Vol. 174, No. 1JOURNAL OF BACTERIOLOGY, Jan. 1992, p. 279-2900021-9193/92/010279-12$02.00/0Copyright © 1992, American Society for Microbiology

Purification and Some Properties of 2-Halobenzoate 1,2-Dioxygenase, a Two-Component Enzyme System from

Pseudomonas cepacia 2CBSSUSANNE FETZNER,l* RUDOLF MULLER,2 AND FRANZ LINGENS1

Institut far Mikrobiologie der Universitat Hohenheim, Garbenstrafie 30, D-7000 Stuttgart 70,1 and Technische UniversitiatHamburg-Harburg, Arbeitsbereich Biotechnologie II, POB 901052, D-2100 Hamburg 90,2 Germany

Received 31 July 1991/Accepted 21 October 1991

The two components of the inducible 2-halobenzoate 1,2-dioxygenase from Pseudomonas cepacia 2CBS werepurified to homogeneity. Yellow component B is a monomer (Mr, 37,500) with NADH-acceptor reductaseactivity. Ferricyanide, 2,6-dichlorophenol indophenol, and cytochrome c acted as electron acceptors. Compo-nent B was identified as an iron-sulfur flavoprotein containing 0.8 mol of flavin adenine dinucleotide, 1.7 molof iron, and 1.7 mol of acid-labile sulfide per mol of enzyme. The isoelectric point was estimated to be pH 4.2.Component B was reduced by the addition of NADH. Red-brown component A (Mr, 200,000 to 220,000) is aniron-sulfur protein containing 5.8 mol of iron and 6.0 mol of acid-labile sulfide. The isoelectric point was withinthe range of pH 4.5 to 5.3. Component A could be reduced by dithionite or by NADH plus catalytic amountsof component B. Component A consisted of nonidentical subunits a (Mr, 52,000) and 1 (Mrs 20,000). Itcontained approximately equimolar amounts of a and (, and cross-linking studies suggested an a303 subunitstructure of component A. The NADH- and Fe2+-dependent enzyme system was named 2-halobenzoate1,2-dioxygenase, because it catalyzes the conversion of 2-fluoro-, 2-bromo-, 2-chloro-, and 2-iodobenzoate tocatechol. 2-Halobenzoate 1,2-dioxygenase exhibited a very broad substrate specificity, but benzoate analogswith electron-withdrawing substituents at the ortho position were transformed preferentially.

Chlorobenzoates are key intermediates in the degradativepathway of chlorobiphenyls (25). Due to their good watersolubility and low toxicity, chlorobenzoates are favorablemodel compounds for studying the degradation of haloge-nated aromatic substances. The ortho-substituted haloben-zoates are of special interest because they are more refrac-tory than the other isomers to biodegradation. Sterichindrance and the effect of chlorine atoms on the electrondensity at the ortho position of the benzene ring weresuggested to be responsible for the resistance of 2-haloben-zoates (other than 2-fluorobenzoate) to hydroxylation by thebenzoate dioxygenase system (50, 52).A number of reports describe the utilization of 2-chlo-

robenzoate by various Pseudomonas strains (20, 33, 62, 77).Sylvestre et al. (62) suggested an initial attack of 2-chlo-robenzoate by a 2-chlorobenzoate dioxygenase in Pseudo-monas sp. strain B-300. Engesser and Schulte (20) postu-lated a 2-chlorobenzoate 1,2-dioxygenase system catalyzingthe conversion of 2-chlorobenzoate to catechol in Pseudo-monas putida CLB 250. However, these authors did notdetect any 2-chlorobenzoate dioxygenase activity in cellextracts.Pseudomonas cepacia 2CBS utilizes 2-chlorobenzoate as

a sole source of carbon and energy. In the first step of2-chlorobenzoate degradation, 2-chlorobenzoate is con-verted to catechol, which is subject predominantly to meta-ring cleavage (23). The enzyme catalyzing the initial degra-dation step was shown to be a two-component dioxygenasesystem, previously termed 2-chlorobenzoate 1,2-dioxygen-ase (23).The benzoate dioxygenases of P. putida (arvilla) C-1 (74)

and P. putida B13 (27, 50, 51) and the isofunctional TOL

* Corresponding author.

plasmid-encoded enzyme toluate 1,2-dioxygenase from P.putida (arvilla) mt-2 (27, 38, 70, 72) are unable to oxygenate2-chlorobenzoate. Here we investigated the dehalogenating2-halobenzoate 1,2-dioxygenase from P. cepacia 2CBS tocompare these multicomponent enzyme systems.

MATERIALS AND METHODS

Materials. All chemicals were of the highest purity com-mercially available.Growth of P. cepacia 2CBS and preparation of crude

extracts. P. cepacia 2CBS (23) was grown in chloride-freemineral salts medium (22) containing 3.5 mM 2-chloroben-zoate as the sole source of carbon and energy. Large-scalegrowth of bacteria was carried out aerobically at 30°C in a200-liter fermenter, which was inoculated with eight 1-litercultures grown for about 26 h in Erlenmeyer flasks on arotary shaker. After 16, 27, and 40 h of fermentation,bacteria were fed with an additional 0.43 mol of 2-chloroben-zoate. During fermentation, the release of chloride ions from2-chlorobenzoate was monitored by measuring the chlorideconcentration in samples taken from the fermenter (Chlor-o-counter; Marius Instrumenten, Utrecht, The Nether-lands). Three hours after the last addition of growth sub-strate, cells were harvested by centrifugation, washed twicewith 50 mM sodium phosphate buffer (pH 7.2), and stored at-20°C until processed. This method yielded about 300 g (wetweight) of cells from 200 liters of medium.For the preparation of crude extracts, frozen cells were

thawed and suspended in 20 mM sodium phosphate buffer(pH 7.2). The cell suspension was sonified for 20 min at 260W with 0.6-s pulses every 0.4 s with cooling to 4°C (BransonSonifier 450; Branson Instruments, Danbury, Conn.). Cen-trifugation, first at 48,000 x g for 1 h and then at 80,000 x g

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for 2 h, at 4°C yielded the crude extracts from P. cepacia2CBS.

Purification of component A of 2-halobenzoate 1,2-dioxy-genase. All purification steps were performed at 4°C.

(i) Step 1: anion-exchange chromatography (23). The crudeextract from 100 g (wet weight) of cells was applied to aDEAE-cellulose DE-52 column (2.5 by 40 cm) that had beenequilibrated with 20 mM sodium phosphate buffer (pH 7.5).The column was washed with 800 to 1,000 ml of the samebuffer, and bound proteins were eluted with a linear gradientof K2SO4 (0 to 0.25 M in 20 mM sodium phosphate buffer[pH 7.5]; gradient volume, 900 ml) at a flow rate of 40 ml/h.Brown fractions contained one of the components of2-halobenzoate dioxygenase system (named component A).Fractions were assayed for this component at 460 nm. Thereductase activity of the second component of 2-haloben-zoate dioxygenase (named component B) was measured asNADH-dependent reduction of 2,6-dichlorophenol indophe-nol (DCPIP). Fractions containing component A were con-centrated by ultrafiltration.

(ii) Step 2a: gel filtration chromatography. The concen-trated pool A from step 1 was divided into two portions,which were then passed through a Sephadex G-150 column(2.5 by 95 cm; flow rate, 10 ml/h) that had been equilibratedwith 100 mM sodium phosphate buffer (pH 7.2). Fractionscontaining component A were pooled and rinsed with 5 mMsodium phosphate buffer (pH 6.9) in an ultrafiltration cell.

(iii) Step 3a: adsorption chromatography. Hydroxyapatitewas prepared by the method of Atkinson et al. (4). Thehydroxyapatite column (2.5 by 8 cm) was equilibrated with 5mM sodium phosphate buffer (pH 6.9). Component A fromstep 2a was applied to the column, which was then washedwith about 100 ml of the same buffer. Proteins were elutedwith a linear gradient of 5 to 200 mM sodium phosphatebuffer (pH 6.9) (gradient volume, 300 ml) at a flow rate of 25ml/h. Red-brown fractions containing component A werepooled, concentrated, and stored at -80°C.

Purification of component B of 2-halobenzoate 1,2-dioxy-genase. (i) Step 1: anion-exchange chromatography. Step 1was performed as described above for component A, exceptthat the crude extract from 140 g (wet weight) of cells wasloaded onto the column.

(ii) Step 2b: hydrophobic interaction chromatography. PoolB from step 1 was adjusted to 1 M (NH4)2SO4 and applied toa column of butyl Sepharose (2.5 by 9 cm) equilibrated with100 mM sodium phosphate buffer (pH 7.2) containing 1 M(NH4)2SO4. The column was washed with 250 to 300 ml ofthe same buffer, and bound proteins were eluted with a lineargradient from 100 mM sodium phosphate buffer-1 M(NH4)2SO4 (pH 7.2) to 5 mM sodium phosphate buffer (pH7.2) (gradient volume, 600 ml; flow rate, 25 ml/h). Fractionscontaining component B were pooled and rinsed with 5 mMsodium phosphate buffer (pH 6.9) in an ultrafiltration cell.

(iii) Step 3b: adsorption chromatography. The hydroxyap-atite column (1.7 by 12 cm) was equilibrated with 5 mMsodium phosphate buffer (pH 6.9). Component B from step2b was loaded onto the column. After the column waswashed with 100 ml of buffer, proteins were eluted with agradient of sodium phosphate buffer (5 to 100 mM [pH 6.9];gradient volume, 160 ml) at a flow rate of 25 ml/h. Yellowfractions containing component B were pooled, rinsed with20 mM sodium phosphate buffer (pH 7.0), and concentrated.For storage at -80°C, purified component B was mixed withglycerol to a final concentration of 20% (vol/vol) glycerol.

Analytical methods. (i) TLC. For thin-layer chromatogra-phy (TLC) analyses, plastic-backed Polygram SilG/UV254

sheets and SiIG-25/UV254 glass plates were used (Macherey& Nagel, Duren, Germany). The mobile phases consisted ofthe following: (i) n-butanol-acetic acid-water (4:1:4, vol/vol/vol) (54); (ii) 5% aqueous Na2HPO4, saturated with isoamylalcohol (63): (iii) tertiary amyl alcohol-formic acid-water(3:1:1, vol/vol/vol) (63); and (iv) toluene-acetic acid (4:1,vol/vol). For the detection of catechol and catechol deriva-tives, the TLC sheets were sprayed with nitrite-molybdatereagent (3).

(ii) HPLC. Reversed-phase high-pressure liquid chroma-tography (HPLC) was performed with a Lichrospher RP18column (Bischoff, Leonberg, Germany). Solvent deliverysystem model 2200 (Bischoff) and UV spectrophotometricdetector SPD-6A and an integrator model C-RlB (ShimadzuCorp., Tokyo, Japan) were used. Flavins were separated byusing 0.1 M formic acid-0.1M ammonium formate-17%(vol/vol) methanol as the eluent at a flow rate of 0.8 ml/min(45).

Determination of metal content. The metal contents ofcomponents A and B were determined by using X-rayfluorescence spectrometry (system 77; Finnigan Interna-tional Inc.). For calibration, standards of FeSO4 were pre-pared. Buffer samples were used for background correc-tions. The iron content of the purified enzyme componentswas also determined by using the iron-chelating reagentFerene S [3-(2-pyridyl)-5,6-bis(2-(5-furylsulfonic acid))-1,2,4-triazine, disodium] as described by Haigler and Gibson(28). A standard curve for calibration of the Ferene S-ironcomplex was obtained by using stock iron solutions.

Determination of acid-labile sulfide. Acid-labile sulfide wasextracted from proteins by zinc acetate treatment and deter-mined by the formation of methylene blue as described byBeinert (8). Spinach ferredoxin was used as a positivecontrol.

Identification of the flavin cofactor and determination offlavin content. The flavin cofactor was extracted from com-ponent B by boiling for 10 min, and protein was removed byultrafiltration. The flavin cofactor in the yellow filtrate wasidentified by TLC with solvents i, ii, and iii. Solutions offlavine adenine dinucleotide (FAD), flavin mononucleotide(FMN), and riboflavin were used as references. The FADcontent of component B was determined spectrophotomet-rically in protein-free ultrafiltrates from boiled component B(E4¼o0 11,30 M-1 cm-1; E375, 9,300 M-1 cm-' [68]). TheFAD content was also determined by HPLC with UVdetection at 265 nm. For calibration of the system, FADstandards were prepared. The identity of the flavin cofactorfrom component B was confirmed by cochromatography onthe RP18 column with authentic references FAD, FMN, andriboflavin.

Determination of NH2-terminal amino acid sequences. TheNH2-terminal amino acid sequence of component B wasdetermined with a protein sample lyophilized in 20 mMsodium phosphate buffer (pH 7.0). The NH2-terminal aminoacid sequences of the subunits of component A were ob-tained with protein bands blotted from sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gelsonto Immobilon membranes. Semidry electroblotting wasperformed as recommended by Pharmacia (48), except thatthe Immobilon membrane was immersed first in methanol(wetting solution) and then in water before equilibration inanode buffer. Proteins were subjected to automated Edmandegradation in a 471A gas-phase protein sequencer (AppliedBiosystems, Weiterstadt, Germany).

Electrophoresis. Purification of components A and B wasmonitored by SDS-PAGE (55). Slab gels used were as

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follows (T denotes the total percentage concentration of bothmonomers [acrylamide and bisacrylamide] and C denotesthe percentage concentration of the cross-linker relative tothe total concentration T): separating gels, 10% T and 3% C;stacking gels, 4% T and 3% C. Components A and B werealso subjected to nondenaturing PAGE. Gel systems withthe following buffers were used: 7.7% T, 2.6% C (pH 7.5)separating gel and 3.1% T, 29% C (pH 5.5) stacking gel (43);8.3% T, 3.6% C (pH 8.3) separating gel and 3.1% T, 3.5% C(pH 6.8) stacking gel (modified from reference 66); and 8.3%T, 3.6% C (pH 8.8) separating gel and 3.1% T. 20% C (pH6.8) stacking gel (66). After the gels were fixed in 12.5%(wt/vol) trichloroacetic acid, they were stained routinely in0.2% (wt/vol) Coomassie brilliant blue R250 dissolved inaqueous 50% (vol/vol) methanol-10% (vol/vol) acetic acidand destained in 20% (vol/vol) methanol-10% (vol/vol) aceticacid.

Cross-linking experiments. To study the subunit structureof component A, subunits of oligomeric component A werecross-linked with dimethyl suberimidate as described byDavies and Stark (19). Under the conditions used (1.0 mg ofcomponent A per ml and 1.0 to 15.0 mg of dimethyl suber-imidate per ml in 0.2 M triethanolamine hydrochloride [pH8.5]), cross-links were formed within the oligomers of com-ponent A but not between oligomers. After denaturation, thecross-linked peptides were resolved by SDS-PAGE with an8 to 18% polyacrylamide gradient gel (49).

Isoelectric focusing. Analytical isoelectric focusing wasperformed in rehydrated gels as recommended by Pharmacia(47). Carrier ampholytes of pH 2.0 to 11.0 and pH 2.5 to 5.0were used. The formation of the pH gradient was monitoredby the aid of marker proteins. Polyacrylamide gels withimmobilized pH gradients (pH 3.5 to 5.0 and pH 4.0 to 6.0)were prepared as recommended by Pharmacia (46).

Determinations of Mr. The Mrs of peptides under denatur-ing conditions were estimated by using SDS-PAGE. The Mrof native component A was estimated by gel filtrationthrough a Sephadex G-200 column (2.5 by 95 cm; flow rate,7 ml/h) and a Superose 12 HR 10/30 column (flow rate, 9ml/h). The calibration proteins were ferritin (Mr, 440,000),catalase (Mr, 232,000), gamma globulin (Mr, 169,000), bovineserum albumin (Mr, 67,000), ovalbumin (Mr, 43,000), andcx-chymotrypsinogen A (Mr, 25,000). The Mr of component Bwas estimated by gel filtration through a Sephadex G-75column (2.5 by 95 cm; flow rate, 25 ml/h) and Superdex 200prep grade 16/60 (flow rate, 40 ml/h). The calibration proteinswere bovine serum albumin, ovalbumin, DNase I (Mr,30,000), a-chymotrypsinogen A, and cytochrome c (Mr,12,500). The void volumes of the columns were estimatedwith blue dextran 2000. In all cases, 100 mM sodiumphosphate buffer-0.1 M NaCl (pH 7.0) was used as theeluent.

Determination of sedimentation coefficients. Before ultra-centrifugation, components A and B were dialyzed against20 mM sodium phosphate buffer (pH 7.0). The dialysis bufferwas used as the reference solution in the ultracentrifugeruns. Sedimentation velocity runs were carried out in aBeckman model E analytical ultracentrifuge. Scanning wasdone at 280 nm. The sedimentation coefficients were calcu-lated as described by Chervenka (17).

Photometric determinations and absorption spectra. Forphotometric determinations, a PMQ III spectrophotometer(Zeiss, Oberkochen, Germany) was used. Absorption spectrawere recorded with an Uvikon 810 or Uvikon 930 spectro-photometer (Kontron Instruments, Eching, Germany).

Determination of protein concentration. Protein concentra-

tions were estimated routinely by the method of Lowry et al.(40) with bovine serum albumin as the standard. For quan-titative determination of flavin, metal, and acid-labile sulfide,purified protein components were lyophilized and weighedagainst freeze-dried buffer blanks.Enzyme assays. (i) Assays for NADH-acceptor reductase.

All assays were performed at 25°C. NADH-acceptor reduc-tase activity was routinely assayed as DCPIP reduction. Thedecrease in A600 was monitored, and an r60 of 21,000 M-'cm1 (2) was used for the calculations. The reaction mixturecontained 20 mM sodium phosphate buffer (pH 8.0), 0.1 mMDCPIP, 0.4 mM NADH, and 0.2 to 2.0 p.g of component B.The reaction was started by the addition of the protein.NADH-acceptor reductase activity was also measured as thereduction of cytochrome c (r550, 21,000 M-1 cm-' [42]),potassium hexacyanoferrate-Ill (ferricyanide) (r420, 1,020M-1 cm-' [57]), and 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2H-tetrazolium chloride (r503, 19,300 M-1 cm-' [6]).The concentrations used were as follows: 0.1 mM cytochromec, 0.2 mM ferricyanide, and 0.2 mM 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2H-tetrazolium chloride. One unit ofreductase activity was defined as the amount of enzyme thatreduced 1 pimol of electron acceptor per min.

(ii) Assays for 2-halobenzoate 1,2-dioxygenase. The activityof 2-halobenzoate 1,2-dioxygenase was assayed at 25°Cspectrophotometrically at 340 nm as NADH consumption(r340, 6,300 M` cm-1 [9]) and polarographically as oxygenuptake with a Clark-type oxygen electrode (YSI 4004; Yel-low Springs Instrument Co.). The reaction was optimized fortemperature, pH, and buffer and for the concentrations ofbuffer, Fe2+, NADH, FAD, and 2-chlorobenzoate. The 50mM succinate buffer (pH 6.5) contained 0.14 mM(NH4)2Fe(SO4)2, 0.2 mM NADH, 2 ,uM FAD, and suitableamounts of components A and B. The reaction was startedby the addition of 0.2 mM 2-chlorobenzoate. All measure-ments were corrected for endogenous NADH or 02 con-sumption recorded in the absence of a substrate. The re-sponse of NADH consumption and oxygen uptake as afunction of protein concentration was nonlinear (23). There-fore, the protein concentration in the enzyme assay wasadjusted arbitrarily to a fixed value to allow comparisons tobe made. To estimnate the activity of component B in thedioxygenase test, the assay contained 0.7 mg of componentA per ml and 10 pLg of component B per ml, corresponding toabout a 12-fold molar excess of component A. To estimatethe activity of component A, the assay contained 0.25 mg ofcomponent A per ml and 0.15 mg of component B per ml,corresponding to about a fourfold molar excess of compo-nent B. This method ensured that the component to bemeasured was rate limiting. One unit of dioxygenase activitywas defined as the amount of enzyme that consumed 1 ,umolof NADH or 1 ,umol of oxygen per min.The reaction of 2-halobenzoate 1,2-dioxygenase was con-

firmed by HPLC measurement of 2-chlorobenzoate con-sumption, by colorimetric measurement of catechol produc-tion, and by measurement of chloride release from2-chlorobenzoate as described previously (23). The substraterange of 2-halobenzoate 1,2-dioxygenase was investigatedby measuring substrate-dependent oxygen uptake andNADH consumption. To exclude false-positive results dueto uncoupling of oxygen uptake and uncoupling of electrontransfer from substrate oxygenation, formation of hydrogenperoxide was monitored with catalase added to the polaro-graphic dioxygenase assay. Here the assay contained limit-ing amounts of NADH, and catalase was added after thetermination of the reaction as described by White-Stevens

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TABLE 1. Purification of 2-halobenzoate 1,2-dioxygenasecomponents A and B

Purification Protein U Recovery Sp act Purificationstep (mg) (%) (U/mg) (fold)

Component A"Crude extract 9,380DEAE-cellulose DE-52 1,216 183 100 0.15 1Sephadex G-150 135 72 39 0.53 3.5Hydroxyapatite 91 24 13 0.26 1.7

Component BbCrude extract 13,200DEAE-cellulose DE-52 768 122 100 0.16 1Butyl Sepharose 4B 51 119 97 2.33 15Hydroxyapatite 13 52 42 4.00 24

" Samples of 100 g (wet weight) of cell paste were used.b Samples of 140 g (wet weight) of cell paste were used.

and Kamin (71) to detect 02 liberation from hydrogenperoxide.To demonstrate the enzymatic conversion of substrates,

assay mixtures were incubated at 25°C for 30 min and thenacidified with HCI; the precipitated proteins were removedby centrifugation. The supernatants were extracted twicewith ethylacetate, and substrate disappearance was moni-tored by TLC with solvent iv. Reference assays containingall ingredients of the test system except components A and Bwere treated the same way.

RESULTSPurification of components A and B of 2-halobenzoate

1,2-dioxygenase. The results of a typical purification proce-dure are summarized in Table 1. The activity of componentA was measured in the presence of excess amounts ofcomponent B and vice versa, as described in Materials andMethods.Because of high endogenous NADH consumption and

oxygen uptake in the crude extracts, enzyme activity wasnot detectable by the photometric and polarographic assays.The specific activity of 2-halobenzoate 1,2-dioxygenase incrude extracts estimated by HPLC analysis of 2-chloroben-zoate consumption was 8.5 mU/mg (23). A 100-g (wetweight) cell sample yielded about 90 mg of component A,and 13 mg of component B was isolated from 140 g (wetweight) of cells.

2-Chlorobenzoate was not converted by crude extractsfrom cells grown in complex medium. When the crudeextract from such cells was resolved by anion-exchangechromatography on the DEAE-cellulose column, no enzymecomponents were detected. We thus propose that 2-haloben-zoate 1,2-dioxygenase is an inducible enzyme system.

Purified component B retained 85% of its initial activityafter storage at -20 and -80°C for 2 weeks in the presenceof glycerol (20%, vol/vol), compared with 30% activity leftwhen component B was stored without additives under thesame conditions. Sucrose (20%, wt/vol) also had stabilizingeffects. After storage with sucrose at -20°C for 2 weeks,75% of the initial component B activity was left. ComponentA retained about 50% of its activity after storage in 20%(vol/vol) glycerol, compared with 15 to 20% activity leftwhen component A was stored in buffer without additives.The addition of dithioerythritol (0.5, 1, or 5 mM) or FAD(0.05 mM, in the case of component B) to the storage bufferdid not stabilize the enzyme components.

I1~X, 0V .0

0

FIG. 1. PAGE of native component A. Lanes: 1, 7.7% T, 2.6% C(pH 7.5) (see Materials and Methods); 2, 8.3% T, 3.6% C (pH 8.3);3, 8.3% T, 3.6% C (pH 8.8). The arrows indicate the margins of theseparating gels.

The homogeneity of purified components A and B wasmonitored by PAGE. PAGE of freshly prepared componentA in native gels with three different buffer systems revealeda single band after staining with Coomassie blue (Fig. 1).When homogeneous component A stored for a few days at-20°C was subjected to native PAGE, three to five bandswere observed. Purified component A showed two majorbands on SDS-PAGE; these bands correspond to the nonidentical subunits a and a (Fig. 2). Component B obviouslyformed aggregates under nondenaturing PAGE, resulting ina seemingly inhomogeneous multibanded pattern (data notshown). However, SDS-PAGE of component B under re-ducing and denaturing conditions gave a single band (Fig. 3).

Physical and chemical properties. (i) Mr values and sedi-mentation coefficients. Gel filtration of the native proteincomponents resulted in MrS of 200,000 (Superose 12) and220,000 (Sephadex G-200) for component A and 38,000(Sephadex G-75) and 37,500 (Superdex 200) for componentB. The Mr of component B determined with SDS-PAGEunder reducing conditions was 37,000 to 37,500. Thus, wepropose this component to be a monomer. The Mrs of thenonidentical subunits of component A were estimated as

FIG. 2. SDS-PAGE of component A. Lanes: 1, purified compo-nent A; 2, Mr standard I, containing phosphorylase b (94,000),bovine serum albumin (67,000), ovalbumin (43,000), carbonic anhy-drase (30,000), trypsin inhibitor (20,100), and ct-lactalbumin(14,400). The arrows indicate the margins of the separating gel.

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FIG. 3. SDS-PAGE of purified componentstandard I (Fig. 2); 2, purified component B.

B. Lanes: 1. Mr'

51,000 to 53,000 (a subunit) and 19,000 to 21,000 (3 subunit)with SDS-PAGE. For components A and B, sedimentationcoefficients (s20.s) of 10.3 and 3.2S, respectively, werecalculated.

(ii) Subunit composition of component A. Integration ofdensitometric scans of Coomassie blue-stained gels revealeda molar subunit al/ ratio of 0.84:1, based on the Mrs of52,000 and 20,000.When oligomeric component A was cross-linked with

dimethyl suberimidate and analyzed by SDS-PAGE, thepattern of protein bands shown in Fig. 4 was obtained. TheMrs of these bands were estimated to be 20,000, 40,000,52,000, 88,000 108,000, 117,000, 126,000, 145,000, 155,000,169,000, 178,000, and 197,000. The values tentatively sug-gested that these bands might correspond to P, 12, a, a42,a2, a3, a21, %a21, a3, a23, a3, and a312, respectively.Based on the molar ratio of about 1:1 of the subunits andbased on the Mr of native component A and on the cross-linking data, a subunit structure of a3P3 is proposed. Somepreparations of component A used for the cross-linkingexperiments contained two minor protein bands at Mr 26,000and Mr 28,000 (Fig. 4) that did not participate in thecross-linking reaction. These protein bands are formed bycleavage of the large subunit (a), since they were not visiblein fresh preparations but occurred during storage of compo-nent A. The addition of the protease inhibitor phenylmeth-ylsulfonyl fluoride to the purification buffers failed to preventthe formation of these peptides. Heat-induced bond cleavageduring sample preparation for SDS-PAGE does not explainthis effect, because the results were the same for samplesprepared with and without heating and with and withoutreducing agents like dithioerythritol or mercaptoethanol.

(iii) Isoelectric points. The pl of purified component B waspH 4.2. The gels revealed one single band that stained forprotein. During isoelectric focusing of component A, whichappeared homogeneous as judged by PAGE and SDS-PAGE, component A resolved as a cluster of bands withinthe range of pH 4.5 to 5.3. The formation of multiple bandshas been observed repeatedly as an artifact due to complexformation of a single protein with carrier ampholytes (16, 24,31, 36). The protein species with multiple isoelectric pointsmight also be due to differential dissociation of subunits orloss of iron rather than to the binding of carrier ampholytes(32). Furthermore, it was not possible to determine the pl ofcomponent A with polyacrylamide gels with immobilized pHgradients without carrier ampholytes, because component Aprecipitated at the application point before entering the gel.

FIG. 4. Cross-linking of component A with dimethyl suberimi-date. Lanes: 1, 2. and 3, cross-linked component A (the arrowsindicate the protein bands explained in the text); 4, Mr standard I(Fig. 2); 5, M, standard Il, containing thyroglobulin (330,000),ferritin (220,000 and 18,500), bovine serum albumin (67,000), cata-lase (60.000), and lactate dehydrogenase (36,000); 6, component Awithout the cross-linking reagent.

Precipitation of the protein probably was due to the lowconductivity of the carrier ampholyte-free gels.

(iv) Absorption spectra. In the oxidized state, componentA had an intensive red-brown color. The UV-visible absorp-tion spectrum of oxidized component A showed maxima at279, 325, and 462 nm and a shoulder at 550 nm. The A279/A462ratio was 11.5 with homogeneous component A. Uponreduction with sodium dithionite, component A bleachedand the spectrum changed (Fig. 5).Component A could be reduced enzymatically by the

addition of NADH and catalytic amounts of component B,but the addition of NADH alone did not reduce componentA. These characteristics are very similar to the propertiesrecorded for the oxygenase components of other dihydrox-ylating two-component enzyme systems, especially to theoxygenases of benzoate dioxygenase (74, 75), o-phthalatedioxygenase (7), and 4-chlorophenylacetate 3,4-dioxygenase(41).

Solutions of purified component B were red-yellow incolor with absorption maxima at 272, 343, and 457 nm andshoulders at 390, about 425, and 550 nm in the oxidized state(Fig. 6). The ratios of the A272 to the A343, A390, and A457were 3.9, 4.0, and 3.4, respectively. Similar data wereobserved for the reductase component of the benzoate1,2-dioxygenase system (73). The addition of a small amountof sodium dithionite resulted in total bleaching of componentB, and the maxima disappeared (Fig. 6). In contrast tocomponent A, component B was reduced directly by theaddition of NADH. This finding indicates that component Bbinds NADH and acts as an NADH-acceptor reductase.

(v) Flavin and iron-sulfur contents. The absorption spec-trum of material extracted from boiled component B wasvery similar to that of FAD, and this tentative identification

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284 FETZNER ET AL.

Waveegth [nmlFIG. 5. Absorption spectra of 1.6 mg of component A per ml in

20 mM sodium phosphate buffer (pH 7.0): oxidized form:reduced with dithionite; ........ reduced with NADH

and catalytic amounts of component B.

was confirmed by TLC with various mobile phases and bycochromatography with authentic references FAD, FMN,and riboflavin in HPLC analysis. The flavin recovered in theheat-prepared extract showed the same chromatographicbehavior as FAD in all chromatographic systems.The amount of FAD determined in different preparations

varied from 0.6 to 0.9 mol of FAD per mol of reductase. Wesuggest that 1 mol of reductase contains 1 mol of FAD, butthe reductase protein of the 2-halobenzoate 1,2-dioxygenasesystem might have lost part of its flavin cofactor during thepurification procedure.Loss of the flavin cofactor in the course of purification of

the reductase also has been reported for the reductasecomponents of toluene dioxygenase (60) and naphthalenedioxygenase (28). Homogeneous component B contained 1.6to 1.8 mol of iron and 1.5 to 1.8 mol of acid-labile sulfide permol of component B. We propose that the reductase com-ponent contains one [2Fe-2S] cluster. Iron and acid-labilesulfide analysis of component A from different preparationsrevealed the presence of 5.4 to 6.1 mol of iron and 5.8 to 6.3mol of acid-labile sulfide per mol of oxygenase component.We tentatively suggest that component A contains three[2Fe-2S] centers, i.e., one [2Fe-2S] cluster per aot subunit.

(vi) NH2-terminal amino acid sequences. The NH2-terminalamino acid sequence determined for component B wasMet-Leu-(Cys)-Ser-Ile-Ala-Leu-Arg-Phe-Glu- Asp- Asp- Val-Thr-Tyr-Phe-Ile-Thr-Ser-(Cys)-. The amino acid sequencesof the NH2 termini of the subunits of component A were asfollows: a, Ser-Thr-Pro-Leu-Ile-Ala-Gly-Thr-Gly-Pro-Ser-Ala-Val-(Cys)-Gln-Leu-Ile-Ser-Asn-Ala-; IS, (Thr/Met)-Ser-Leu-Glu-Ser-Ser-Tyr-Leu-Asp-Val-Val-Ala-Phe-Ile-. (Theresidues within parentheses have not been confirmed.)

Catalytic properties. (i) Artificial electron acceptors. Com-ponent B was identified as the reductase component of the2-halobenzoate 1,2-dioxygenase system by its NADH-ac-ceptor reductase activity with artificial electron acceptors.Specific activities of 2, 76, 237, and 320 U/mg of protein wereobserved with 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl-

_aelwh 1M11FIG. 6. Absorption spectra of 1.1 mg of component B per ml in

20 mM sodium phosphate buffer (pH 7.0): , oxidized form;----- reduced with dithionite.

2H-tetrazolium chloride, DCPIP, cytochrome c, and ferricy-anide, respectively. Component A showed no oxidoreduc-tase activity with these acceptors.NADPH was ineffective as an electron donor for the

reductase (less than 1% activity with NADPH in the DCPIPreductase assay compared with the activity with NADH).

Since component A was reduced in the presence ofNADH and NADH-acceptor reductase, an electron transferis proposed from NADH via component B to component A,which is considered as the oxygenase component of the2-halobenzoate 1,2-dioxygenase system.

(ii) Cofactor requirements. Several cofactors were exam-ined for their effects on 2-halobenzoate 1,2-dioxygenaseactivity. The enzyme system required components A and B,NADH, and exogenous Fe2+ for activity. NADPH could notreplace NADH. The addition of 2 ,uM FAD (and, to a lesserextent, the addition of FMN) led to an increase in activity(Table 2). As mentioned above, component B presumablylost part of its flavin cofactor during the purification proce-dure. We suggest that exogenous FAD added to the assaysystem might replace the missing FAD cofactors.None of the metal cations examined (Fe3+, Ca2 , Mg2+,

Mn2+, and Co2+; 5, 50, and 500 ,uM each) could replaceFe2+. However, the addition of 50 p.M Ni2+, Zn2+, or Cu2+resulted in 25, 40, or 100% inhibition of activity, respec-tively, compared with the activity without any metal.

(iii) Inhibitors. The effects of various compounds onDCPIP reduction by component B and on 2-halobenzoate1,2-dioxygenase activity are summarized in Table 3. Al-though these test systems are not directly comparable, sincethe NADH-DCPIP-reductase assay contained much lessprotein than did the dioxygenase assay, we examined bothreductase and dioxygenase activities for the effect of puta-tive inhibitors.

All reactions were started by the addition of 2-chloroben-zoate to assay mixtures preincubated for 2 min with eachcompound listed, and the initial reaction velocities weredetermined. Sodium azide at 2 and 4 mM showed no effect.

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P. CEPACIA 2-HALOBENZOATE 1,2-DIOXYGENASE 285

TABLE 2. Requirement of components A and B and cofactorsfor the activity of 2-halobenzoate 1,2-dioxygenase

RelativeConstituents of the assay mixture activity

(%c)

A, B, NADH, Fe2+," ......................100A, NADH, Fe2+..................... 0B, NADH, Fe2+ ..................... 0A, B, NADHa ..................... <10"A, B, NADPH, Fe2+ab..................... <1A, B, NADH, Fe2+, riboflavin ..................... 100A, B, NADH, Fe2 , FMNb ..................... 115A, B, NADH, Fe2+, FADb ..................... 130

" Determined with excess amounts of component B.b Determined with excess amounts of component A.' The relative activity without Fe2+ ions added to the assay mixture varied

from 3 to 10% in different preparations of component A.

The metal-chelating agents EDTA, 2,2-dipyridyl, and 1,10-phenanthroline did not influence reductase activity in theconcentrations tested, but 2-halobenzoate 1,2-dioxygenaseactivity was severely inhibited. Similarly, the reductaseactivity, unlike the dioxygenase activity, was not affected by1 to 2 mM KCN. Substances that inhibit sulfhydryl groups(N-ethylmaleimide, iodoacetate, and p-chloromercuriben-zoate) affected both NADH-DCPIP-reductase activity and 2-halobenzoate 1,2-dioxygenase activity. Similarly, the reduc-tase components of benzoate 1,2-dioxygenase (73) and4-methoxybenzoate monooxygenase (12) were inhibited bysulfhydryl reagents, whereas metal-chelating agents ap-peared to have no significant effect.

(iv) Km values. From Lineweaver-Burk plots, the apparentKms of component A for the reductase and for 2-chloroben-zoate were estimated as approximately 22 and 23 ,uM,respectively. The apparent K,,,s of component B for DCPIPand NADH were calculated as approximately 94 and 79 p.M,respectively.

(v) Substrate stoichiometry. Polarographic determinationsof 02 uptake with limiting concentrations of 2-chloroben-zoate or NADH revealed a 1:1:1 stoichiometry for 2-chlo-robenzoate-02-NADH.

(vi) Substrate specificity. The substrate range of 2-haloben-zoate 1,2-dioxygenase was investigated by measuring sub-strate-dependent 02 uptake and NADH consumption and bymonitoring substrate disappearance with TLC. In all assays,excess amounts of component B were used. The relativerates of dioxygenation of a number of substituted benzoateanalogs and other aromatic compounds are given in Table 4.The results obtained with the polarographic assay corre-sponded to the reaction rates measured with the spectropho-tometric assay. When catalase was added to the assaymixtures at the end of the reaction, the evolution of oxygenwas not detected polarographically. For all compounds thatcaused 02 uptake and NADH consumption, substrate disap-pearance and the appearance of product(s) were observedwith TLC. These data indicate that it was rather unlikely thatsome of the substrate analogs tested uncoupled electrontransfer from substrate hydroxylation. Oxygen uptake due tosubstrate-dependent uncoupling of electron transfer ratherthan true oxygenase activity has been reported for a numberof oxygenases, e.g., 4-methoxybenzoate monooxygenase(10, 11, 67), phthalate dioxygenase (7), and salicylate hy-droxylase (71).

2-Halobenzoate 1,2-dioxygenase exhibited a very broadsubstrate specificity (Table 4). All substituted analogs of

TABLE 3. Effects of various compounds on NADH-DCPIPreductase activity and on 2-halobenzoate 1,2-dioxygenase activity"

91 Inhibition of activityReagent Concn

(M) NADH-DCPIP 2-Halobenzoatereductase 1,2-dioxygenase

Sodium azide 2 x 10-' 0 04x10-3 0 0

KCN 1 x 10-3 0 152 x 10-3 0 40

o-Phenanthroline 2 x 10-4 NDh 95 x 10-4 0 100

2,2'-Dipyridyl 2 x 10-4 ND 61 x 10-3 0 100

EDTA 2 x 10-5 ND 62 x 10-4 ND 871 x 10-3 0 100

lodoacetate 5 x 10-4 12 192 x 10-3 50 55

N-Ethylmaleimide 2 x 10-4 13 452 x 10-3 55 100

p-Chloromercuribenzoate 2 x 10-7 25 ND2 x 106 70 05 x 10-6 100 17

"The protein concentration in the NADH-DCPIP reductase assay was 2,ug/ml. For measuring 2-halobenzoate 1,2-dioxygenase activity, 800 p.g ofcomponent A per ml and 10 ,ug of component B per ml were used.

" ND, not determined.

benzoate examined were transformed, except for the dihy-droxybenzoates. Aromatic compounds like benzene, chlo-robenzene, toluene, phenol, and chlorophenylacetate iso-mers, which lack a carboxyl group directly attached to thearomatic ring, did not serve as substrates. Some oxygenases,e.g., toluene dioxygenase (65), catalyze the oxidation oftrichloroethylene. Trichloroethylene oxidation, however, isnot a common property of broad-specificity microbial oxy-genases (64). When 2-halobenzoate 1,2-dioxygenase activitywas assayed with trichloroethylene as the substrate, nooxygen uptake was observed. Benzoate analogs with elec-tron-withdrawing substituents like halogens at the orthoposition were converted at considerable rates by 2-haloben-zoate 1,2-dioxygenase, especially when the substituent wasnot too bulky.

2-Fluoro-, 2-chloro-, 2-bromo-, and 2-iodobenzoate (andalso anthranilate) were transformed to catechol. Catecholwas identified by using TLC and cochromatography withauthentic catechol in HPLC analyses as described previ-ously (23).

Since the two-component enzyme system we isolatedcatalyzes the conversion of all 2-halobenzoates to catechol,we suggest the designation 2-halobenzoate 1,2-dioxygenase(1,2-hydroxylating, dehalogenating, and decarboxylating).

DISCUSSION

The first step in the microbial degradation of an aromatichydrocarbon like benzene or benzoate typically is catalyzedby a multicomponent dioxygenase, which yields a stabledihydrodiol derivative. For instance, 3,5-cyclohexadiene-1,2-diol-1-carboxylic acid is formed from benzoate in adioxygenase-catalyzed reaction (53). The rearomatization ofsuch a dihydrodiol derivative to the corresponding vicinaldiphenol requires the activity of an NAD+-dependent dehy-drogenase.The two-component 2-halobenzoate 1,2-dioxygenase sys-

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TABLE 4. Substrate specificity of 2-halobenzoate 1.2-dioxygenase"

RelativeSubstrate analog activity Reaction product(s)(%a)

2-Chlorobenzoate 100 Catecholh2-Fluorobenzoate 105 Catecholt2-Bromobenzoate 78 Catecholh2-Iodobenzoate 38 Catecholh2-Aminobenzoate 90 Catechol"2-Methoxybenzoate 55 Catechol"2-Hydroxybenzoate 12 Catechol'2-Methylbenzoate 44 o-Cresol'Benzoate 45 3,5-Cyclohexadiene-1,2-

diol-1-carboxylicacid'

2-Chloro-4-fluorobenzoate 130 +"2,4-Difluorobenzoate 100 +2,4-Dichlorobenzoate 9 +2-Amino-5-chlorobenzoate 60 +2,6-Difluorobenzoate 30 +2-Chloro-6-fluorobenzoate 10 +3-Chlorobenzoate 35 +4-Chlorobenzoate 8 +3,4-Difluorobenzoate 25 +3-Hydroxybenzoate 15 2,5-Dihydroxybenzoate'4-Hydroxybenzoate 17 Hydroquinone'2,3-, 2,4-, 2,5-, 3,4-. or 3,5- <2 -,

dihydroxybenzoateNaphthalene-1-carboxylic acid 8 +Naphthalene-2-carboxylic acid 3 +

" The following compounds were not converted: N-formylanthranilate.N-acetylanthranilate, 2-chlorobenzamide, o-phthalate, 2-. 3-. and 4-chlo-rophenylacetate, benzene, chlorobenzene, toluene, 2-chlorotoluene. 1-chloro-2-methoxybenzene, phenol, 2-aminophenol, biphenyl, and naphthalene."Products were tentatively identified with TLC.The reaction product was identified by thermal decomposition (pH 2,

50'C) to salicylate and phenol, which were identified with TLC.dThe formation of a reaction product(s) was shown with TLC.The product was identified with TLC and gas chromatography-mass

spectroscopy.f"No product shown with TLC.

tem from P. cepacia 2CBS catalyzes the formation ofcatechol from 2-fluoro-, 2-chloro-, 2 bromo-, and 2-iodoben-zoate. With gas chromatography-mass spectrometry of anextract of an enzyme assay, no intermediate was detected inthe conversion of 2-chlorobenzoate to catechol (23). Wetentatively suggest that the hypothetical dihydrodiol inter-mediate 2-chloro-3,5-cyclohexadiene-1,2-diol-1-carboxylicacid is highly unstable and, in the course of an energeticallyfavorable rearomatization, spontaneously loses carbon diox-ide and halogenide (Fig. 7). Thus, no dehydrogenase activityis required to yield the aromatic product catechol. Thedioxygenation reaction seems to be highly regioselective,since 6-chloro-3,5-cyclohexadiene-1,2-diol-1-carboxylic acidand 3-chlorocatechol were not detectable in the enzymeassays or in the culture supernatant of strain 2CBS (23). Ananalogous scheme was proposed for the dehalogenative4-chlorophenylacetate 3,4-dioxygenase from Pseuidomonassp. strain CBS3 (41, 58), the desulfonative 4-sulfobenzoate3,4-dioxygenase from Comamonas testosteroni T-2 (39), andthe anthranilate dioxygenase from Pseudomonas sp. (37).Components A and B of 2-halobenzoate 1,2-dioxygenase

were purified to apparent homogeneity and partially charac-terized. Only an about twofold purification of component Awas achieved, and we failed in developing a more productivepurification procedure. We assume that component A lost

COO-

NADH NAWy

[0,/VoL i

OH

sportareous ,OH

C02+X-

FIG. 7. Proposed mechanism for the dihydroxylation reactionscatalyzed by 2-halobenzoate 1,2-dioxygenase. x, = F, Cl, Br, l, orNH,.

activity mainly because of subunit dissociation of the nativeoligomer and loss of iron. Subunit dissociation yieldingvarious ot3,P oligomers possibly produced the multiplebanding observed in isoelectric focusing gels. This wouldalso explain the formation of a number of bands fromhomogeneous component A when frozen and thawed proteinsamples were subjected to native PAGE. In addition, theproposed fission of the ox subunit, which may cause theformation of two additional bands in SDS-PAGE, maycontribute to the activity losses.

In Table 5, 2-halobenzoate 1,2-dioxygenase is comparedwith some other multicomponent enzyme systems. A com-mon feature of these enzyme systems is the use of a shortelectron transport chain to catalyze electron transfer fromNAD(P)H to a terminal iron-containing oxygenase compo-nent. Two redox centers, a flavin and a ferredoxin, areemployed in most systems described, except for naphthalenedioxygenase, which as a unique case uses a flavin and twoiron-sulfur clusters to transfer electrons from the externaldonor to the oxygenase component. In two-componentsystems, flavin and a [2Fe-2S] center both are localized onthe monomeric reductase. Systems containing three compo-nents mostly use a flavoprotein-type reductase and a smallferredoxin-type protein to transfer the electrons from thedonor to the terminal oxygenase component. All oxygenasesdescribed so far are iron-sulfur proteins (Table 5).Among the multicomponent enzyme systems listed in

Table 5, 2-halobenzoate 1,2-dioxygenase obviously appearsto be very similar to benzoate 1,2-dioxygenase from P.pittida (ar'ilIa) C-1 (73-75) (Table 6). The oxygenase com-ponents of these two enzyme systems exhibit almost identi-cal absorption spectra, and the MrS of the native oxygenasesand of the subunits are very similar. For both oxygenases, anLL433 subunit structure is proposed.

In the oxygenase component of benzoate 1,2-dioxygenase,about 8 mol of iron and 6 mol of acid-labile sulfide werefound, indicating the presence of three [2Fe-2S] clusters andadditional iron (75). Apart from the cluster-bound iron,additional iron also was detected in phthalate dioxygenase(7) and naphthalene dioxygenase (21). In the case of theoxygenase component of the 0-demethylating 4-methoxy-benzoate monooxygenase (putidamonooxin), this additionalmononuclear nonheme iron was demonstrated to mediatethe electron transfer from the Rieske-type [2Fe-2S] cluster tomolecular oxygen, thus activating the dioxygen as iron-peroxo complex. [FeO2]+ as the active oxygenating speciesin the active site of putidamonooxin is proposed to attack thesubstrate to yield the 0-demethylated product (1, 14, 15).However, in component A of 2-halobenzoate 1,2-dioxy-

genase, we detected approximately equimolar amounts ofiron and acid-labile sulfide. In none of the preparations wasadditional iron found. If component A also had containedmononuclear iron, this iron cofactor must have been com-pletely lost in our preparations.

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P. CEPACIA 2-HALOBENZOATE 1,2-DIOXYGENASE 287

TABLE 5. Properties of some multicomponent enzyme systems

Oxygenase componentReductase component

Enzyme system Ferredoxin Mr (1)SubunitMr(103) Flavin Fe/S NvMre1bs) structure

Mr (10') cofactor center Native Subunit(S)

Two-component systems4-Sulfobenzoate 3,4-dioxygenase (39) 36 FMN 12Fe-2S] None 85-105 50 Oa24-Methoxybenzoate monooxygenase (11-13) 42 FMN [2Fe-2S] None 126 40 a34-Chlorophenylacetate-3,4-dioxygenase (41, 58) 35 FMN [2Fe-2S] None 140-144 46-52 a3o-Phthalate dioxygenase (7) 33-34 FMN [2Fe-2S] None 217 58 a4Benzoate-1,2-dioxygenase (73-75) 37-38.3 FAD [2Fe-2S] None 201 50, 20 aA332-Halobenzoate 1,2-dioxygenase (this study) 37-38 FAD [2Fe-2S] None 200-220 52, 20 a,3P3

Three-component systemsBenzene dioxygenase (5, 18, 26, 78) 60/81 FAD None 12 168/215 54.5, 23.5 (X2P2Toluene 2,3-dioxygenase (59-61) 46 FAD None 15.3 151 52.5, 20.8 a212Pyrazon dioxygenase (54) 67 FAD None 12 180 NR" NRNaphthalene dioxygenase (21, 28, 29) 36.3 FAD [2Fe-2S] 13.6 158 55, 20 at212Toluene-4-monooxygenase (69) NR (FAD) NR 23 >500 50, 32 NR

" NR, not reported.

2-Halobenzoate 1,2-dioxygenase required exogenousFe2+ for activity. Similarly, the activities of a number ofmulticomponent enzyme systems, e.g., toluene dioxygenase(76), benzene dioxygenase (5, 26), o-phthalate dioxygenase(7), 4-chlorophenylacetate 3,4-dioxygenase (41, 58), and4-sulfobenzoate 3,4-dioxygenase (39), depend on the pres-ence of Fe2+ ions. In contrast, enzyme systems like ben-zoate dioxygenase or naphthalene dioxygenase, which obvi-ously retain the mononuclear iron cofactor completelyduring the purification procedure, do not require Fe2+ ionsfor activity (21, 74). The dependency of enzyme activity onexogenous Fe2+ tentatively might be explained as the re-placement of mononuclear iron lost in the course of thepurification procedure by the Fe2+ ions added (7, 26, 39).Not only the oxygenases but also the reductase compo-

nents of benzoate 1,2-dioxygenase and 2-halobenzoate 1,2-dioxygenase appear to be structurally related (Tables 5 and6). Both reductases belong to the class of FAD- and [2Fe-2S]-containing reductases, in contrast to the reductases ofother two-component enzyme systems listed, which areFMN-containing iron-sulfur flavoproteins (Table 5). More-over, the reductases of benzoate and 2-halobenzoate 1,2-dioxygenase seem to have almost identical Mrs and pIs (73)(Table 6).Although benzoate 1,2-dioxygenase from P. putida (ar-

villa) C-1 and 2-halobenzoate 1,2-dioxygenase from P. cepa-cia 2CBS appear to be structurally related, our resultsindicate that they differ fundamentally in their substratespecificities. Benzoate 1,2-dioxygenase catalyzes the dioxy-genation of benzoate and various benzoate analogs withsubstituents in the meta position. Benzoate analogs withortho and para substituents were converted with low reac-

tion rates. For instance, relative activities of 11, 1, and 25%were observed for this enzyme with 2-chlorobenzoate,2-methoxybenzoate, and anthranilate, respectively (the ac-tivity with benzoate is taken as 100%) (74).The TOL plasmid-encoded toluate 1,2-dioxygenase from

P. pitida (arvilla) mt-2 is isofunctional with benzoate diox-ygenase. This enzyme system has not been characterized,but genetic data indicate a multicomponent structure similarto those of the benzoate and 2-halobenzoate dioxygenasesystems (30). Besides, the reductase component of thechromosomally encoded benzoate dioxygenase from P.plitida (arvilla) C-1 was shown to be immunologically ho-mologous with the xvlD gene product (toluate dioxygenase)from P. pitida (arvilla) mt-2 (44). Toluate 1,2-dioxygenase isconsidered to be an enzyme system with broad substratespecificity, since a number of meta- and/or para-substitutedbenzoate analogs are converted to the corresponding dihy-drodiol derivatives (27, 38, 50, 51, 72). However, benzoateanalogs substituted in the ortho position do not serve assubstrates. 2-Halobenzoate 1,2-dioxygenase has an ex-tremely broad substrate range, but it shows a markedpreference for ortho-substituted benzoate analogs, espe-cially with electron-withdrawing substituents.The NH,-terminal amino acid sequence we obtained for

component B of 2-halobenzoate 1,2-dioxygenase was com-pared with the amino-terminal sequences reported for thereductases of naphthalene dioxygenase (28) and 4-sulfoben-zoate 3,4-dioxygenase (39) and with the amino acid se-quences derived from genetic data for P4 protein frombenzene dioxygenase (35), the nahA gene product (56), andthe todA gene product (79). The NH2-terminal sequences ofthe subunits of component A were compared with the NH2

TABLE 6. Comparison of some properties of benzoate 1,2-dioxygenase from P. piutida (arvilla) C-1 (73-75) and of 2-halobenzoate1,2-dioxygenase from P. cepacia 2CBS

Content (mol/mol of enzyme) AbsorptionEnzyme Component s20, ,,. (S) pI maimIron Sulfide FAD maxima

Benzoate 1,2-dioxygenase Oxygenase 10.0 8.2 5.9 4.5 279, 325, 464Reductase 3.3 2.1 1.7 1.0 4.2 273, 340, 402, 467

2-Halobenzoate 1,2-dioxygenase Oxygenase 10.3 5.4-6.1 5.8-6.3 4.5-5.3 279, 325, 462Reductase 3.2 1.6-1.8 1.5-1.8 0.6-0.9 4.2 272, 343, 457

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terminus of the oxygenase component of 4-sulfobenzoate3,4-dioxygenase (39), with the gene products P1 and P2 frombenzene dioxygenase genes (35), and with todCIC2 geneproducts (79). The NH2 terminus of 2-halobenzoate 1,2-dioxygenase was also compared with the NH2-terminalsequence predicted for the first gene product of the xvID(toluate dioxygenase) operon (34). However, we did not findany homologies between the NH2-terminal sequence of the2-halobenzoate 1,2-dioxygenase components and the se-quences reported for the other multicomponent enzymesystems mentioned above.Our results indicate that oxygenolytic dehalogenation of

2-halobenzoates is catalyzed by a new two-component,broad-specificity 2-halobenzoate 1,2-dioxygenase, whichseems to be structurally related to benzoate 1,2-dioxygenasefrom P. putida (arvilla) C-1 and to toluate 1,2-dioxygenasefrom P. putida (arvilla) mt-2 but is not identical with theseenzyme systems.

ACKNOWLEDGMENTS

We thank A. Merz for technical assistance. We are grateful to B.Hauer, BASF AG. Ludwigshafen, Germany, for performing NH,-terminal amino acid sequence analyses. Thanks are also due to H.Schreiber for recording X-ray fluorescence spectra and to K.Kapassakalis for fermentation. We thank K.-H. van Pee for criticalreading of the manuscript.

This work was supported by grant 0319416 AO from theBundesministerium fur Forschung und Technologie, Germany.

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