost substitutes for PheA2 to give substrate conversion. Asimilar observation was made for 4-hydroxyphenylacetate3-monooxygenase from E. coli W (30). During purification ofPheA1, phenol hydroxylase activity was determined with andwithout complementation with PheA2. The phenol hydroxylaseactivity determined in the absence of PheA2 decreased afterevery purification step (Table I). The specific activity of purifiedPheA1 determined in the presence of PheA2 was 0.32 units/mg.SDS-PAGE analysis of the purified PheA1 protein revealed thepresence of a single polypeptide chain corresponding to a mo-lecular mass of �57 kDa (Fig. 2). This value is in good agree-ment with the molecular mass predicted from gene sequenceanalysis (24).

Purified PheA1 did not contain any chromophore as judgedby absorption spectral analysis and eluted from an analyticalSuperdex 200 HR10/30 column in a single symmetrical peakwith an apparent molecular mass of 120 � 5 kDa. This suggeststhat the PheA1 protein is a dimer composed of identicalsubunits.

Isoelectric focusing of PheA1 revealed a main protein bandwith an isoelectric point of 5.2 � 0.1. This value is considerablylower than the theoretical value of 6.29 calculated from theamino acid sequence. Purified PheA1 was stable for 2 h at60 °C, as no significant decrease in phenol hydroxylase activitywas observed in complementation experiments with PheA2. Incontrast, at 70 °C, inactivation of PheA1 was complete after 10min of incubation. The purified PheA1 protein was not verystable when stored at �70 °C because it formed aggregatesafter thawing. Therefore, PheA1 was stored as a protein pre-cipitate in 80% ammonium sulfate at 4 °C.

Purification of PheA2—The PheA2 protein expressed inE. coli cells containing the plasmid with the pheA2 gene waspurified to apparent homogeneity using three chromatographicsteps (Table II). The specific NADH:FAD reductase activity ofthe purified PheA2 protein at 53 °C was 800 units/mg. Analysisby SDS-PAGE revealed the presence of a single band corre-sponding to a polypeptide chain molecular mass of �18 kDa(Fig. 3). Again, this value is in good agreement with the mo-

TABLE IPurification of the PheA1 oxygenase component of phenol hydroxylase from B. thermoglucosidasius A7 expressed in E. coli PH2

Phenol hydroxylase activity was measured at 50 °C. The reaction mixture contained 0.1 mM phenol, 1 mM NADH, and 10 �M FAD in 50 mM

potassium phosphate (pH 7.0).

Step Protein Activitya Specific activitya Activityb Specific activityb Yield

mg units units/mg units units/mg %

Cell extract 764 43 0.056 46 0.060 100Q-Sepharose 302 2.4 0.008 16 0.053 35Hydroxyapatite 27 0.11 0.004 8.6 0.320 18

a In the absence of 1 mM PheA2.b In the presence of 1 mM PheA2.

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lecular mass predicted from gene sequence analysis (17,660Da) (24). The purified PheA2 protein was bright yellow andeluted from an analytical Superdex 200 HR10/30 column in asingle symmetrical peak with an apparent molecular mass of35 � 3 kDa, indicative of a homodimeric structure. WhenPheA2 was mixed with PheA1 and analyzed by gel filtration, nointeraction between both proteins was observed.

The isoelectric point of PheA2 was determined by isoelectricfocusing to be 5.2 � 0.1. This value agrees with the theoreticalvalue (5.36) estimated from the amino acid sequence. PheA2was stable in the frozen state because no significant loss ofactivity was observed during 4 months of storage at �70 °C.When purified PheA2 was incubated for 2 h at 65 °C, �65% ofthe NADH:FAD reductase activity was maintained. Time-de-pendent analysis showed that the partial loss of activity at65 °C occurred within the first 10 min of incubation. When theenzyme was incubated at 75 or 85 °C, the activity decreasedfaster, but a similar behavior was observed. After 2 h of incu-bation at 85 °C, still 15–20% of the NADH:FAD reductaseactivity remained.

PheA2 contains a single cysteine residue (Cys112) perpolypeptide chain (24). Absorption measurements at 412 nmrevealed that this cysteine reacted rapidly with 5,5�-dithio-bis(2-nitrobenzoate) when the protein was unfolded with 0.5%SDS. In the native enzyme, the side chain of Cys112 is notaccessible for solvent. This is concluded from the lack of reac-tivity of native PheA2 with Ellman’s reagent.

Spectral Properties of PheA2—The optical spectrum ofPheA2 was typical of a flavoprotein with maxima at 376 and455 nm and characteristic shoulders around 355 and 485 nm(Fig. 4A). The A270/A455 ratio for the purified enzyme was 4.1.This value is in good agreement with the low amount of aro-matic amino acid residues (24) and suggests that each PheA2subunit binds one molecule of flavin. Extraction of the cofactorrevealed that the flavin was noncovalently bound. Moreover,the HPLC retention time and fluorescence properties of theextracted flavin were identical to those of authentic FAD. Fromthe absorption spectra of PheA2 in the absence and presence of0.5% SDS (Fig. 4A), a molar absorption coefficient of �455 � 11.8mM�1 cm�1 for protein-bound FAD was estimated.

The fluorescence quantum yield of the protein-bound FAD ofPheA2 was rather high. Upon excitation at 450 nm, a maxi-mum fluorescence emission was observed around 525 nm (datanot shown). Upon unfolding of PheA2 in 0.5% SDS, the fluo-rescence intensity of the flavin decreased by almost an order ofmagnitude. This indicates that the flavin fluorescence quan-tum yield of PheA2 is comparable to that of free FMN (38) andin the same range as that of lipoamide dehydrogenase, one ofthe most fluorescent flavoproteins (49).

Fig. 4B shows the flavin circular dichroism spectrum ofPheA2 in the visible region. This spectrum resembles those offlavin reductase from Streptomyces viridifaciens (27), ferredox-in:NADP� oxidoreductase (50), and the enzyme-substrate com-plex of p-hydroxybenzoate hydroxylase (51) by displaying neg-ative circular dichroism in the 450 nm region and positiveoptical activity in the near-UV band.

When PheA2 was (photo)chemically reduced under anaero-bic conditions, the flavin hydroquinone was formed, and nothermodynamic stabilization of semiquinone species was ob-served. Upon titration with NAD�, the two-electron reducedenzyme formed a complex with NAD�, as evidenced by the for-mation of a long wavelength absorption band. Anaerobic reduc-tion of PheA2 with NADH also led to the reduced enzyme-NAD�

complex (Fig. 5). At pH 8.0, the enzyme could not be fully reducedwith NADH, in contrast to reduction at pH 6.0 (Fig. 5).

Catalytic Properties of PheA2—In addition to NADH:FAD re-ductase activity, PheA2 exhibited also NADH:cytochrome c re-ductase and NADH:dichlorophenolindophenol and NADH:AcPyAD� transhydrogenase activities (Table III). However,PheA2 displayed no ferric reductase activity. PheA2 was alsoactive with oxygen as an electron acceptor (Table III). In contrastto the aromatic substrate-stimulated NADH oxidase activity ob-served for the reductase component of 4-hydroxyphenylacetate3-hydroxylase from Acinetobacter baumannii (52), phenol did notinfluence the rate of NADH oxidation or oxygen consumption.Moreover, the oxygen consumed by PheA2 in the presence andabsence of phenol was halved in the presence of catalase, con-firming that, in both cases, hydrogen peroxide was the onlyreduced form of molecular oxygen that was generated. The hy-drogen peroxide formed results from the reaction of the reducedprotein-bound flavin with oxygen, which presumably involves theintermediate formation of a flavin semiquinone-superoxide rad-ical pair and a highly unstable flavin C4a-peroxide (7).

The pH and temperature optima of the NADH:FAD reduc-tase activity of PheA2 were pH 6.7 and 55 °C (Figs. 6 and 7).Determination of kinetic parameters revealed that NADH is amuch better coenzyme for PheA2 than NADPH (Table IV). TheFAD reductase activity exhibited a linear dependence onNADPH concentration over a range 10–500 �M, and no indica-tion of saturation was observed. Thus, Km and kcat values forNADPH could not be determined under these conditions. How-ever, from the slope of the kinetic plot, a second-order rateconstant of k�cat/K�m � 0.006 s�1 could be estimated. This value

FIG. 2. SDS-PAGE analysis of protein samples obtained duringthe purification of the PheA1 component of phenol hydroxylasefrom B. thermoglucosidasius A7. Lanes A and E, molecular massmarkers (in kilodaltons); lane B, cell extract; lane C, Q-Sepharose pool;lane D, hydroxylapatite pool.

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is at least two orders of magnitude lower than the correspond-ing value for NADH (Table IV).

When the NADH:FAD reductase activity of PheA2 was stud-ied under anaerobic conditions, all free FAD was rapidly re-duced, confirming that the free FAD acts as a true substrate. Inaddition to FAD, PheA2 was also active with FMN and ribofla-vin. The turnover rate of PheA2 with free flavins was stronglydependent on temperature. At 25 °C, the activity with FMNand riboflavin was much higher than with FAD (Table III).However, when the temperature was raised to 53 °C, the turn-over rates with the different flavins became nearly identical(Table IV). On the other hand, the Michaelis constants forNADH, FAD, riboflavin, and FMN did not vary with tempera-ture (Tables III and IV).

Initial velocity measurements in dependence of both FADand NADH were performed to provide insight into the kineticmechanism of PheA2. When the NADH concentration was var-ied at a fixed FAD concentration, a series of parallel lines wasobserved in a double-reciprocal plot (Fig. 8A). A similar patternwas observed when the FAD concentration was varied at afixed NADH concentration (Fig. 8 B). This suggests that PheA2acts according to a Ping Pong Bi Bi reaction mechanism inwhich NADH reduces the FAD cofactor, which in turn transferselectrons to the FAD substrate. A similar kinetic behavior wasobserved when the NADH:AcPyAD� transhydrogenase activityof PheA2 was determined as a function of AcPyAD� concentra-tion at several constant levels of NADH and as a function ofNADH at several constant levels of AcPyAD� (data not shown).The NADH:FAD reductase activity of PheA2 was inhibited byNAD�. In line with the above-described mechanism, the inhi-

bition was noncompetitive to NADH and competitive to FAD(data not shown).

Phenol Hydroxylase Activity—Purified PheA1 showed almostno phenol hydroxylase activity when assayed at 50 °C and pH7.0 in the absence of PheA2. The PheA1-mediated conversion ofphenol to catechol was strongly stimulated in the presence ofcatalytic amounts of PheA2. At relatively low concentrationsof PheA1, phenol hydroxylase activity was not linear with time.The addition of catalase to the reaction mixture to avoid the

TABLE IIPurification of the PheA2 reductase component of phenol hydroxylase from B. thermoglucosidasius A7 expressed in E. coli PH3

Step Protein Activity Specific activitya Yield

mg units unit/mg %

Cell extract 1474 165,214 112 100Q-Sepharose 402 71,720 179 43.4Phenyl-Sepharose 142 48,482 341 29.3Superdex 200 26.5 21,253 802 12.9

a NADH:FAD reductase activity was measured at 53 °C. The assay mixture contained 25 mM potassium phosphate (pH 7.0), 150 mM KCl, 200�M NADH, and 10 �M FAD.

FIG. 3. SDS-PAGE analysis of protein samples obtained duringthe purification of flavin reductase (PheA2) from B. thermoglu-cosidasius A7. Lane A, cell extract; lane B, supernatant after prota-mine sulfate treatment; lane C, HiLoad Q-Sepharose pool; lane D,phenyl-Sepharose pool; lane E, Superdex 200 pool; lane F, molecularmass markers (in kilodaltons).

FIG. 4. Spectral properties of flavin reductase (PheA2) fromB. thermoglucosidasius A7. The spectra were recorded in 50 mM

sodium phosphate (pH 7.0). A, optical spectrum of 16 �M PheA2 (OO)and 5 min after the addition of 0.5% SDS (– – –); B, circular dichroismspectrum of 32 �M PheA2. deg, degrees.

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accumulation of hydrogen peroxide had no effect. At a constantconcentration of PheA2 and increasing concentrations of PheA1in the presence of phenol, the oxygen consumption increased,and the relative amount of hydrogen peroxide formed de-creased. When the dependence of the hydroxylation reaction onthe concentration of free FAD at constant concentrations ofPheA1 and PheA2 (200:1 molar ratio) was studied, the oxygenconsumption became faster with increasing concentrations offree FAD, but the amount of uncoupling increased (Table V). Incontrast to the NADH:flavin reductase activity of PheA2, thehydroxylase activity of PheA was strictly FAD-dependent, andneither FMN nor riboflavin could replace FAD in this reaction.

Under optimal assay conditions with PheA1 in large excessover PheA2, maximum phenol hydroxylase activity wasreached at 55 °C and pH 7.0. HPLC analysis revealed thatPheA from B. thermoglucosidasius A7 also catalyzed the con-version of 4-methylphenol, 4-chlorophenol, and 4-fluorophenolto the corresponding catechols. No monooxygenase activity wasobserved with 4-hydroxybenzoate, 4-hydroxyacetophenone,and 4-hydroxyphenylacetate.

To test whether a nonspecific FAD reductase might substi-tute for PheA2 to give substrate conversion, the phenol hydrox-ylase activity of purified PheA1 was measured in the presenceof E. coli NADH oxidoreductase, which is physiologically asso-

ciated with the so-called hybrid cluster protein (53). At 50 °C,this iron-sulfur and FAD-containing reductase initiated theformation of catechol. However, the E. coli reductase was not

FIG. 5. Spectral changes upon anaerobic reduction of PheA2from B. thermoglucosidasius A7. Trace 1 is the absorption spectrumof 16 �M oxidized PheA2 in 25 mM potassium phosphate (pH 8.0) and150 mM KCl. The spectrum of oxidized PheA2 is identical at pH 6.0.Spectrum after anaerobic reduction by 25 �M NADH at pH 8.0 (trace 2),after reduction by 25 �M NADH at pH 6.0 (trace 3), and after reductionby sodium dithionite at pH 8.0 (trace 4).

TABLE IIINADH-dependent activities of PheA2 with different electron acceptors

Electron acceptor k�cata K�m k�cat/K�m

s�1 �M s�1 �M�1

FAD 43 1.6 26.9FMN 180 5.5 32.7Riboflavin 147 5.5 26.7DCPIPb 7.1 22 0.32AcPyAD� 8.0 1270 0.006Oxygenc 1.4 ND

a NADH-dependent reduction was measured at 25 °C. The assaymixture contained 25 mM potassium phosphate (pH 7.0), 150 mM KCl,and 200 �M NADH.

b DCPIP, dichlorophenolindophenol; ND, not determined.c Activity was measured in air-saturated buffer.

FIG. 6. Dependence of the NADH:FAD reductase activity offlavin reductase (PheA2) from B. thermoglucosidasius A7 on pH.The assay mixture contained 25 mM potassium phosphate buffer atdifferent pH values, 150 mM KCl, 200 �M NADH, and 20 �M FAD. Theassay temperature was 53 °C.

FIG. 7. Dependence of the NADH:FAD reductase activity offlavin reductase (PheA2) from B. thermoglucosidasius A7 ontemperature. The assay mixture contained 25 mM potassium phos-phate buffer (pH 7.0), 150 mM KCl, 200 �M NADH, and 20 �M FAD. Theassay temperature was varied from 18 to 64 °C.

TABLE IVKinetic parameters of PheA2 with flavin analogs

Kinetic experiments were performed at 53 °C. The assay mixturecontained 25 mM potassium phosphate (pH 7.0) and 150 mM KCl.Values of kinetic parameters have a maximum mean S.E. of 5%.

Substrate Second substrate k�cat K�m k�cat/K�m

s�1 �M s�1 �M�1

NADH FAD 225 8.8 26NADPH FAD �500 0.006FAD NADH 252 1.5 168FMN NADH 279 5.4 52Riboflavin NADH 276 5.6 49

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very stable under these assay conditions, and several aliquotsof enzyme were needed to consume all of the phenol.

Sequence Homology—N-terminal amino acid sequence analy-sis of the purified recombinant PheA1 and PheA2 proteins re-vealed sequences Met-Lys-Asp-Met-Met and Met-Asp-Asp-Arg-Leu, respectively. These sequences are identical to those deducedfrom the nucleotide sequences of the pheA1 and pheA2 genes (24).Multiple sequence alignment revealed that PheA1 shares themost sequence identity (up to 54%) with the oxygenase compo-nent of (i) phenol hydroxylase from Bacillus thermoleovorans A2(23), (ii) chlorophenol 4-monooxygenase from Ralstonia picketii(54) and Burkholderia cepacia (55), and (iii) several (putative)bacterial 4-hydroxyphenylacetate 3-hydroxylases (30, 56, 57).

PheA2 resembles a larger number of proteins than PheA1.Except for the reductase components of the above-mentionedtwo-component enzymes, PheA2 shares considerable sequenceidentity (up to 57%) with the reductase component of (i) styrene

monooxygenase (58); (ii) dibenzothiophene monooxygenase(59); (iii) actinorhodin-polyketide dimerase (25); (iv) nitrilotri-acetate monooxygenase (60); (v) VlmR, an NADPH:FAD oxi-doreductase from S. viridifaciens (27); (vi) SnaC, the FMNreductase component of pristinamycin IIA synthase fromStreptomyces pristinaespiralis (26); and (vii) a number of hy-pothetical flavin-binding proteins, including the putative re-ductase component of phenol hydroxylase from Oceanobacillusiheyensis (61) and the NimA protein (GenBankTM/EBI DataBank accession number Q8VQV3).

Structure Homology Modeling of PheA2—So far, no crystalstructures of members of the family of two-component flavopro-tein monooxygenases have been reported. However, the PheA2-related flavin reductases are homologous to the NAD(P)H-de-pendent FeR from the hyperthermophilic Archaea A. fulgidus(62), for which the three-dimensional structure was recentlysolved (46). FeR reduces FAD and FMN, but not riboflavin,whereas PheA2 shows no ferric reductase activity. Anotherremarkable difference between PheA2 and FeR is that thelatter enzyme was purified as an apoprotein (62). Furthermore,in the FeR crystal structure, only one subunit contains FMN,and NADP� was found to bind only to the subunit that harborsFMN (46).

PheA2 shares 46% sequence homology (24% sequence iden-tity) with FeR (Fig. 9), which prompted us to build a model ofthe three-dimensional structure of PheA2 (Fig. 10). As foundfor FeR, each PheA2 subunit consists of a single domain that isorganized around a six-stranded antiparallel �-barrel that ishomologous to the FMN-binding protein from Desulfovibriovulgaris (63). The FMN part of FAD was fit in the modelstructure at the same position and orientation as the FMN inFeR. The AMP part of FAD was docked into the PheA2 modelin a groove elongating the FMN cofactor-binding site. However,because of steric constraints and the poor alignment with aflexible loop in the FeR structure comprising residues 85–100,the location of this part of the FAD molecule remained elusive.

DISCUSSION

PheA is a novel type of phenol hydroxylase that is induced inthe thermophilic bacterium B. thermoglucosidasius A7 whenthe strain is grown on phenol as a source of carbon and energy.The phenol hydroxylase system consists of two homodimericproteins with subunit molecular masses of 57 and 18 kDa,respectively. The large PheA1 component contains no cofactorsand has no enzyme activity on its own. The small PheA2component is an NADH-dependent FAD-containing reductasethat supplies PheA1 with reduced FAD to catalyze the ortho-hydroxylation of simple phenols to the corresponding catechols.

During the past few years, several two-protein componentaromatic hydroxylases relying on reduced flavin as a substrate

TABLE VEffect of FAD concentration on efficiency of phenol

hydroxylation by PheAEnzymatic reactions were performed in air-saturated solutions at

50 °C. The reaction mixtures contained 50 mM sodium phosphate (pH7.0), 0.1 mM phenol, 0.1 mM NADH, 0.5-10 �M FAD, 100 nM PheA1, and0.5 nM PheA2.

Catechola H2O2b

% %FAD

0.5 �M 78 � 3 20 � 51.0 �M 66 � 4 32 � 82.5 �M 54 � 6 47 � 45.0 �M 43 � 4 56 � 610.0 �M 18 � 5 83 � 2

a Determined colorimetrically.b Determined with catalase.

FIG. 8. NADH:FAD reductase activity mechanism of PheA2from B. thermoglucosidasius A7. A, double-reciprocal plots of en-zyme activity as a function of NADH concentration at different FADconcentrations: 0.5 (●), 0.75 (f), 2 ( Œ), 4 (�), and 16 (E) �M. B, double-reciprocal plots of enzyme activity as a function of FAD concentration atdifferent NADH concentrations: 1 (●), 2 ( f), 4 (Œ), 8 (�), 16 (E), 32 (�),64 (ƒ), and 128 (‚) �M. The assay temperature was 40 °C.

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have been identified. These enzymes show a remarkable vari-ety in protein size and flavin specificity (52). So far, no struc-tural data of these enzymes are available, and their mecha-nisms of reduced flavin transfer and substrate hydroxylationremain to be elucidated. For 4-hydroxyphenylacetate 3-hydrox-ylase from E. coli (64) and A. baumannii (52), it was reportedthat the oxygenase component is able to use chemically reducedflavin for 4-hydroxyphenylacetate monooxygenation and that aphysical interaction between the oxygenase and reductase pro-tein components is not required for substrate hydroxylation(30, 65). Here we have shown that the purified dimeric PheA1and PheA2 proteins do not strongly interact, but that for the invitro conversion of phenol to catechol, a large excess of PheA1over PheA2 is needed to prevent the unproductive formation ofhydrogen peroxide.

Another important finding described in this study is the factthat PheA2 harbors a tightly bound FAD. The interaction be-tween PheA2 and FAD is much stronger than found for othershort-chain flavin reductases (30, 64, 66). From this and thePing Pong Bi Bi kinetic mechanism observed, we propose that,in PheA2, the reducing equivalents are transferred fromNADH via protein-bound FAD to exogenous free FAD. Thisproposal is supported by the inhibition of NAD� that is com-petitive with FAD. After reduction by PheA2, the FADH2 prod-uct is transferred to the PheA1 oxygenase component, in which

the hydroxylation of phenol to catechol takes place (Scheme 1).Our studies with PheA2 suggest a direct role for the protein-

bound FAD in the mediation of electron transfer fromNAD(P)H to exogenous free flavin. Such a role has also beenproposed for the flavoprotein component of sulfite reductase(67) and for flavin reductase I, the major NAD(P)H:FMN oxi-doreductase from Vibrio fisheri (68).

The kinetic mechanism and flavin-binding properties ofPheA2 are clearly different from those of the related ActVBNADH:flavin reductase participating in the last step of acti-norhodin synthesis in Streptomyces coelicolor (66). This ho-modimeric FMN-binding enzyme acts according to an orderedsequential mechanism and is not classified as a flavoprotein.The mechanism depicted in Scheme 1 also differs from theternary complex mechanism proposed for Fre, the prototype ofthe class I flavin reductases (69–71). In this monomeric en-zyme, which does not contain any prosthetic group, flavins arerecognized mainly through hydrophobic interactions with theisoalloxazine ring, providing an explanation of why Fre usesriboflavin as a substrate rather than as a cofactor (72, 73). Themechanism depicted in Scheme 1 is more consistent with themechanism proposed for the homodimeric FMN-containing fla-vin reductase P from Vibrio harveyi, which is involved in bi-oluminescence by providing reduced FMN to luciferase (74, 75).With the latter enzyme, a shift in the kinetic mechanism in theluciferase-coupled assay was taken as evidence that the re-duced FMN cofactor is transferred to luciferase via protein-protein complex formation (76). Subsequent studies with thebioluminescence enzymes from V. fisherii (77) and V. harveyi(78, 79) have indicated that the mechanism of reduced flavintransfer may depend on the constituent protein partners in thereductase-luciferase couple and that, under in vitro reconstitu-tion conditions, direct flavin product transfer is feasible as well.

PheA2 belongs to a newly recognized family of short-chainflavin reductases (30). These enzymes are considerably smallerin size than members of the Fre family (71, 72) and the flavin

SCHEME 1

FIG. 9. Sequence alignment of PheA2 from B. thermoglucosidasius and FeR from A. fulgidus. Dark-gray boxes indicate identicalresidues, and light-gray boxes show similar residues. Numbering is according to the PheA2 sequence.

FIG. 10. Structural model of PheA2. The subunits of the ho-modimer are shown in light and dark gray. Arrow A, FAD cofactor;Arrow B, loop region possibly involved in the binding of the adeninepart of FAD; arrow C, 2�-phosphate of NADP�. The figure was madewith PyMol (80).

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reductases from luminous bacteria (68, 74, 75). Our studiesshow that PheA2 is structurally related to FeR, a ferric reduc-tase from the hyperthermophilic Archaea A. fulgidus that usesits single domain to provide both the flavin- and NAD(P)H-binding sites (46). However, PheA2 displays no ferric reductaseactivity, binds FAD instead of FMN, and is specific for NADH.

For FeR, it was proposed that the substrate binds in a pocketnear the flavin isoalloxazine ring and that Thr31, Leu35, Cys45,and His126 might serve as protein ligands for the metal ion.Except for the conserved histidine (His123), these residues arereplaced in PheA2, disfavoring iron ligandation. In FeR, theFMN ribityl phosphate interacts with Asp51, Thr52, Arg82,Ser84, and Lys89. These residues are not conserved or lacking inthe PheA2 sequence. This suggests that the specificity for FADbinding is related to the shorter loop (region 84–92, PheA2numbering) (Fig. 9).

For FeR, it was found that the 2�-phosphate of NADP� doesnot make strong interactions with the polypeptide chain. Thismight explain why FeR shows no preference for NADH orNADPH. From the three-dimensional model of PheA2, no in-dication was obtained as to why this enzyme is very specific forNADH. This suggests that FeR and PheA2 must differ in theirmode of coenzyme binding.

Finally, we have shown here that the monooxygenase activ-ity of PheA1 is strictly FAD-dependent. This is indicative of aspecific interaction of PheA1 with the AMP moiety of FAD.Therefore and especially because both PheA1 and PheA2 haveno signature sequences for FAD binding, it is of great interestto obtain more insight into the structural and mechanisticfeatures of this novel type of two-protein component phenolhydroxylase in which FAD acts both as a substrate and as aprosthetic group.

Acknowledgments—We thank Dr. Fiona Duffner for valuable discus-sions, Dr. Walter van Dongen for the gift of E. coli NADH oxidoreduc-tase, and Bert Janssen for help in some of the experiments.

REFERENCES

1. Timmis, K. N., and Pieper, D. H. (1999) Trends Biotechnol. 17, 200–2042. Gupta, S. S., Stadler, M., Noser, C. A., Ghosh, A., Steinhoff, B., Lenoir, D.,

Horwitz, C. P., Schramm, K. W., and Collins, T. J. (2002) Science 296,326–328

3. Moiseeva, O. V., Solyanikova, I. P., Kaschabek, S. R., Groning, J., Thiel, M.,Golovleva, L. A., and Schlomann, M. (2002) J. Bacteriol. 184, 5282–5292

4. Moonen, M. J. H., Fraaije, M. W., Rietjens, I. M. C. M., Laane, C., and vanBerkel, W. J. H. (2002) Adv. Synth. Catal. 344, 1023–1035

5. Eppink, M. H. M., Overkamp, K. M., Schreuder, H. A., and van Berkel, W. J. H.(1999) J. Mol. Biol. 292, 87–96

6. Wang, J., Ortiz-Maldonado, M., Entsch, B., Massey, V., Ballou, D. P., andGatti, D. L. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 608–613

7. Massey, V. (1994) J. Biol. Chem. 269, 22459–224628. Entsch, B., and van Berkel, W. J. H. (1995) FASEB J. 9, 476–4839. Palfey, B. A., and Massey, V. (1996) in Comprehensive Biochemical Catalysis

(Sinnott, M., ed) pp. 83–154, Academic Press, Inc., New York10. van der Bolt, F. J. T., van den Heuvel, R. H. H., Vervoort, J., and van Berkel,

W. J. H. (1997) Biochemistry 36, 14192–1420111. Xu, D., Enroth, C., Lindqvist, Y., Ballou, D. P., and Massey, V. (2002) Bio-

chemistry 41, 13627–1363612. Schreuder, H. A., Mattevi, A., Obmolova, G., Kalk, K. H., Hol, W. G., van der

Bolt, F. J. T., and van Berkel, W. J. H. (1994) Biochemistry 33, 10161–1017013. Gatti, D. L., Palfey, B. A., Lah, M. S., Entsch, B., Massey, V., Ballou, D. P., and

Ludwig, M. L. (1994) Science 266, 110–11414. Palfey, B. A., Moran, G. R., Entsch, B., Ballou, D. P., and Massey, V. (1999)

Biochemistry 38, 1153–115815. Enroth, C., Neujahr, H., Schneider, G., and Lindqvist, Y. (1998) Structure 6,

605–61716. Arunachalam, U., Massey, V., and Vaidyanathan, C. S. (1992) J. Biol. Chem.

267, 25848–2585517. Arunachalam, U., Massey, V., and Miller, S. M. (1994) J. Biol. Chem. 269,

150–15518. Arunachalam, U., and Massey, V. (1994) J. Biol. Chem. 269, 11795–11780119. Kim, I. C., and Oriel, P. J. (1995) Appl. Environ. Microbiol. 61, 1252–125620. Mutzel, A., Reinscheid, U. M., Antranikian, G., and Muller, R. (1996) Appl.

Microbiol. Biotechnol. 46, 593–59621. Reinscheid, U. M., Bauer, M. P., and Muller, R. (1996) Biodegradation 7,

455–46122. Duffner, F. M., Reinscheid, U. M., Bauer, M. P., Mutzel, A., and Muller, R.

(1997) Syst. Appl. Microbiol. 20, 602–61123. Duffner, F. M., and Muller, R. (1998) FEMS Microbiol. Lett. 161, 37–4524. Duffner, F. M., Kirchner, U., Bauer, M. P., and Muller, R. (2000) Gene (Amst.)

256, 215–22125. Kendrew, S. G., Harding, S. E., Hopwood, D. A., and Marsh, E. N. (1995)

J. Biol. Chem. 270, 17339–1734326. Thibaut, D., Ratet, N., Bisch, D., Faucher, D., Debussche, L., and Blanche, F.

(1995) J. Bacteriol. 177, 5199–520527. Parry, R. J., and Li, W. (1997) J. Biol. Chem. 272, 23303–2331128. Gray, K. A., Pogrebinsky, O. S., Mrachko, G. T., Xi, L., Monticello, D. J., and

Squires, C. H. (1996) Nat. Biotechnol. 14, 1705–170929. Uetz, T., Schneider, R., Snozzi, M., and Egli, T. (1992) J. Bacteriol. 174,

1179–118830. Galan, B., Dıaz, E., Prieto, M. A., and Garcıa, J. L. (2000) J. Bacteriol. 182,

627–63631. Studier, F. W., Rosenberg, A. H., Dunn, J. J., and Dubendorff, J. W. (1990)

Methods Enzymol. 185, 60–8932. Fromm, H. J. (1979) Methods Enzymol. 63, 467–48633. Gurujeyalakshmi, G., and Oriel, P. J. (1989) Appl. Environ. Microbiol. 55,

500–50234. Bradford, M. M. (1976) Anal. Biochem. 72, 248–25435. Laemmli, U. K. (1970) Nature 227, 680–68536. Schagger, H., and von Jagow, G. (1987) Anal. Biochem. 166, 368–37937. de Jong, E., van Berkel, W. J. H., van der Zwan, R. P., and de Bont, J. A. M.

(1992) Eur. J. Biochem. 208, 651–65738. Mayhew, S. G., and Wassink, J. H. (1980) Methods Enzymol. 66, 217–22039. Eppink, M. H. M., Boeren, S. A., Vervoort, J., and van Berkel, W. J. H. (1997)

J. Bacteriol. 179, 6680–668740. Ellman, G. L. (1959) Arch. Biochem. Biophys. 82, 70–7741. Habeeb, A. F. S. A. (1972) Methods Enzymol. 25, 457–46442. Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller, W.,

and Lipman, D. J. (1997) Nucleic Acids Res. 25, 3389–340243. Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F., and Higgins,

D. G. (1997) Nucleic Acids Res. 25, 4876–488244. Sali, A., and Blundell, T. L. (1993) J. Mol. Biol. 234, 779–81545. Dauber-Osguthorpe, P., Roberts, V. A., Osguthorpe, D. J., Wolff, J., Genest, M.,

and Hagler, A. T. (1988) Proteins 4, 31–4746. Chiu, H.-J., Johnson, E., Schroder, I., and Rees, D. C. (2001) Structure 9,

311–31947. Laskowski, R. A., MacArthur, M. W., Moss, D. S., and Thornton, J. M. (1993)

J. Appl. Crystallogr. 26, 283–29148. Sippl, M. J. (1993) Proteins 17, 355–36249. van Berkel, W. J. H., Benen, J. A. E., and Snoek, M. C. (1991) Eur. J. Biochem.

197, 769–77950. Medina, M., Martınez-Julvez, M., Hurley, J. K., Tollin, G., and Gomez-Moreno,

C. (1998) Biochemistry 37, 2715–272851. van Berkel, W. J. H., and Muller, F. (1989) Eur. J. Biochem. 179, 307–31452. Chaiyen, P., Suadee, C., and Wilairat, P. (2001) Eur. J. Biochem. 268,

5550–556153. van den Berg, W. A. M., Hagen, W. R., and van Dongen, W. M. A. M. (2000)

Eur. J. Biochem. 267, 666–67654. Takizawa, N., Yokoyama, H., Yanagihara, K., Hatta, T., and Kiyohara, H.

(1995) J. Ferment. Bioeng. 80, 318–32655. Hubner, A., Danganan, C. E., Xun, L., Chakrabarty, A. M., and Hendrickson,

W. (1998) Appl. Environ. Microbiol. 64, 2086–209356. May, B. J., Zhang, Q., Li, L. L., Paustian, M. L., Whittam, T. S., and Kapur, V.

(2001) Proc. Natl. Acad. Sci. U. S. A. 98, 3460–346557. Waterfield, N. R., Bowen, D. J., Fetherston, J. D., Perry, R. D., and ffrench-

Constant, R. H. (2001) Trends Microbiol. 9, 185–19158. Beltrametti, F., Marconi, A. M., Bestetti, G., Colombo, C., Galli, E., Ruzzi, M.,

and Zennaro, E. (1997) Appl. Environ. Microbiol. 63, 2232–223959. Xi, L., Squires, C. H., Monticello, D. J., and Childs, J. D. (1997) Biochem.

Biophys. Res. Commun. 230, 73–7560. Xu, Y., Mortimer, M. W., Fisher, T. S., Kahn, M. L., Brockman, F. J., and Xun,

L. (1997) J. Bacteriol. 179, 1112–111661. Takami, H., Takaki, Y., and Uchiyama, I. (2002) Nucleic Acids Res. 30,

3927–393562. Vadas, A., Monbouquette, H. G., Johnson, E., and Schroder, I. (1999) J. Biol.

Chem. 274, 36715–3672163. Liepinsh, E., Kitamura, M., Murakami, T., Nakaya, T., and Otting, G. (1997)

Nat. Struct. Biol. 4, 975–97964. Xun, L., and Sandvik, E. R. (2000) Appl. Environ. Microbiol. 66, 481–48665. Louie, T. M., Xie, X. S., and Xun, L. (2003) Biochemistry, 42, 7509–751766. Filisetti, L., Fontecave, M., and Nivicre, V. (2003) J. Biol. Chem. 278, 296–30367. Eschenbrenner, M., Coves, J., and Fontecave, M. (1995) J. Biol. Chem. 270,

20550–2055568. Koike, H., Sasaki, H., Kobori, T., Zenno, S., Saigo, K., Murphy, M. E., Adman,

E. T., and Tanokura, M. (1998) J. Mol. Biol. 280, 259–27369. Fieschi, F., Nivicre, V., Frier, C., Decout, J. L., and Fontecave, M. (1995)

J. Biol. Chem. 270, 30392–3040070. Nivicre, V., Vanoni, M. A., Zanetti, G., and Fontecave, M. (1998) Biochemistry

37, 11879–1188771. Nivicre, V., Fieschi, F., Decout, J. L., and Fontecave, M. (1999) J. Biol. Chem.

274, 18252–1826072. Ingelman, M., Ramaswamy, S., Nivicre, V., Fontecave, M., and Eklund, H.

(1999) Biochemistry 38, 7040–704973. Louie, T. M., Yang, H., Karnchanaphanurach, P., Xie, X. S., and Xun, L. (2002)

J. Biol. Chem. 277, 39450–3945574. Lei, B., Liu, M., Huang, S., and Tu, S.-C. (1994) J. Bacteriol. 176, 3552–355875. Tanner, J. J., Lei, B., Tu, S.-C., and Krause, K. L. (1996) Biochemistry 35,

13531–1353976. Lei, B., and Tu, S.-C. (1998) Biochemistry 37, 14623–1462977. Jeffers, C. E., and Tu, S.-C. (2001) Biochemistry 40, 1749–175478. Jeffers, C. E., Nichols, J. C., and Tu, S.-C. (2003) Biochemistry 42, 529–53479. Low, J. C., and Tu, S.-C. (2003) Photochem. Photobiol. 74, 446–45280. DeLano, W. L. (2002) PyMol, DeLano Scientific, San Carlos, CA

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Ulrike Kirchner, Adrie H. Westphal, Rudolf Müller and Willem J. H. van BerkelComponent Monooxygenase with a Dual Role for FAD

A7, a Two-proteinBacillus thermoglucosidasiusPhenol Hydroxylase from

doi: 10.1074/jbc.M307397200 originally published online September 10, 20032003, 278:47545-47553.J. Biol. Chem. 

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