JouRNAL o0 BACTURIOLIGY, Mar. 1976, p. 985-998Copyright 0 1976 American Society for Microbiology

Vol. 125, No. 3Printed in U.S.A.

Metabolism of Resorcinylic Compounds by Bacteria:Alternative Pathways for Resorcinol Catabolism in

Pseudomonas putidaPETER J. CHAPMAN AND DOUGLAS W. RIBBONS*

Department ofBiochemistry, College ofBiological Sciences, University of Minnesota, St. Paul, Minnesota55108, and Department ofBiochemistry, University ofMiami School of Medicine, and Howard Hughes

Medical Institute, Miami, Florida 33152*

Received for publication 6 October 1975

Two strains of Pseudomonas putida isolated by enrichment cultures withorcinol as the sole source ofcarbon were both found to grow with resorcinol. Dataare presented which show that one strain (ORC) catabolizes resorcinol by ametabolic pathway, genetically and mechanistically distinct from the orcinolpathway, via hydroxyquinol and ortho oxygenative cleavage to give maleylace-tate, but that the other strain (01) yields mutants that utilize resorcinol. Onemutant strain, designated 010C, was shown to be constitutive for the enzymesof the orcinol pathway. After growth of this strain on resorcinol, two enzymes ofthe resorcinol pathway are also induced, namely hydroxyquinol 1,2-oxygenaseand maleylacetate reductase. Thus hydroxyquinol, formed from resorcinol, un-dergoes both ortho and meta diol cleavage reactions with the subsequent forma-tion of both pyruvate and maleylacetate. Evidence was not obtained for theexpression of resorcinol hydroxylase in strain 01OC; the activity of orcinolhydroxylase appears to be recruited for this hydroxylation reaction. P. putidaORC, on the other hand, possesses individual hydroxylases for orcinol andresorcinol, which are specifically induced by growth on their respective sub-strates. The spectral changes associated with the enzymic and nonenzymicoxidation of hydroxyquinol are described. Maleylacetate was identified as theproduct of hydroxyquinol oxidation by partially purified extracts obtained fromP. putida ORC grown with resorcinol. Its further metabolism was reducednicotinamide adenine dinucleotide dependent.

The microbiological dissimilation of natu-rally occurring and synthetic benzenoid deriva-tives has been extensively studied, and the ma-jor metabolic routes from these to common cel-lular metabolites have been clarified (6). Alarge group of 1,3-dihydroxybenzene com-pounds are formed as secondary plant products(15), but studies on the microbial catabolism ofthese resorcinols have been limited. Since, withrare exceptions, the presence of at least twohydroxyl groups in the benzene nucleus is anecessary condition for enzymic ring cleavage,and the substitution pattern of these so fardescribed is 1,2 and/or 1,4 (1), it was of interestto know how microorganisms catabolize 1,3-dihydroxybenzenes. Ofthe simple mononuclearresorcinols, only resorcinol and orcinol (3,5-dih-ydroxytoluene) catabolism by pseudomonadshave been documented in preliminary commu-nications (P. Larway and W. C. Evans, Bio-chem. J. 95:52P, 1965). Larway and Evans con-cluded that their pseudomonad introduced athird hydroxyl group into resorcinol to give hy-droxyquinol, and that this was the substrate for

an ortho ring cleavage enzyme. The product ofring cleavage had the absorption spectral char-acteristics of maleylacetate, and it was sug-gested that fumarate and acetate were formedafter isomerization and hydrolysis (Fig. 1). Inthe accompanying paper (2), we present evi-dence that orcinol is catabolized by other flu-orescent pseudomonads via the intermediate2,3,5-trihydroxytoluene, but that this com-pound undergoes a meta cleavage between car-bon atoms 1 and 2, with consequent formationof acetate and pyruvate by two successive hy-drolytic reactions of the ring fission product(Fig. 2).We have previously observed that orcinol-

grown cells of two strains ofPseudomonas pu-tida were also able to oxidize resorcinol and,reciprocally, resorcinol-grown cells oxidized or-cinol. It was not known whether orcinol andresorcinol were nonspecific substrates of theenzymes of a single metabolic route that couldtransform both compounds or whether orcinoland resorcinol specifically induce enzymes ofseparate catabolic pathways for their own ca-

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986 CHAPMAN AND RIBBONS

HO OH

OH

HO OH

Resorcinol Hydroxyquinol

COOHCOOH HOOC

0

MaleylacetateFIG. 1. Resorcinol catabolism by Pseudomonas sp. (Larway and Evans, Biochem. J. 95:52P, 1965).

CH3

HO © OHORCINOL

4 a

CH3IDOH

HO O-I H

TRIHYDROXYTOLUENE

CH3

COOH

ACETATE

HO 2 OHRESORCINOL

a'

",OH

HO..,

OH

HYDROXYQUINOL

bJ

CHOCOOH

w c~0; COOH \o'AJXo FORMATE

ACETYLPYRUVATE

IdCH3 COOH

+ gICOOH ,CO

CH3

FIG. 2. Metabolic sequences for resorcinol catabolism used by P. putida ORC and P. putida 010C. Lightarrows indicate enzymes specifically induced for resorcinol catabolism. Heavy arrows indicate the constitutiveenzymes of the orcinol pathway present in P. putida OlOC, which are also induced in P. putida ORC and 01

by orcinol and which catalyze pyruvate formation from resorcinol. (a) Orcinol hydroxylase; (a') resorcinolhydroxylase; (b) 2,3,5-trihydroxytoluene 1,2-oxygenase; (c) 2,4,6-trioxoheptanoate hydrolase; (d) acetylpyru-vate hydrolase; (e) hydroxyquinol 1,2-oxygenase; (f) maleylacetate reductase.

tabolism, either of which might show overlap-ping specificity for the analogous substratesand intermediates. The present study is con-

cerned with this question. Evidence for the ex-istence of both possibilities is provided by astudy of these two different strains ofP. putida.

MATERIALS AND METHODSStrains of bacteria. Two strains of P. putida des-

ignated 01 (by D. W. Ribbons) and ORC (by P. J.Chapman) were obtained independently by us by

streaking orcinol enrichments on selective agar me-dia and isolating single colonies. P. putida O1OCwas selected from strain 01 by streaking on mineralsalts agar supplemented with resorcinol as the car-bon source. All strains were maintained on nutrientagar slants. Zymomonas mobilis was obtained fromthe American Type Culture Collection (ATCC no.10988) and used as a source of pure glucose 6-phos-phate dehydrogenase (unpublished observations).An unidentified species of Bacillus isolated with

4-hydroxyphenylpropionate as the sole carbon source

(R. L. Crawford and P. J. Chapman, Abstr. Annu.

COOH

+ COOHCH3

eCOOH

lo+< COOH

MALEYLACETATE

IfCOOH

COOH/3.- KETOADIPATE

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RESORCINOL CATABOLISM IN P. PUTIDA 987

Meet. Am. Soc. Microbiol. 1975, 03, P. 192) wasused for the preparation of extracts used in experi-ments on hydroxyquinol autooxidation.

Growth of bacteria. Liquid cultures of P. putidawere obtained by inoculating mineral media of thefollowing composition (g/liter): KH2PO., 5.4;(NH.)2504, 1.2; MgSO .7H20, 0.2; FeSO4'7H2O, 0.01;and NaOH to pH 6.8 to 7.1. The carbon sources usedwere orcinol and resorcinol (0.05-0.1%) and succi-nate (0.1%). Cultures were usually incubated at 30to 33 C for 16 to 20 h before harvest in the late-logarithmic phase of growth. Solid media were pre-pared by the addition ofOxoid lonagar no. 2 (Consol-idated Laboratories Inc., Chicago Heights, Ill.) tothe above formulation. Bacteria were sedimented atapproximately 20,000 x g for 5 min and were washedonce with 50 mM KH2PO4-NaOH buffer at pH 6.8 to7.1. The final pellets were weighed and suspended inbuffer to give cell densities of 25 mg (dry weight) ofcells/ml for manometric experiments.

Cell disintegration. Extracts of cells were pre-pared by suspending washed cell pellets in 2 to 3volumes of the standard buffer followed by one pas-sage through a French Press (American InstrumentCo., Silver Spring, Md.) or by exposure to ultra-sound at 5 to 20 C. The crude extracts employed forall the experiments were the supernatant fractionsobtained after centrifugation at 20,000 x g for 20 to30 min at 0 to 5 C.

Respiratory studies. Respiration was measuredby oxygen consumption both manometrically withconstant volume manometers (Braun, Melsungen,Germany) and polarographically with a Clark oxy-gen electrode (Yellow Springs Instrument Co., Yel-low Springs, Ohio) at 30 C.Enzyme assays. Orcinol and resorcinol hydroxyl-

ase activities and dioxygenase activities were meas-ured by standard polarographic and spectrophoto-metric assays as detailed in the figure legends. Ace-tylpyruvate hydrolase was determined by the disap-pearance of acetylpyruvate at 290 nm (8). Maleyl-acetate reductase was assayed by the rate ofreducednicotinamide adenine dinucleotide (NADH) con-sumption at 340 nm or at 243 nm due to maleylace-tate disappearance. Details of individual assays ap-pear in the figure legends.

Chemical determinations. The following stand-ard methods were used: protein by the method ofLowry et al. (22); pyruvate by the Friedemann andHaugen method (13) and lactate dehydrogenase(Sigma Chemical Co., St. Louis, Mo.). Mafeylace-tate was assayed by its absorbance at 243 nm.

Chemicals. Acetylacrylic acid (melting point,125 C) was from PCR Inc. (Gainesville, Fla.). Bovineserum albumin was from Nutritional BiochemicalsCorp. (Cleveland, Ohio). Solutions of maleylacetatewere prepared enzymically from hydroxyquinol us-ing partially purified hydroxyquinol 1,2-oxygenasefrom resorcinol-grown P. putida ORC. Sources ofchemicals not available commercially have been de-scribed elsewhere (2).

Physical techniques. Optical measurements weremade with either Perkin-Elmer model 124 or Uni-cam SP 800 spectrophotometers.Mass spectrometry was carried out using an LKB

9000A gas chromatograph-mass spectrometer.

RESULTSComparative growth studies of P. putida 01

and ORC. It has been observed previously thatboth strains ofP. putida, 01 and ORC, isolatedfrom enrichment cultures containing orcinol asthe sole source of carbon, were able to grow onresorcinol. A more detailed investigation of thisgrowth of strain 01 was erratic, and thatORC grew readily at the expense of resorcinol,growth of strain 01 was erratic, and thatgrowth of the latter strain occurred only afterlong lag periods. Resorcinol culture media re-mained dark brown after inoculation withstrain 01, and the cells obtained were tanned.Only transient darkening of media was seenwith cultures of strain ORC, and the packedcells were pink. When suspensions of strain 01,after growth in nutrient broth, were plated onresorcinol minimal media, colonies appearedonly after 3 to 5 days, whereas colonies ap-peared after 1 day on orcinol media. Colonyformation on resorcinol media occurred with afrequency of 1 in 105 to 10c of that observed onorcinol plates. Subsequent transfer ofthe bacte-ria from the resorcinol plates to fresh resorcinolmedia gave new colonies within 24 h; this alsooccurred after an intermediate passage throughsuccinate minimal media. It was concludedthat strain 01 was able to grow on resorcinol bythe selection of mutants from the wild type,and one of these, designated as strain OlOC,was used in the work described here.

Respiratory activities of washed suspen-sions of P. putida. The data given in Fig. 3 andTable 1 show that after growth of the bacteriaon orcinol or resorcinol as sole carbon source,washed suspensions ofP. putida ORC were ableto oxidize orcinol and resorcinol, but differencesexisted both in the relative rates of oxidationand the amount ofoxygen consumed per mole ofsubstrate. Orcinol-grown P. putida ORC res-pired faster when orcinol was provided as sub-strate than when resorcinol was provided. Res-piration of resorcinol-grown P. putida ORC onresorcinol was considerably more rapid on re-sorcinol than orcinol in the presence of chlor-amphenicol. The stoichiometry of oxygen con-sumption by P. putida ORC also indicated thatorcinol was oxidized more completely than re-sorcinol by orcinol-grown cells, and the con-verse was apparent from resorcinol-grown cells(Table 2, Fig. 3). In particular, the oxidation oforcinol by resorcinol-grown cells in the pres-ence of chloramphenicol led to the accumula-tion of a red compound with Xmax = 485 nmcharacteristic of a 2-hydroxy-1,4-benzoquinonelacking a 3-alkyl group (5). When this productwas reduced by dithionite it was readily oxi-dized by extracts of orcinol-grown cells, with

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988 CHAPMAN AND RIBBONS

the expected equimolar oxygen consumption forthe ring cleavage of 2,3,5-trihydroxytoluene.P. putida 01 oxidized orcinol approximately

eight times faster than resorcinol after growthwith orcinol (Table 1). The derived mutantOlOC showed a similar respiratory pattemwhether grown with orcinol or resorcinol. Sig-nificant rates of oxidation of orcinol were foundwith succinate-grown cells of the mutant OlOCbut not the parent strain 01.

Hydroxyquinol was more rapidly oxidized byresorcinol-grown P. putida ORC than by orci-nol-grown cells. 3-Methylcatechol was oxidizedby orcinol-grown cells with the accumulation ofa yellow product but was not oxidized by resor-cinol-grown cells (not shown for whole cells).Maleate and fumarate were not oxidized by

any strain grown either with orcinol or resorci-nol, possibly because they failed to enter thecells. Acetylpyruvate was oxidized as rapidly aswas resorcinol by orcinol-grown cells of P. pu-

tida ORC but not by resorcinol-grown cells(Fig. 3).Hydroxylase and dioxygenase activities of

cell-free extracts of P. putida. NADH oxida-tion by extracts of cells was markedly stimu-lated by orcinol and resorcinol (Table 3) and to alesser extent by m-cresol (data not shown).However, the relative rates ofoxygen consump-tion (Table 3) (and also of NADH utilization[data not shown]) in the presence of orcinolparalleled the observations made with cell sus-pensions of P. putida ORC, 01, and OlOC fordifferent growth substrates. As before, the stoi-chiometric relationships for these oxidationswere quite different, and for P. putida ORCthey were dependent upon the growth sub-

strate. For example, with extracts of orcinol-grown P. putida ORC the ratio of oxidation oforcinol versus resorcinol was approximately6:1, whereas the ratio was 1:3.5 with extracts ofresorcinol-grown cells. Extracts of orcinol-grown cells oxidized hydroxyquinol poorly, con-

J. BACTERIOL.

firming the observation made with intact cells.Aged extracts of resorcinol-grown P. putidaORC accumulated a product from resorcinol(Fig. 4) that had the spectral properties of theautooxidation product ofhydroxyquinol (see be-low).Table 4 compares the ability of extracts to

oxidize several catechols. Thus hydroxyquinolwas oxidized more rapidly than 3-methylcat-echol by extracts of resorcinol-grown cells ofstrain ORC, but the rates were similar withextracts derived from orcinol-grown cells. Bothhydroxyquinol and 3-methylcatechol were read-ily oxidized by extracts of orcinol-grown strain01 and extracts of strain OlOC. The technicaldifficulties associated with determining accu-rate values for hydroxyquinol oxidation are de-scribed below.Oxidation of hydroxyquinol. Hydroxyquinol

rapidly autooxidizes in slightly acidic and basicaqueous solutions, and this can be assayed byeither 02 consumption or spectral changes.Consequently the absorbance changes recordedduring hydroxyquinol oxidation in the presenceof crude extracts from either orcinol- or resorci-

TABLE 2. Stoichiometry of resorcinol and orcinoloxidation by washed suspensions ofP. putida ORC

after growth on resorcinol or orcinola

Substrate O2 con- °2 con-Growth conditions supplied sumed sumed/

(nmol) (nmol) substrate(mol/mol)

OrcinolOrcinol 3,000b 13,900 4.63Resorcinol 3,000b 7,250 2.32

Resorcinol grownOrcinol 30 100 3.3Resorcinol 30 147 4.9a Reaction mixtures were as in the footnote to Table 1

with the exception of substrate concentrations as indicated.bManometric determinations; reaction conditions were

as given in the legend to Fig. 1.

TABLE 1. Respiratory rates of orcinol- and resorcinol-grown P. putidaa

Respiratory rate (nmol of 02 consumed/min/ml of suspension

Substrate supplied P. putida ORC P. putida 01 P. putida O1OC

Orcinolb Resorcinol Orcinol Succinate Orcinol Resorcinol Succinate

None 20 29 24 20 14 17 11Orcinol 550 280 420 23 467 266 88Resorcinol 145 615 50 24 46 57 26

a Reaction mixtures contained: 50 mM phosphate buffer, pH 7.0 (2.7 ml); cell suspension (20 to 50 ,ul); 10mM substrate (0.3 ml). Temperature, 30 C. Values presented are initial oxidation rates measured polaro-graphically during the first 2 min of the reactions. Cell suspensions contained approximately 25 mg (dryweight) of cells per ml.

b Growth substrate.

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VOL. 125, 1976

co

M.CD

RESORCINOL CATABOLISM IN P. PUTIDA

MINUTESFIG. 3. Respiratory activities of orcinol- and resorcinol-grown P. putida ORC. Each Warburg flask

contained: 20 mMphosphate buffer, pH 7 (1 ml); cell suspension (1 ml, 20 mg [wet weight]); 10 mM substrate(0.3 ml); and water to 2.8 ml. The center well contained 20% KOH (0.2 ml). Chloramphenicol (50 pglflask)was added as indicated. The temperature was 30 C. (A) Orcinol-grown cells respiring on orcinol (0),resorcinol (O.), resorcinol plus chloramphenicol (4), and acetylpyruvate plus chloramphenicol (0). Endoge-nous respiration (40 pl of021h) was subtracted. (B) Resorcinol-grown cells respiring on resorcinol (O), orcinol(A), orcinol plus chloramphenicol (0), and acetylpyruvate plus chloramphenicol (0). Endogenous respiration(43 of 021h) was subtracted.

TABLE 3. Stimulation ofNADH oxidation in crude cell-free extracts ofP. putida by orcinol,resorcinol and m-cresola

Oxidation rate (Jtmol of °2 consumed/min/ml of extract)

Substrate P. putida ORC P. putida 01 P. putida OIOC

OrcinoPb Resorcinol Orcinol Succinate Orcinol Resorcinol Succinate

NADH + orcinol 20.9 13.8 18.0 0.6 20.1 16.1 20.5NADH + resorci- 3.38 43.0 2.0 0.6 2.5 1.9 3.5

nola Reaction mixtures contained: 50 mM phosphate buffer, pH 7.0 (2.9 ml); cell-free extract (20 ,ul); 10 mM

NADH (40 p,l); 10 mM aromatic substrate (10 p1L). Temperature, 30 C. Values given are initial oxidationrates measured polarographically. NADH oxidase activities were between <0.1 and 0.9 ,umol of 02 con-sumed/min per ml of extract. For P. putida OlOC the value did not exceed 0.36.

b Growth substrate.

nol-grown cells are difficult to interpret une-

quivocally. Figure 5 shows the absorbancechanges that occurred when hydroxyquinol wasallowed to autooxidize in buffer at pH 6.8. Thepeak at 285 nm due to hydroxyquinol was re-

placed by a new absorption maximum at 260nm (of higher absorbance) and a broad weakerband at 485 nm (not shown). These spectralchanges are attributable to the formation of 2-hydroxy-1,4-benzoquinone (5, 23).

Additions of sodium borohydride or sodiumdithionite reversed this change by reducing thequinone to quinol. The quinone product alsoreacts readily with a number of thiols such as

mercaptoethanol and glutathione to form addi-tion products having Ax = 345 nm (26). Dur-ing hydroxyquinol autooxidation a third peakwith X,r = 325 nm was also seen. The rate offormation of this component increased with in-creasing concentration of hydroxyquinone, and

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990 CHAPMAN AND RIBBONS

260 3WAVELENGTH (nmj

100

FIG. 4. Formation of hydroxyquinone from resor-cinol with extracts of P. putida ORC (resorcinolgrown). The reaction mixture contained: 50 mMphosphate buffer, pH 7.1 (3.0 ml); aged cell extract(50 p1); 10 mM NADH (3 pi); 2 M ethanol (50 1d);alcohol dehydrogenase (2 pd); and 10 mM resorcinol(20 p). Resorcinol was omitted from the referencereaction mixture. Repeat scans were made at theintervals indicated (minutes). Temperature, 26 C.

its formation continued as the quinone disap-peared (Fig. 6). It would appear that the mate-rial responsible was formed by further reactionof hydroxybenzoquinone to give a dihydroxy-benzoquinone and other products, as is the casewith 2-hydroxy-6methyl-1,4-benzoquinone (4).When hydroxyquinol oxidation was rapidly

catalyzed by extracts ofresorcinol-grown P. pu-

tida ORC, the product absorbed maximally at245 nm (Fig. 7); spectral changes at 260 nm, 325nm, and 485 nm were scarcely seen. If extractshaving low enzymic activity towards hy-droxyquinol were used, however, then spectralchanges indicated the occurrence of both en-zymic and nonenzymic oxidation. Complicatingthe interpretation of these changes is the factthat the presence of bacterial cell extract itselfalso slowed the rate of nonenzymic oxidation.These findings are summarized in Table 5. Itcan be seen (lines 1, 2, 3, and 5) that the pres-ence of increasing amounts of crude bacterialextract (from a succinate-grown Bacillus spe-cies) slowed the rate of appearance of autooxi-dation products. Extracts from succinate-grownP. putida ORC and from other bacteria also hadthis effect.By contrast, bovine serum albumin (Table 5,

line 6) accelerated autooxidation. After holdingcrude Bacillus extract at 100 C for 3 min itsprotective effect was decreased by approxi-mately one-half (line 4), suggesting that hy-droxyquinol autooxidation may be retarded byan enzymic process. With extracts of resorcinol-grown P. putida ORC (line 7), a compound hav-ing Xmax = 245 nm was the major product ob-served. Use of smaller amounts of such extracts(line 8) led to a significant increase in hy-droxyquinone formation that obscured the en-zymic product at 245 nm. In the presence ofBacillus crude extract (line 9), considerably lessautooxidation occurred, and the change in ab-sorbance at 245 nm (when corrected for autooxi-dation by subtraction of line 3) reflects moreappropriately the decreased quantity ofenzymeused.The spectral changes observed during the

rapid enzymic oxidation of hydroxyquinol areentirely consistent with its conversion to mal-eylacetate, i.e., an ortho-oxygenative cleavage,as reported earlier by Larway and Evans.The absorption spectrum at 245 nm was abol-

ished upon acidification to pH 3.5 or less andrestored by readjustment to pH 7.0. This ab-sence of absorption in acid solution is a featureobserved with other maleyl-substituted ring fis-sion products, notably maleylacetoacetate (19)and maleylpyruvate (20). It would appear thatthese cis acids, unlike their trans isomers, canform cyclic pseudo acids on acidification of theiracyclic carboxylate anions, as is the case withformylacrylic acid (26, 28), maleylacetone (12),and cis- 3-acetylacrylic acid (29). Further evi-dence for maleylacetate is provided by its reac-tion with p-nitrobenzene-diazohydroxide togive an N,N'-diphenylformazan (X = 450nm) spectrally indistinguishable from that

220

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RESORCINOL CATABOLISM IN P. PUTIDA

TABLE 4. Respiratory rates of"catechol" oxidations catalyzed by cell-free extracts oforcinol-, resorcinol-, andsuccinate-grown P. putidaa

Oxidation rate (,umoI of 02 consumed/min/ml of extract)

Substrate oxidized P. putida ORC P. putida 01 P. putida 010C

Orcinolb Resorcinol Orcinol Succinate Orcinol Resorcinol Succinate

Hydroxyquinol 1.34c 9.55 15.0 0.2d 6.0 8.0 2.5c2,3,5-Trihydroxy- NDe ND 60.0 1.03 ND 15.2 ND

toluenec3-Methylcatechol 1.0 0.14 6.5 0.01 7.0 3.5 3.7Catechol 0.07 0.01 0.6 0.01 ND ND ND4-Methylcatechol 0.04 0.01 0.9 0.01 ND ND ND

a Reaction mixtures contained: 50 mM phosphate buffer, pH 7.0 (2.9 ml); extract (25 to 100 1.l); 10 mMsubstrate as indicated (0.1 ml). Temperature, 30 C.

b Growth substrate.c Nonenzymic oxidation rate, 0.6 ,umol/min.d Nonenzymic oxidation rate, 0.4 ,umol/min.e ND, Not determined.

0-8C 0.6.*

250 215 300WAVELENGTH Inml

FIG. 5. Spectral changes during rapid nonenzymic oxidation of hydroxyquinol. The reaction mixturecontained: 50 mM phosphate buffer, pH 6.8 (3 ml); and 25 mM hydroxyquinol (20 j.). Repeat scans weremade after 0, 1.5, 3, 6, 10, 13, and 25 min (numbered 0 to 6, respectively). Temperature, 25 C.

formed by compounds containing a reactivemethylene group such as acetoacetate (31) and,3-ketoadipate. Attempts to isolate maleylaceticacid (see below) lead to its decarboxylation.Extracts from orcinol-grown cells of eitherstrain of P. putida, on the other hand, did notcatalyze the formation of maleylacetate fromhydroxyquinol (Fig. 8A and B), in that only

ultraviolet-end absorbance was observed afteroxygen uptake had ceased and a transient in-crease at 285 nm was seen, similar to thatobserved when 2,3,5-trihydroxytoluene is thesubstrate for the meta cleavage oxygenase (2).The nature of hydroxyquinol oxidation is not soevident with extracts ofresorcinol-grown strain010C since the time course of the spectral

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992 CHAPMAN AND RIBBONS

hydroxyquinol was complete (1 h), the reaction0.8 260 nm mixture was acidified to pH 2.0 with 6 N HCl

and centrifuged for 15 min at 8,000 x g. Thesupernatant was then continuously extractedwith diethylether for 48 h. The ether was driedover anhydrous MgSO4 and evaporated, giving

/245 nm 71 mg of off-white solid. After crystallization06 from ether-petroleum ether it had a melting

point of 122 to 123 C and in aqueous solutionshowed Xmax = 220 nm. Mass spectral analysis(direct probe; 70 electron volts) gave a parent

1.AJ ion electronic mass of 114 with a fragmentationz / / pattern identical to that of authentic acetyl-Q04 / acrylic acid. The trans isomer of this compound

0

< 285nm 5

0.2 485nm

0.8-325 nm

0 6 12 18 240\6-MINUTES

FIG. 6. The time course of spectral changes at dif- w 0ferent wavelengths during nonenzymic oxidation of 0 \zhydroxyquinol. Reaction was initiated by addition of ./5 i1 of 50 mM hydroxyquinol to 3 ml of 50 mM cD Iphosphate buffer (pH 7.2) and was followed at the Xwavelengths indicated. Temperature of incubation, o 0423 C. co

changes at 240 and 285 nm illustrated in Fig. 38C (bottom) indicates that possibly both orthoand meta fission of hydroxyquinol occurred. Itwas consistently observed that the final absorb- \ance at 240 nm reached higher values that re- 02 5mained stable when extracts derived from re- 0sorcinol-grown cells were compared with thosefrom orcinol-grown cells (see Fig. 8A and C).Experiments to support this view are de-

scribed below.Identification of maleylacetate as the prod- 0 L I ,

uct of hydroxyquinol oxidation. Extracts of 220 260 300resorcinol-grown P. putida ORC were allowed WAVELENGTH(nm)to oxidize hydroxyquinol with the accumulation FIG. 7. Spectral changes during enzymic oxida-of the 245 nm species. The incubation mixture tion of hydroxyequinol by extracts of P. putida ORCcontained: 200 nM phosphate buffer, pH 7.0 (300 (resorcinol grown). Hydroxyquinol (10 id; 50 mM)ml); crude extract derived from resorcinol- was added to 3.0 ml of phosphate buffer (pH 7.0)grown P. putida ORC (8 ml); and 100 mM hy- containing 10 pl of crude extract of P. putida ORCdroxyquinol (10 ml) added, in portions of 0.5 t (resorcinol grown) to initiate the reaction. Repeatdroxqunl, (1 ml) added,

r tion poxturtinso 05 scans were made at 1, 3 and 5 min against a control1.0 ml, to the stirred reaction mixture as sub- reaction lacking hydroxyquinol. The zero time spec-strate was depleted. This was monitored by trum was obtained by scanning 10 p2 of 50 mMdisappearance of dissolved oxygen with an oxy- hydroxyquinol in 3 ml of distilled water. Tempera-gen electrode. After oxidation of all the added ture of incubation, 23 C.

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RESORCINOL CATABOLISM IN P. PUTIDA 993

TABLE 5. Spectral changes during enzymic and nonenzymic oxidation of hydroxyquinola

Absorbance changes (per min) at:Additions

245 nm 260 nm 325 nem 485 nm

None 0.150 0.20 0.013 0.0570 ug ofBacillus crude extract protein 0.060 0.080 0.010 0.02140 ,ug of Bacillus crude extract protein 0.030 0.040 0.007 0.01140 jig ofBacillus crude extract protein (heated 3 min at 0.060 0.080 0.02

100 C)280 jg ofBacillus crude extract protein 0.028 0.036 0.007 0.01400 pg of bovine serum albumin 0.190 0.245 0.014 0.06590 pig of P. putida crude extract protein (resorcinol 0.150 0.105 0.003 0.002grown)

18 pg of P. putida crude extract protein (resorcinol 0.110 0.140 0.007 0.03grown)

18 pg of P. putida crude extract protein + 140 jg of 0.060 0.060 0.003 0.01Bacillus crude extract proteina Reaction mixtures contained the additions shown in 3 ml of 50 mM phosphate buffer, pH 7.2. Hy-

droxyquinol (12.5 ,ul, 50 mM) was added to initiate reactions, which were followed at the wavelengthsindicated against control miartures lacking hydroxyquinol. Temperature of incubation, 23 C.

b Maximum rate observed. For other wavelengths this is the initial rate.

(melting point, 125 C) had a X., = 220 nm inboth neutral and acidic solution, whereas its cisisomer can exist either as its cyclic pseudo acid(melting point, 33 to 36 C) with X.,a = 195 nmor as its acyclic anion (Xmax = 240 nm) (29). Theproperties of the isolated product are thereforethose ofthe trans isomer formed presumably byboth acid-catalyzed decarboxylation and isom-erization of maleylacetate during its isolation.Metabolism of maleylacetate. When hy-

droxyquinol was oxidized by extracts of resorci-nol-grown P. putida ORC with formation ofmaleylacetate (Fig. 7), the Xma_ at 245 nm wasalmost completely abolished upon subsequentaddition of NADH, and NADH was concomi-tantly oxidized. For each mole ofhydroxyquinolused to generate maleylacetate under the con-ditions given in Fig. 7, 1 mol of NADH wasconsumed (as calculated from absorbance at340 nm changes due to NADH consumption)during maleylacetate reduction. This enzymicreaction is referred to as maleylacetate reduc-tase.Figure 9 shows that when hydroxyquinol oxi-

dation was catalyzed by extracts of resorcinol-grown P. putida ORC in the presence of anNADH-generating system, the spectral band at245 nm characteristic of maleylacetate ini-tially appeared and later disappeared.

Maleylacetate reductase activity could not bedemonstrated in extracts of orcinol-grown P.putida ORC or in those of 010C. There was nochange in the spectrum of maleylacetate onaddition of these same extracts. Extracts of re-sorcinol-grown P. putida strain OOC, how-ever, like those of strain ORC, were shown tocontain maleylacetate reductase activity.

Acetylpyruvate hydrolase activity of ex-

tracts. Table 6 shows the relative rates of ace-tylpyruvate hydrolysis by extracts of orcinol-,resorcinol-, and succinate-grown P. putida010C and ORC. The results clearly indicatethat acetylpyruvate hydrolase was not presentin extracts of resorcinol-grown P. putida ORCbut was readily demonstrated in extracts oforcinol-grown cells of P. putida 01 and P. pu-tida ORC. Similar specific activities were foundin extracts of P. putida 010C with all growthsubstrates examined.Pyruvate and maleylacetate formation by

extracts. Confirmation that pyruvate isformed, as proposed in Fig. 2, after meta cleav-age of hydroxyquinol by P. putida 010C isgiven in Table 7. Pyruvate was formed in 70 to80% of the yield with extracts of 01 (notshown), 010C, and ORC after growth onorcinol. Pyruvate was not formed when ex-tracts of resorcinol-grown P. putida ORC wereused. Instead, maleylacetate accumulated inalmost equimolar proportions from hydroxy-quinol when such extracts were used. Extractsof resorcinol-grown P. putida 010C, however,oxidized hydroxyquinol to both maleylacetateand pyruvate, as suspected from the spectralchanges observed (Fig. 8), and in these experi-ments ortho cleavage giving maleylacetate wasthe predominant route; only about 20 to 30% ofthe substrate supplied was transformed topyruvate. Pyruvate was formed quantitatively,however, from acetylpyruvate by extracts ofresorcinol-grown P. putida 010C (data notshown).

DISCUSSIONOne of our interests in the catabolic path-

ways employed by microorganisms for the utili-

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U.

0-08

250 215 300 325WAVELENGTH (nmj

FIG. 9. Effects ofNADH on the spectral changes during enzymic oxidation of hydroxyquinol. Reactionmixtures contained: 50 mM phosphate buffer, pH 6.8 (3 ml); 20 mM glucose 6-phosphate (0.4 ml); 25 mMNADH (1.5 pl); cell extract from resorcinol-grown P. putida ORC (10 ,ul); and glucose 6-phosphate dehydro-genase from Z. mobilis (10 p1). The reaction was initiated with 25 mM hydroxyquinol (20 p1) added to thesample cuvette; spectral changes were recorded at 0, 4, 8, 14, 18, 22 and 30 min. Temperature, 26 C.

zation of the wide variety of products of plant,microbial and industrial origin is the specificityof the enzymes involved as well as the specific-ity of the control of their synthesis. It has beenamply demonstrated that several catabolicroutes, or portions ofthem, are nonspecific withrespect to the transformations their respectiveenzymes catalyze and to the inducers requiredto elicit their synthesis (25). Notable examplesare the meta cleavage pathways for the com-plete degradation of monohydric phenols, thegentisate pathways for the metabolism of simi-lar compounds (7, 17, 18, 27), and the metabo-lism of aromatic hydrocarbons (14). It seemedlikely that orcinol and resorcinol were metabo-lized by the same sequence of enzymes in P.putida ORC and 01, but there are some incon-sistensies to such a proposal. The more detailedcomparison of the enzymic activities of orcinol-and resorcinol-grown cells made here supportsthe view that these two compounds are metabo-lized by distinct pathways (Fig. 2) by P. putidaORC, but that the enzymes of the orcinol path-way in P. putida 01 may be used also for the

metabolism of resorcinol since, for growth ofstrain 01 to occur, a mutation to constitutivityof the orcinol pathway enzymes is necessary.Table 8 summarizes the differences found be-

tween P. putida ORC and the mutant P. putidaOlOC. Thus the respiratory activities of wholecells and the reactions catalyzed by extracts ofP. putida 010C show that enzymes of the or-cinol pathway are constitutive in P. putida010C. Furthermore, the homologous primarysubstrate resorcinol is oxidized, although muchless efficiently, and pyruvate is formed fromthis substrate (not shown quantitatively) byextracts ofP. putida 010C. Similarly, the firstintermediate of resorcinol catabolism, hy-droxyquinol, is converted to pyruvate; however,maleylacetate, which is the sole product of hy-droxyquinol oxidation by extracts of resorcinol-grown P. putida ORC, is also a product whenextracts from resorcinol-grown but not orcinol-or succinate-grown cells ofP. putida 010C areused. Extracts of P. putida 010C also hydro-lyze acetylpyruvate to pyruvate irrespective ofthe growth substrate. These observations sug-

FIG. 8. Comparison ofthe polarographic and spectral events that occur during hydroxyquinol oxidation byorcinol-grown P. putida strains O10C (A) and ORC (B) and resorcinol-grown P. putida strains OIOC (C)and ORC (D) at 240 and 285 nm. The reaction mixtures contained: 50 mM phosphate buffer, pH 6.8 (3 ml);cell extract (10 p1); and 100 mM hydroxyquinol (3 p). Temperature, 26 C.

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996 CHAPMAN AND RIBBONS

gest that the four enzymes of the orcinol path-way in P. putida 01 are under coincidentcontrol in that they are all expressed by theconstitutive mutant obtained. However, duringgrowth on resorcinol, mechanistically distinctenzymes are also induced in P. putida OlOC,namely hydroxyquinol 1,2 - oxygenase andmaleylacetate reductase. These enzymes arealso induced by the wild type ofP. putida ORCafter exposure to resorcinol. The single analo-gous reaction of the orcinol and resorcinol path-ways depicted in Fig. 1 and 2 is the initialhydroxylation of the benzene nucleus. The datafrom Tables 1, 3, and 4 show that differenthydroxylases are probably induced in P. putidaORC but that in P. putida OlOC a single en-zyme is possibly responsible for both orcinoland resorcinol hydroxylation, and that this pro-vides the selective pressure for the expressionof constitutive mutants when resorcinol is pro-

TABLE 6. Acetylpyruvate hydrolase activity inextracts oforcinol-, resorcinol-, and succinate-grown

P. putidaa

Strain Growth substrate Sp act (U/mgof protein)

P. putida ORC Orcinol 0.2Resorcinol 0

P. putida 01 Orcinol 0.18Succinate 0

P. putida 010C Orcinol 0.16Resorcinol 0.13Succinate 0.12

a Reaction mixtures obtained: 20 mM KH2PO4-NaOH buffer, pH 7.1 (2.9 ml); 25 mM acetylpyru-vate (25 pl); and cell extract (10 ,ul containingapproximately 0.25 mg of protein). Activity wasmeasured by the change in absorbance at 290 nmand converted to units (micromoles/minute). Tem-perature, 27 C.

vided as the sole source of carbon. The route ofhydroxyquinol oxidation is then determined bythe relative activities of the constitutive metaring cleavage enzyme of the orcinol pathwayand the inducible ortho cleavage enzyme of theresorcinol pathway in P. putida 010C. Fromthe known properties of orcinol hydroxylasefrom P. putida 01 (24), it might be the ratelimiting enzyme in this organism duringgrowth on resorcinol. The growth rates and theappearance of the harvested cells observed sup-port this idea. Growth of P. putida OlOC onresorcinol is relatively slow, the medium turnsbrown, and harvested cells are tanned. This isnot so for P. putida ORC, which clearly pos-sesses the genes for two hydroxylases, onewhich prefers orcinol and the other whichprefers resorcinol as a substrate, although they

TABLE 7. Yields ofpyruvate and maleylacetate fromhydroxyquinola

Hydroxy- 02 Pyru- Maleyl-Strain quinol con- vate acetate

supplied sumed formed formed(nmol) (nmol) (nmol) (nmol)

P. putida O1OCOrcinol grown 225 206 160 0

375 342 241 ND°

Resorcinol 225 206 53 111grown

375 340 106 207

P. putida ORCOrcinol grown 225 214 159 ND

375 359 276 0

Resorcinol 225 211 ND 193growngTown__________ 435 410 0 425a Composition of reaction mixtures was as described in

the footnote to Table 4 with substrate (10 mM) added asindicated.

b ND, Not determined.

TABLz 8. Summary of the differences between orcinol- and resorcinol-grown P. putida ORC and OlOCa

P. putida ORC P. putida O1OCProperty Resorcinol ResorcinolOrcinol grown gRown Orcinol grown grown

Whole cell respiration Orc > Res Res > Orc Orc > Res Orc > Res3-MC > HQ HQ > 3-MC 3-MC > HQ 3-MC > HQ

Product of orcinol oxidation Pyruvate Hydroxytolu- Pyruvate Pyruvatequinone

Hydroxylase activities Orc > Res Res > Orc Orc > Res Orc > ResDioxygenase activities 3-MC > HQ HQ > 3-MC 3-MC > HQ HQ > 3-MCAcetylpyruvate hydrolase + - + +Maleylacetate reductase - + - +Maleylacetate from HQ - + - +Pyruvate formation from HQ + + +

a Orc, Orcinol; 3-MC, 3-methylcatechol; Res, resorcinol; HQ, hydroxyquinol; +, present; -, absent.

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have overlapping specificities for each sub-strate (unpublished observations).The catabolic pathways for resorcinol and or-

cinol metabolism suggested from these studiesand other reports have not been rigorously de-fined. However, the evidence presented is con-sistent with the proposals summarized in Fig. 2for P. putida ORC, 01, and OlOC. The demon-stration ofhydroxyquinol formation from resor-cinol by washed cell suspensions of resorcinol-grown P. putida ORC cultures (Fig. 4) and thecharacterization of maleylacetate as a productof hydroxyquinol oxidation by extracts providenew evidence for the reactions shown in Fig. 2.It was also shown that 2,3,5-trihydroxytolueneis the only product of orcinol oxidation by resor-cinol-induced cells of P. putida ORC; unlikeresorcinol-grown P. putida 010C, these cellsdo not contain 2,3,5-trihydroxytoluene 1,2-oxy-genase. It must be concluded from this that hy-droxyquinol 1,2-oxygenase does not oxidize2,3,5-trihydroxytoluene showing a substratespecificity in common with certain other orthofission dioxygenases (30). By contrast, 2,3,5-trihydroxytoluene 1,2-oxygenase cleaves thering of some substrate analogues, in this casehydroxyquinol and 3-methylcatechol, as ob-served for other meta fission oxygenases (1, 6).As a consequence of the relaxed specificity ofsubsequent enzymes of this meta cleavagepathway, pyruvate can also be formed fromhydroxyquinol.The evidence presented here provides an-

other example of the metabolic diversity of theaerobic pseudomonads and the acquisition ofnew nutritional capabilities by the recruitmentof an enzyme of a related metabolic pathway;e.g., orcinol hydroxylase is recruited for resor-cinol hydroxylation by P. putida 010C. Evolu-tion of P. putida 01 to P. putida 010C occursby a regulatory mutation to constitutivity forthe orcinol pathway enzymes. Mutations inregulatory genes of catabolic routes have beenshown to expand the nutritional spectra of sev-eral bacterial species. Thus, for Escherichia colito grow on xylitol a mutation in the regulatorygenes for ribitol dehydrogenase occurs; other"fitter" derivatives are obtained in which thearabitol transport system is derepressed, andthis facilitates xylitol uptake (21). Similarly,constitutive synthesis of acetamidase in P.aeruginosa allows growth on some noninducingsubstrates (3). Some strains of P. putida havethe genetic potential for both ortho and metacleavage of catechol; synthesis of the enzymesinvolved is determined by the growth sub-strates. Feist and Hegeman (11), however,have shown that suppression of mutantsblocked in the meta pathway can occur by an-

other mutation to block an earlier reaction ofthat sequence, so that accumulation of its sub-strate allows the expression and use of anothermetabolic route, thus restoring the wild pheno-type.The nature of the product of maleylacetate

reduction by NADH remains to be established,although preliminary experiments suggest thatit is 3-ketoadipate. Maleylacetate has beenidentified as a metabolite of the herbicide 4-chlorophenoxyacetic acid in species of Pseu-domonas (10) and Arthrobacter (9). In the latterorganism its further metabolism appears alsoto involve a reductive step. A similar activityhas also been observed in other pseudomonadsgrown at the expense of 2,4-dihydroxybenzoate(unpublished observations).

ACKNOWLEDGMENTSWe are grateful to Edye E. Groseclose, John L. Michal-

over, Yoshiyuki Ohta, and Placida Venegas for help withthese experiments, and to Tom Krick for skilled technicalassistance in the operation of the mass spectrometry facili-ties provided and maintained by the Minnesota Agricul-tural Experiment Station.

This research was supported by grant no. GB4422S fromthe National Science Foundation to P. J. C. and by PublicHealth Service grant no. GM 20172 from the National Insti-tute of General Medical Sciences to D. W. R.

LITERATURE CITED1. Chapman, P. J. 1972. An outline of reaction sequences

used for the bacterial degradation of phenolic com-pounds, p. 17-55. In Degradation of synthetic organicmolecules in the biosphere. Printing and PublishingOffice, National Academy of Sciences, Washington,D.C.

2. Chapman, P. J., and D. W. Ribbons. 1976. Metabolismof resorcinylic compounds by bacteria: orcinol path-way in Pseudomonas putida. J. Bacteriol. 125:975-984.

3. Clarke, P. H. 1970. The aliphatic amidases ofPseudom-onas aeruginosa. Adv. Microb. Physiol. 4:179-222.

4. Corbett, J. F. 1970. The chemistry ofhydroxy-quinones.V. The oxidation of 5-alkyl and 2,5-dialkyl-3-hydroxy-benzoquinones in the presence of alkali. J. Chem.Soc. C, p. 1912-1916.

5. Corbett, J. F. 1970. The chemistry of hydroxyquinones.VI. Formation of 2-hydroxysemiquinones during theautoxidation of benzene 1,2,4-triols in alkaline solu-tion. J. Chem. Soc. C, p. 2101-2106.

6. Dagley, S. 1971. Catabolism of aromatic compounds bymicroorganisms. Adv. Micro. Physiol. 6:1-46.

7. Dagley, S., P. J. Chapman, D. T. Gibson, and J. M.Wood. 1964. Degradation of the benzene nucleus bybacteria. Nature (London) 202:775-778.

8. Davey, J., and D. W. Ribbons. 1975. Metabolism ofresorcinylic compounds by bacteria. Purification andproperties of acetylpyruvate hydrolase. J. Biol.Chem. 250:3826-3830.

9. Duxbury, J. M., J. M. Tiedje, M. Alexander, and J. E.Dawson. 1970. 2,4D metabolism: enzymatic conver-sion of chloromaleylacetic acid. J. Agric. Food Chem.18:199-201.

10. Evans, W. C., B. S. W. Smith, P. Moss, and H. N.Fernley. 1971. Bacterial metabolism of 4-chlorophen-oxyacetate. Biochem. J. 122:509-517.

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12. Fowler, J., and S. Seltzer. 1970. The synthesis of modelcompounds for maleylacetoacetic acid. Maleylace-tone. J. Org. Chem. 36:3529-3532.

13. Friedemann, T. E., and G. E. Haugen. 1943. Pyruvicacid. II. The determination of keto acids in blood andurine. J. Biol. Chem. 147:415-42.

14. Gibson, D. T. 1973. Initial reactions in the degradationof aromatic hydrocarbons, p. 116-135. In Degradationof synthetic organic molecules in the biosphere.Printing and Publishing Office, National Academy ofSciences, Washington, D.C.

15. Harborne, J. B., and N. W. Simmonds. 1964. The natu-ral distribution ofphenolic aglycones, p. 77-128. In J.B. Harborne (ed.), Biochemistry of phenolic com-pounds. Academic Press Inc., London.

16. Hellstrom, N. 1960. Structure of P-acylacrylic acids.Nature (London) 187:146.

17. Hopper, D. J., and P. J. Chapman. 1971. Gentisic acidand its 3- and 4-methyl substituted homologues asintermediates in the bacterial degradation of m-cre-sol, 3,5 xylenol and 2,5 xylenol. Biochem. J. 122:19-28.

18. Hopper, D. J., P. J. Chapman, and S. Dagley. 1971. Theenzymic degradation of alkyl-substituted gentisates,maleates and malates. Biochem. J. 122:29-40.

19. Knox, W. E., and S. W. Edwards. 1955. The propertiesof maleylacetoacetate, the initial product of homo-gentisate oxidation in the liver. J. Biol. Chem.216:489-498.

20. Lack, L. 1959. The enzymic oxidation of gentisic acid.

Biochim. Biophys. Acta 34:117-123.21. Lin, E. C. C. 1970. Evolution of catabolic pathways in

bacteria, p. 89-102. In D. W. Ribbons, K. Savard, W.J. Whelan, and J. F. Woessner (ed.), Homologies inenzymes and metabolic pathways. 2nd Miami WinterSymposia, vol. 1. North Holland, Amsterdam.

22. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J.Randall. 1951. Protein measurement with the Folinphenol reagent. J. Biol. Chem. 193:265-275.

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24. Ohta, Y., I. J. Higgins, and D. W. Ribbons. 1975. Me-tabolism of resorcinylic compounds by bacteria. Puri-fication and properties of orcinol hydroxylase fromPseudomonas putida 01. J. Biol. Chem. 250:3814-3825.

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