Vol. 176, No. 11JOURNAL OF BACrERIOLOGY, June 1994, p. 3368-33740021-9193/94/$04.00+0Copyright ©D 1994, American Society for Microbiology

Pseudomonas aeruginosa 142 Uses a Three-Component ortho-Halobenzoate 1,2-Dioxygenase for Metabolism of

2,4-Dichloro- and 2-ChlorobenzoateVLADIMIR ROMANOV" 2 AND ROBERT P. HAUSINGER2,3*

Institute for Biochemistry and Physiology of Microorganisms, Russian Academy of Science, Pushchino, Moscow Region142292, Russia,1 and Center for Microbial Ecology2 and Departments of Microbiology and Biochemistiy,3

Michigan State University, East Lansing, Michigan 48824

Received 10 December 1993/Accepted 25 March 1994

Cell extracts of Pseudomonas aeruginosa 142, which was previously isolated from a polychlorinatedbiphenyl-degrading consortium, were shown to degrade 2,4-dichlorobenzoate, 2-chlorobenzoate, and a varietyof other substituted ortho-halobenzoates by a reaction that requires oxygen, NADH, Fe(II), and flavin adeninedinucleotide. By using extracts that were chromatographically depleted of chlorocatechol and catechol1,2-dioxygenase activities, products of the initial reaction with 2,4- or 2,5-dichlorobenzoate and 2-chloroben-zoate were identified by mass spectrometry as 4-chlorocatechol and catechol. In contrast to the well-characterized benzoate dioxygenases or the recently described 2-halobenzoate 1,2-dioxygenase from P. cepacia2CBS (S. Fetzner, R Muller, and F. Lingens, J. Bacteriol. 174:279-290, 1992) that possess two proteincomponents, the P. aeruginosa enzyme was resolved by ion-exchange chromatography into three components,each of which is required for activity. To verify the distinct nature of this enzyme, we purified, characterized,and identified one component as a ferredoxin (Mr, -13,000) containing a single [2Fe-2S] Rieske-type cluster(electron paramagnetic resonance spectroscopic values ofg. = 1.82, gy = 1.905, and gz = 2.02 in the reducedstate) that is related in sequence to ferredoxins found in the naphthalene and biphenyl three-componentdioxygenase systems. By analogy to these enzymes, we propose that the P. aeruginosa ferredoxin serves as anelectron carrier between an NADH-dependent ferredoxin reductase and the terminal component of theortho-halobenzoate 1,2-dioxygenase. The broad specificity and high regiospecificity of the enzyme make it apromising candidate for use in the degradation of mixtures of chlorobenzoates.

Pseudomonas aen*ginosa 142, isolated from a polychlorinatedbiphenyl-degrading consortium, was recently shown to be able togrow on 2,4-dichlorobenzoate (24DCB) or 2-chlorobenzoate(2CB) as the sole source of carbon and energy (31); however, thebiodegradative pathways for these compounds in this microorgan-ism were not established. On the basis of precedents in theliterature, two reasonable pathways can be considered: a reduc-tive-hydrolytic mechanism and an oxidative mechanism for chlo-robenzoate degradation. Reductive dehalogenation of 24DCB inthe ortho position followed by hydrolysis of 4-chlorobenzoate(4CB) at the para position to yield p-hydroxybenzoate is sug-gested to occur in strain NTB-1 (a coryneform bacterium that wasmisidentified as Alcaligenes denitrificans) (39) and possibly inCorynebacterium sepedonicum KZ-4 (42). Reductive eliminationof chloride from the ortho position of 2CB also has beendescribed in anaerobic enrichment cultures (18), but nothing isknown about the mechanism of the dehalogenating reductase. Incontrast, an ATP- and coenzyme A-dependent 4CB hydrolasesystem from Pseudomonas sp. strain CBS3 has been characterized(7, 33). The inability of P. aeruginosa 142 to degrade 4CB (31),however, is most consistent with 24DCB metabolism by a differ-ent pathway. Oxidative elimination of chloride from the orthoposition of 24DCB and 2CB to yield 4-chlorocatechol and cate-chol offers an alternate possible degradative scheme for strain142. For example, degradation of 2CB to yield catechol has beendemonstrated in several Pseudomonas strains (5, 11, 14, 37). This

* Corresponding author. Mailing address: 160 Giltner Hall, Depart-ment of Microbiology, Michigan State University, East Lansing, MI48824. Phone: (517) 353-9675. Fax: (517) 353-8957. Electronic mailaddress: 23206MGR@MSU.EDU.

reaction is catalyzed by a two-component 2-halobenzoate 1,2-dioxygenase that is composed of an NADH-dependent oxi-doreductase flavoprotein and a terminal oxygenase (15). Thisenzyme has properties very similar to those of classical two-component benzoate dioxygenases (such as that from P. arvilla[40]) that catalyze the incorporation of both atoms of oxygen intothe substrate to form 3,5-cyclohexadiene-1,2-diol-1-carboxylate(30). Benzoate degradation requires a dehydrogenase for furthermetabolism, whereas the 2-halobenzoate 1,2-dioxygenase-cata-lyzed reaction yields an unstable intermediate that spontaneouslydegrades by decarboxylation coupled to chloride release to yieldcatechol. In Pseudomonas sp. strain CBS3 and other systems,however, additional or alternative oxidative products of 2CB,such as 2,3-dihydroxybenzoate (14) and 3-chlorocatechol (21, 22,37), also have been identified, indicating a lack of absoluteregiospecificity in these reactions. Finally, it was possible that P.aeruginosa 142 degrades 24DCB and/or 2CB by a novel pathwaythat has not been characterized.

Here, we establish the presence of a broad substrate speci-ficity and high-regiospecificity oxidative dehalogenation path-way involving an ortho-halobenzoate 1,2-dioxygenase as theprimary step for degradation of 24DCB and 2CB in P. aerugi-nosa 142. Further, we demonstrate that this is a three-compo-nent enzyme that includes a ferredoxin which is likely tofunction as an intermediate electron carrier.

MATERIALS AND METHODS

Growth conditions and preparation of cell extract. P. aerugi-nosa 142 was cultivated at 30°C by using minimal H medium(35) containing a 10 mM organic substrate concentration (5

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mM for 24DCB) in 2-liter Erlenmeyer flasks at 200 rpm or ina 10-liter fermentor (MG-114; New Brunswick Scientific, NewBrunswick, N.J.) at 400 rpm with air supplied at 2 liters/min.Cells were harvested at the end of the exponential phase bycentrifugation (8,000 x g) at 4°C, washed three times with 20mM potassium phosphate buffer (pH 7.5), and stored at-20°C. Frozen biomass was thawed and resuspended in 3volumes of 20 mM potassium phosphate buffer (pH 7.5)containing 15% (vol/vol) glycerol, 10% (vol/vol) ethanol, and 2mM dithiothreitol (termed PGE buffer), passed twice througha French pressure cell at 18,000 lb/in2, and centrifuged for 40min at 140,000 x g to obtain cell extracts.Enzyme assays. The rate of chlorobenzoate degradation in

cell extracts was assayed by using a high-pressure liquidchromatography (HPLC) method. The standard assay wascarried out at 30°C and contained, in a volume of 3.6 ml, 100mM Na * MES (morpholinoethanesulfonic acid) buffer (pH6.5), 0.1 mM Fe(NH4)2(SO4)2, 2 ,uM flavin adenine dinucle-otide (FAD), 2 mM NADH, the aromatic substrate at 1 mM,and 0.5 ml of the protein sample. At selected time points, thereactions were terminated by addition of sulfuric acid to a finalconcentration of 0.5 M and samples were extracted three timesby addition of equal volumes of ethyl acetate. The combinedextraction mixture was evaporated, dissolved in 0.2 ml ofmethanol, and subjected to reversed-phase HPLC on a Lichro-sorb C18 column (4 by 250 mm; Merck, Rahway, N.J.) by usinga 9% (vol/vol) solution of acetonitrile in 100 mM ammoniumacetate buffer (pH 5.5) as the eluent at a flow rate of 1 ml/min.The A230 was monitored, and the amount of the substrateremaining was determined by comparison to calibration curvesprepared with appropriate standards. Control experimentsincluded the use of mixtures that did not contain NADH,elimination of oxygen, and substitution of NADH by mixturescontaining 1 mM Mg * ATP and 0.2 mM coenzyme A.A spectrophotometric assay for measuring chlorobenzoate

dioxygenase activity was based on monitoring of the decreasein the NADH A340. The standard assay mixture (1 ml) con-tained 50 mM Na . MES buffer (pH 6.5), 0.1 mM Fe(NH4)2(SO4)2, 2 ,uM FAD, 0.2 mM NADH, and a 50- to 150-,uprotein sample. The reactions were started by addition of thecorresponding (halo)benzoate to a final concentration of 0.7mM. The reaction rate was corrected by subtracting theNADH oxidation rate found in the absence of the substrate.

Catechol 1,2-dioxygenase activity was assayed in 50 mMTris * HCl buffer (pH 7.5), in the presence of 10 mM EDTA bymeasuring the A257 and using an £257 of 17,300 M1 cm-1 forthe product of the reaction, cis-,cis-muconate (10). Similarly,for 4-chlorocatechol 1,2-dioxygenase we monitored the A267where the product, 3-chloro-cis,-cis-muconate, has an 6267 of18,300 M- cm-' (10). The assay was initiated by addition ofthe substrate to a final concentration of 100 jiM. Muconatecycloisomerase, dienelactone hydrolase, protocatechuate-3,4-dioxygenase, catechol 2,3-dioxygenase, and 4-hydroxybenzoate3-monooxygenase were assayed spectrophotometrically by es-tablished procedures (13, 16, 29, 32).

Metabolite identification experiments. Since a high level ofcatechol 1,2-dioxygenase activity would prevent accumulationof catechol intermediates, cell extracts were chromatographi-cally depleted of this enzyme. Cell extracts were subjected toMono Q HR 10/10 ion-exchange chromatography in PGEbuffer with elution at a flow rate of 0.5 ml/min and a linear100-ml gradient of increasing NaCl concentrations (0 to 0.5M). All fractions that were free of catechol 1,2-dioxygenaseactivity were pooled and concentrated by PM10 membrane(Amicon, Beverly, Mass.) filtration. The catechol dioxygenase-depleted cell extracts retained the ability to degrade chloro-

benzoates as measured by the HPLC method described above.From these reaction mixtures (containing 5.8 mg of protein perml), the reaction products were extracted three times by equalvolumes of ethyl acetate, evaporated, and derivatized by 20min of heating with 10 p1l of pyridine and 40 [lI of bis-(trimethylsilyl)-trifluoroacetamide containing 1% trimethyl-chlorosilane at 80°C. Analyses made use of an HP 5890 gaschromatograph coupled with a JEOL JMS AXSOSH massspectrometer using electron impact ionization. Three-microli-ter volumes of derivatized sample solutions were injected ontoa DB-SMS column (0.32 mm by 30 m; J & W Scientific,Folsom, Calif.) with an injector temperature of 300°C. Thecompounds were eluted with helium gas at a flow rate of 1ml/min and a 10°C/min temperature gradient starting at 50°C.Catechol and 4-chlorocatechol were derivatized in the samemanner and used as standards.

Resolution of chlorobenzoate dioxygenase into three poolsand component C purification. A 140-ml volume of cellextracts, prepared from 45 g (wet weight) of frozen biomasswas applied to a DEAE-Sepharose CL-6B column (2.5 by 15cm) equilibrated in PGE buffer at 4°C. The column was washedwith 150 ml of PGE buffer, and the proteins were eluted byusing a 400-ml gradient of increasing NaCl concentrations (O to0.6 M) in this buffer. (Alternatively, cell extracts were appliedto a Mono Q HR 10/10 column as described above.) Thisprocedure resolved the chlorobenzoate dioxygenase into threepools (designated components A, B, and C) and separatedthese components from two overlapping catechol 1,2-dioxyge-nase activities. Fractions containing component C activity(eluting at 0.35 to 0.40 M NaCl) were pooled, concentrated to2.3 ml, and chromatographed in three portions on a Superose12 column (1.6 by 50 cm) at 1.5 ml/min with PGE buffer atroom temperature. Fractions containing component C wereapplied to a Mono Q HR10/10 column in this same buffer andeluted by using a 100-ml linear gradient of increasing NaClconcentrations (0.15 to 0.40 M). Appropriate fractions wereconcentrated and applied to a Superose 12 column (1 by 30cm), and the proteins were eluted at 1 ml/min in PGE buffer.Component C characterization studies. Protein concentra-

tion was determined by using the commercial bicinchonic acidmethod (Pierce Chemical Co., Rockford, Ill.) or by the methodof Bradford (4) or Lowry et al. (25). The iron content ofsamples was determined after hydrolysis in 1 M nitric acidovernight, drying, dissolution in 50 mM HNO3, and analysis byusing a computer-interfaced Varian SpectraAA-400Z atomicabsorption spectrometer equipped with an autosampler, agraphite furnace, and Zeeman background correction. Theacid-labile sulfur content of samples was quantitated by a zincacetate method (3). Electrophoretic analysis of peptide molec-ular weights was carried out by the method of Laemmli (23)and by a protocol developed for small peptides that wasprovided by Sigma Chemical Co. (St. Louis, Mo.). Amino acidcompositional analysis and amino-terminal sequence determi-nation were carried out in the Macromolecular Structure,Sequence, and Synthesis Facility in the Biochemistry Depart-ment at Michigan State University. Untreated samples andperformic acid-oxidized samples were hydrolyzed in vacuo at110°C in 6 N HCl, the residues were converted to the phenyl-thiohydantoin-derivatized amino acids, and an HPLC proce-dure was used to obtain the amino acid composition (PICO-TAG system; Millipore, Milford, Mass.). Examination of therelationships between amino acid compositions made use ofthe method of Cornish-Bowden (8). N-terminal sequenceinformation was obtained by using an Applied Biosystems477A automated sequencer. Optical electronic spectra wererecorded on a Gilford Response spectrophotometer. Electron

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TABLE 1. Enzyme activities in cell extracts of P. aeruginosa 142

Activity (nmol/min/mg)a of:

Carbon source Benzoate 1,2-dioxygenase Catechol 1,2- Muconate cycloisomeraseduring growth dioxygenase Dienelactone

2GB 24 Ben- Cate- 4-Chloro- Mu t 2-Chloro- 3-Chloro- 2,4-Dichloro- hydrolaseDCB zoate chol catechol uconate muconate muconate muconate

2CB 4.7 4.3 0.2 285 60 353 31 45 23 6224DCB 1.0 1.4 0 114 48 73 228 227 88 152Benzoate 3.9 4.4 1.6 861 82.5 576 11 7 10 76Glutamate 0 0 0 <0.1 <0.1 12 NDb 16 25 53a Spectrophotometric assays are described in Materials and Methods.b ND, not determined.

paramagnetic resonance spectra were obtained on a BrukerER-200D spectrometer and recorded under the followingconditions: temperature, 10 K; scan time, 200 s; time constant,200 ms; scan range, 1,000 G; field set, 3,550 G; modulationfrequency, 9,468 GHz; power, 20 mW; receiver gain, 3.2 x 106.

RESULTS

Elucidation of the P. aeruginosa chlorobenzoate biodegrada-tive pathway. Cell extracts of P. aeruginosa 142 were found byHPLC analysis to be capable of degrading 24DCB and 2CB inthe presence of NADH (data not shown). Extracts lackingNADH but provided with Mg - ATP and/or coenzyme A failedto degrade these substrates. Furthermore, oxygen was found tobe absolutely required for decomposition of both substrates(data not shown).The level of benzoate 1,2-dioxygenase activity, as monitored

by a spectrophotometric assay, in cell extracts from 2CB- and24DCB-grown cells (Table 1) corresponded well to the re-ported growth rates of P. aeruginosa 142 on these substrates(doubling times of 3.3 and 13 h, respectively) (31). Althoughbenzoate serves as an excellent growth substrate (doublingtime of 2.2 h) (31), only low values of benzoate oxidation weredetected by this assay. This result can be explained by themasking effect of the next enzyme in the pathway (NAD-dependent 1,2-dihydroxy-1-carboxycyclohexadiene dehydroge-nase) that rereduces the NADH that is consumed in the first

1.0 r

0.8

0.6 ;

z 0.4 F

0.2 [

0.0 L

1.2

1.0

Ec 0.80

cOD

0.68e 0.40

.0< 0.2

0.00 10 20 30

Fraction number

0.10

0.08 c

0.06 E

0-04w*2

1:

0.02

0.0040 50

FIG. 1. Mono Q column chromatography of extracts from P.aeruginosa 142 cells grown on 2CB. The column was developed with agradient of increasing concentrations of NaCl (--- -) while monitoringthe A280 ( ). Activities of components A (0), B (0), and C (V)were determined in the presence of excess levels of the other compo-nents.

reaction. Measurements of activities for other key enzymes ofaromatic metabolism (Table 1) demonstrated the presence ofcatechol 1,2-dioxygenase, muconate cycloisomerase, anddienelactone hydrolase in cells grown on 2CB, 24DCB, andbenzoate. Interestingly, the catechol 1,2-dioxygenase and thebenzoate 1,2-dioxygenases were not induced in cells grownwith glutamate as the carbon source. These activities, as well asthat of muconate cycloisomerase, also were shown to be able totransform chlorinated substrates. It is important to note thatthe ratio of catechol 1,2-dioxygenase activity for catecholversus 4-chlorocatechol varies for extracts from cells grownwith different carbon sources (e.g., the ratios are 4.8, 2.3, and10 for cells grown on 2CB, 24DCB, and benzoate, respective-ly). These results are consistent with the presence of at leasttwo distinct enzymes (possibly with overlapping specificities)that appear to be regulated differentially. Indeed, we haveconfirmed published work (31) reporting that one enzyme witha greater affinity toward catechol and a second enzyme moreactive with 4-chlorocatechol can be resolved by ion-exchangechromatography of extracts from these cells (data not shown).The data for muconate cycloisomerase clearly indicate thepresence of two distinct enzymes: one enzyme appears to beinduced in the presence of 24DCB and has greater specificity

TABLE 2. Component and cofactor requirements forchlorobenzoate 1,2-dioxygenase activity'

SubstrateSubstrate Reaction mixture consumption Product found

(%)

2CB Complete 94 Catechol2CB Minus component A 42CB Minus component B 16 Catechol2CB Minus component C 02CB Minus NADH 02CB Minus Fe21 59 Catechol2CB Minus Fe2+ + 1 mM 0

EDTA2CB Minus FAD 54 Catechol24DCB Complete 69 4-Chlorocatechol25DCB Complete 29 4-ChlorocatecholBenzoate Complete 46 NDC

a Extracts from 2CB-grown P. aenrginosa 142 cells were resolved into threecomponents (A, B, and C) by Mono Q column chromatography as illustrated inFig. 1, and equal volumes of the indicated pools were incubated under standardassay conditions. The total protein concentration in each assay was 3 mg/ml.

b Substrate consumption was calculated as the percent difference between theconcentration of the substrate remaining after 24 h (the extended time periodallows easy measurement of the substrate concentration by the HPLC assay) andthe initial concentration (1 mM) for samples incubated at 30°G in the standardassay.'ND, not determined.

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PSEUDOMONAS AERUGINOSA HALOBENZOATE DIOXYGENASE 3371

TABLE 3. Activity of 2-halobenzoate 1,2-dioxygenase withdifferent substrates

Mean rate of NADH RelativeSubstrate' consumptionb activity

(p.M/min) + SD (%)

Benzoate 0.48 ± 0.19 372CB 1.32 + 0.19 1002-Bromobenzoate 1.41 + 0.17 1072-Iodobenzoate 1.72 ± 0.33 13024DCB 0.65 ± 0.13 502,5-Dichlorobenzoate 0.50 + 0.16 382-Chloro-5-bromobenzoate 0.65 + 0.13 502,6-Dichlorobenzoate 0.26 + 0.09 193,5-Dichlorobenzoate 0.21 + 0.13 163,4-Dichlorobenzoate 0.14 + 0.14 103-Chlorobenzoate 0.19 + 0.13 154CB 0.27 + 0.18 212-Amino-4-chlorobenzoate 0.37 ± 0.14 282-Amino-5-chlorobenzoate 0.35 + 0.18 274-Amino-2-chlorobenzoate 0.27 ± 0.24 212-Chloro-4-nitrobenzoate 0 0

a Substrates were provided at a concentration of 0.7 mM.b Rates were calculated by spectrophotometric assay for mixtures containing

70 p.1 of each component obtained by ion-exchange chromatography in a totalassay volume of 1.3 ml.

for chloromuconates, while a second enzyme appears to beinduced by growth on 2CB or benzoate and is more specific forunsubstituted muconate. The activity of dienelactone hydro-lase, an enzyme involved in chlorocatechol (but not catechol)metabolism, is two to three times higher in cells grown on24DCB than the uniformly lower activity levels found in cellsgrown on the other substrates. Catechol 2,3-dioxygenase, pro-tocatechuate 3,4-dioxygenase, and 4-hydroxybenzoate hydrox-ylase activities were not detected in P. aeruginosa 142 cellextracts.

Cell extracts that were depleted of catechol 1,2-dioxygenaseactivity were used for detection of the immediate products ofchlorobenzoate oxidation. Gas chromatography-mass spec-trometry analyses revealed that 2CB is oxidized to catechol and24DCB or 2,5-dichlorobenzoate is oxidized to 4-chlorocat-echol, as shown by comparison to trimethylsilyl derivatives ofauthentic standards. No adducts of 2,3-dihydroxybenzoate,dichlorocatechol, or 3-chlorocatechol were observed.

Resolution of chlorobenzoate 1,2-dioxygenase into threecomponents. The chlorobenzoate-dependent NADH oxidationactivity was separated by Mono Q chromatography into threecomponents, designated A, B, and C in order of elution (Fig.1). As shown by HPLC analysis of substrate consumption, eachcomponent is required for (chloro)benzoate 1,2-dioxygenaseactivity (Table 2). The limited substrate consumption observedin the absence of added component B is due to trace level

contamination of B in pool C. Ultrafiltration analysis of eachpool with a PM10 membrane (able to retain proteins with Mrsgreater than 10,000) established that each component was

likely to be proteinaceous. Pool A exhibited reductase activitywith 2,6-dichlorophenolindophenol in the presence of NADH(data not shown). Similar chromatographic patterns were

observed in experiments with cell extracts of 24DCB- andbenzoate-grown cells; hence, the same three-component en-

zyme may be induced by growth on each substrate.The cofactor requirements for chlorobenzoate 1,2-dioxyge-

nase are also shown in Table 2. EDTA completely inhibited thereaction, consistent with a divalent metal ion requirement forcatalysis. Addition of Fe2+ to the reaction mixture resulted inan increase in the reaction rate, suggesting that the requiredmetal ion is ferrous ion. Similarly, FAD stimulated the reac-

tion, consistent with involvement of FAD in the reaction.Flavin mononucleotide and riboflavin were unable to substi-tute for FAD (data not shown). Finally, NADPH was unable tosubstitute for NADH in the 2-halobenzoate oxidation reaction.

Substrate specificity of the three-component chlorobenzoate1,2-dioxygenase. The enzyme reconstituted from the threecomponents was able to oxidize a wide spectrum of halosub-stituted benzoates, as measured by the spectrophotometricassay (Table 3). Although substrate-dependent uncoupling ofNADH oxidation must be considered possible, several obser-vations from these preliminary data are worth noting. Theenzyme exhibits a higher level of activity with o-halobenzoatesthan with m- orp-substituted compounds. Monohalobenzoateswere found to be better substrates then dihalobenzoates.Among the ortho-halobenzoates tested, the enzymic ratesincreased with increasing size and decreasing electronegativityof the substituent (i.e., the rate increased in the order chloro-,bromo-, and iodobenzoate). Benzoates possessing amino sub-stituents at position 2 or 4 were also substrates, but 2-chloro-4-nitrobenzoate (tested as a possible chromophoric assaysubstrate) was not degraded by the enzyme.

Purification and characterization of component C. Startingwith cell extracts containing 4.6 g of protein, component C was

purified 481-fold to yield 2.8 mg of electrophoretically homo-geneous protein with a specific activity of 1.57 U/mg of protein(Table 4). Denaturing gel electrophoretic analysis revealedthat component C had an Mr of 13,000. Solutions of the proteinin the oxidized form were reddish brown (absorbance maximaat 321 [e321 = 7.8 mM-1 cm-1] and 455 [E455 = 3.5 mM 1

cm-1] nm in addition to a maximum at 278 nm), and thesample was bleached by addition of sodium dithionite (Fig. 2).The iron and acid-labile sulfide contents were determined tobe 1.64 molecules of iron and 2.1 molecules of sulfide permolecule of protein. Although the protein lacked an electronparamagnetic resonance signal in the oxidized form, a sodiumdithionite-reduced sample was found to possess an electronparamagnetic resonance signal with gx = 1.82, gy = 1.905, and

TABLE 4. Purification of ortho-halobenzoate 1,2-dioxygenase component C

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

Cell extract prepn 4,632 15.15 100 0.00327 1Chromatography on:DEAE-Sepharose CL-6B 115 33.26 220 0.29 89Superose 12 column (1.6 by 50 cm) 24.1 8.41 55.5 0.35 107Mono Q HR 10/10 6.19 6.39 42.2 1.03 316Superose 12 column (1 by 30 cm) 2.83 4.45 29.4 1.57 481

a 2CB 1,2-dioxygenase activity was measured in the presence of excess amounts of components A and B. One unit of activity is the enzyme amount needed to oxidize1 p.mol of NADH per min per mg of component C in the presence of 2CB.

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Co

0.2

300 350 400 450 500 550Wavelength (nm)

FIG. 2. Absorbance spectra of component C. The sample (1.23mg/ml) was examined as isolated ( ) and after reduction withsodium dithionite (- -).

g. = 2.02 (Fig. 3). The amino-terminal sequence of P. aerugi-nosa 142 component C was determined (Fig. 4). Amino acidanalysis revealed the protein to be composed of the followingresidues (assuming an Mr of 13,000): 12.1 D/N, 17.1 E/Q, 1.9 S,9.9 G, 3.9 H, 3.8 R, 4.1 T, 9.1 A, 5.5 P, 2.7 Y, 11.3 V, 1.8 M, 8.1I, 9.0 L, 4.2 F, 1.1 K, 2.9 C (on the basis of cysteic acid analysisafter performic acid oxidation), and -1 W (on the basis of thesequence shown in Fig. 4).

DISCUSSION

The P. aeruginosa 142 chlorobenzoate biodegradation path-way. We propose that P. aeruginosa 142 metabolizes substi-tuted ortho-halobenzoates by the pathway indicated in Fig. 5on the basis of (i) the requirement for both NADH and oxygenfor 2CB and 24DCB degradation in cell extracts as monitoredby HPLC; (ii) the failure to observe degradation in extractslacking NADH but containing Mg * ATP and coenzyme A; (iii)the presence of benzoate 1,2-dioxygenase activity capable oftransforming 2CB and 24DCB in extracts of benzoate-, 2CB-,and 24DCB-grown cells; (iv) the presence of catechol 1,2-dioxygenase activity able to utilize catechol and 4-chlorocat-echol, muconate cycloisomerase activity able to utilize mu-conate and chloromuconates, and dienelactone hydrolaseactivity in the same extracts; (v) the formation of catechol and4-chlorocatechol, but not 3-chlorocatechol or 3,5-dichlorocat-echol, from 2CB and 24DCB or 2,5-dichlorobenzoate in (chlo-ro)catechol 1,2-dioxygenase-depleted extracts; and (vi) theabsence of several other enzymes that participate in themetabolism of aromatic compounds by alternate pathways.Substituted ortho-halobenzoates are subjected to a highlyregiospecific 1,2-dioxygenase (not 1,6-dioxygenase) attack fol-lowed by spontaneous chloride release from the ortho positionaccompanying decarboxylation, yielding catechol or 4-chloro-catechol. The catechols are subjected to ortho cleavage, cata-lyzed by (chloro)catechol 1,2-dioxygenases, to form the corre-sponding muconates. Unsubstituted muconate is convertedto 3-oxoadipate enol-lactone by muconate cycloisomerase,whereas, 3-chloromuconate is transformed into the cis-dienelactone by action of chloromuconate cycloisomerase. The

2400

2200

*mCn 2000

1800

3200 3400 3600 3800Field (Gauss)

4000

FIG. 3. Electron paramagnetic resonance spectrum of dithionite-reduced component C. The signal illustrated is an average of ninescans for a sample at 0.75 mg/ml.

latter compound is hydrolyzed by dienelactone hydrolase toform maleylacetate. Such a pathway involving early dehaloge-nation of a chlorinated substrate is advantageous to theorganism because the downstream metabolic intermediatescan be readily degraded by enzymes that function in aromaticcompound degradation and the cell does not require separateenzymes that act on the chlorinated intermediates. The P.aeruginosa 142 pathway makes use of oxidative halide elimina-tion chemistry, similar to that previously reported for the2-halobenzoate 1,2-dioxygenase from P. cepacia 2CBS (15) andfor the 4-chlorophenylacetate 3,4-dioxygenase ofPseudomonassp. strain CBS3 (26). No evidence for chloride eliminationfrom the chlorobenzoates by either a 4CB hydrolase activity ora reductive reaction, as found in certain other microbes (7, 33,39, 42), was observed in P. aeruginosa 142.The P. aeruginosa 142 ortho-halobenzoate dioxygenase is a

three-component enzyme. The first enzyme in the pathway forchlorobenzoate metabolism in P. aeruginosa 142 is a three-component ortho-halobenzoate 1,2-dioxygenase. The substratespecificity of this enzyme is distinct from that of the similarlynamed P. cepacia 2CBS enzyme (15). Whereas the P. cepacia2CBS 2-halobenzoate 1,2-dioxygenase exhibits decreasing rel-

Component C NN- T E -- W D V L A A D ? V P E ? D V

NahAbBphA3BnzCTodB

NN-(M) T £ K W I 1 A \/ A 1 S D P .9 G I) V INH2--- M K F T R v C D R R D P £ G J A I

NH-(M) IT W T YI L R9 S - D L P I -- G JE M QN-(M) T W T Y L RQ -DLPP--G

FIG. 4. Comparison of the amino-terminal sequence of componentC to those of ferredoxins associated with three-component dioxygen-ases. The ferredoxin sequences shown are those from dioxygenasesystems involved in the oxidation of naphthalene (NahAb of P. putidaG7 [34]), biphenyl (BphA3 of P. pseudoalcaligenes Fl [38]), benzene(BnzC of P. putida NCIB 12190 [28]), and toluene (TodB of P. putidaFl [43]). The amino-terminal methionine residue is known to beremoved during synthesis of the naphthalene, benzene, and toluenedioxygenase ferredoxins (28, 34, 43), and this residue is enclosed inparentheses to indicate this processing. Residues that are identical tothose in P. aeruginosa component C are underlined and in boldface,conservative substitutions are underlined, and gaps are represented byhyphens.

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PSEUDOMONAS AERUGINOSA HALOBENZOATE DIOXYGENASE

COOH

0

OH

NX I

NADH

OH

OH 2

HX+ y °2CO2

COOH

V COOHHY05 COO/H

HOO 0

FIG. 5. Proposed pathway for ortho-halobenzoate metabolism in P. aeruginosa 142. X can be Cl, Br, or I, whereas Y at position 4 or 5can be H or Cl. Enzymes: 1, ortho-halobenzoate 1,2-dioxygenase; 2, (chloro)catechol 1,2-dioxygenase; 3, muconate cycloisomerase; 4,chloromuconate cycloisomerase; 5, dienelactone hydrolase.

ative activities for the series 2CB, 2-bromobenzoate, and2-iodobenzoate, the P. aeruginosa 142 (chloro)benzoate 1,2-dioxygenase appears to exhibit increasing relative activities forthis series. Also, the P. aeruginosa enzyme exhibits relativelygreater activity toward 24DCB (65% of that observed with2CB) than does the P. cepacia enzyme (9% of the 2CBoxidation rate). In addition, the P. aeruginosa 142 enzyme isdistinct in its number of required components from thetwo-component 2-chlorobenzoate 1,2-dioxygenase isolatedfrom P. cepacia 2CBS (15), as well as from the well-character-ized two-component benzoate 1,2-dioxygenases, such as thatfrom P. arvilla (40). These enzymes lack a separate ferredoxincomponent and are composed of only NADH-dependentreductase and terminal dioxygenase components. In contrast,P. aeruginosa 142 ortho-halobenzoate 1,2-dioxygenase compo-nent C appears to be a ferredoxin on the basis of the small sizeof this component, the presence of approximately two mole-cules of iron and inorganic sulfide per protein molecule, andthe spectroscopic properties of the oxidized and reducedspecies that are characteristic of a Rieske-type cluster'. Ferre-doxins containing such a [2Fe-2S] metallocenter are known tobe components in several three-component dioxygenase sys-tems (reviewed in reference 27), such as those found innaphthalene (20), biphenyl, benzene (9, 17, 28), and toluene(36) dioxygenases. Indeed, P. aeruginosa component C exhibitsa high degree of similarity to these ferredoxins at the amino-terminal sequence level (Fig. 4). Furthermore, the relatednessindices comparing the amino acid composition of strain 142ferredoxin with that of NahAb (34) meet the "strong" test ofrelatedness and similar comparisons to BphA3 (38), BnzC(28), or TodB (43) meet the "weak" test of relatedness (8).Hence, it is reasonable to conclude that, analogous to otherthree-component systems, P. aeruginosa 142 component Cfunctions as an intermediate electron carrier from an NADH-dependent ferredoxin reductase (likely to be component A onthe basis of its dichlorophenolindophenol oxidoreductase ac-tivity) and the terminal ortho-halobenzoate 1,2-dioxygenase(probably component B), neither of which has been purified.

It is interesting that until now, three-component dioxygen-ases were known to use only hydrophobic, uncharged sub-strates (naphthalene [12], benzene [1], toluene [41], biphenyl[19], dibenzofuran [6], etc.), whereas hydrophilic substratespossessing a charged side chain (benzoate [40], phthalate [2],4-chlorophenylacetate [26], 4-sulfobenzoate [24], etc.) wereoxidized by two-component dioxygenases. The P. aeruginosa142 enzyme demonstrates that this apparent dichotomy is not

universal. It is reasonable to speculate that the genes encodingthe P. aeruginosa 142 ortho-halobenzoate 1,2-dioxygenase areevolutionarily related to the gene clusters encoding thesethree-component dioxygenases. Indeed, preliminary DNA se-quence analysis of the genes encoding the oxygenase compo-nent of the P. aeruginosa 142 ortho-halobenzoate 1,2-dioxyge-nase has revealed greater similarities to the three-componentdioxygenase genes than to the two-component systems (38a).The broad specificity and high regioselectivity of the P.

aeruginosa 142 chlorobenzoate 1,2-dioxygenase make it apromising candidate for use in the degradation of mixtures ofchlorobenzoates, key products of polychlorinated biphenylmetabolism in soils.

ACKNOWLEDGMENTSWe thank Michelle Mac and John McCracken for assistance with

electron paramagnetic resonance spectroscopy, Doug Gage for help incollecting mass spectrometry data, Olga Maltseva for assistance withassays, Tamara Tsoi for helpful discussions, and Pat Oriel for sugges-tions regarding the manuscript.

This work was supported by the Michigan State Agricultural Exper-iment Station and the State of Michigan Research Excellence Funds-Hazardous Waste Bioremediation Program.

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