JouRNAL OF BACrmoILGY, Oct. 1974, p. 159-167Copyright 0 1974 American Society for Microbiology

Vol. 120, No. 1Printed in U.S.A.

Bacterial Degradation of 4-Hydroxyphenylacetic Acid andHomoprotocatechuic Acid

VELTA L. SPARNINS, PETER J. CHAPMAN, AND STANLEY DAGLEY

Department of Biochemistry, College of Biological Sciences, University of Minnesota,St. Paul, Minnesota 55101

Received for publication 15 July 1974

A species of Acinetobacter and two strains of Pseudomonas putida when grown

with 4-hydroxyphenylacetic acid gave cell extracts that converted 3,4-dihydroxy-phenylacetic acid (homoprotocatechuic acid) into carbon dioxide, pyruvate, andsuccinate. The sequence of enzyme-catalyzed steps was as follows: ring-fission bya 2,3-dioxygenase, nicotinamide adenine dinucleotide-dependent dehydrogena-tion, decarboxylation, hydration, aldol fission, and oxidation of succinic semial-dehyde. Two new metabolites, 5-carboxymethyl-2-hydroxymuconic acid and2-hydroxyhepta-2,4-diene-1,7-dioic acid, were isolated from reaction mixturesand a third, 4-hydroxy-2-ketopimelic acid, was shown to be cleaved by extracts togive pyruvate and succinic semialdehyde. Enzymes of this metabolic pathwaywere present in Acinetobacter grown with 4-hydroxyphenylacetic acid but were

effectively absent when 3-hydroxyphenylacetic acid or phenylacetic acid servedas sources of carbon.

Two metabolic pathways are employed bymicroorganisms for the aerobic degradation of4-hydroxyphenylacetic acid. The first reactionsequence is initiated by hydroxylation to givehomogentisic acid (3, 20), which is then con-verted into fumarate and acetoacetate (7) byreactions similar to those found in mammalianliver (18). In the second pathway (1, 5, 19),hydroxylation of 4-hydroxyphenylacetic acidgives rise to 3,4-dihydroxyphenylacetic acid(homoprotocatechuic acid), and the benzenenucleus is then cleaved by a dioxygenase, whichhas been isolated and crystallized (26). Beforethe present work was undertaken, no furtherstudies of this second pathway had been re-ported beyond the observation that the ring-fis-sion product is oxidized by nicotinamide ade-nine dinucleotide (NAD; 26). We now present acomplete sequence of enzymatic reactions serv-ing to convert 4-hydroxyphenylacetic acid intocarbon dioxide, succinate, and pyruvate (Fig. 1)and describe some properties of two new me-tabolites (compounds HII and IV).

MATERIALS AND METHODSOrganisms and cell extracts. The following orga-

nisms were employed. Pseudomonas U (British NCIB10015) is a strain of Pseudomonas putida that wasisolated from mud in a creek in Urbana, Ill., in 1963and has been used in several investigations (2, 8, 10,12, 25). Pseudomonas T is a gram-negative motile rodwith all the characteristics of P. putida except that it

159

fails to elaborate fluorescent pigment on King Bmedium (28). It was isolated from garden soil inMinneapolis by enrichment, with tropine as carbonsource, in a mineral salts medium at 30 C. The thirdorganism was isolated from agricultural soil in St.Paul by enrichment with phenylacetic acid at 30 C. Itis a gram-negative, oxidase-negative coccobacillusthat cannot utilize glucose and is tentatively identi-fied as Acinetobacter. The three strains were grownwith aeration at 30 C in a medium that contained (perliter): Na2HPO,, 4.33 g; KH,PO,, 2.65 g; NH,Cl, 2.0g; nitrilotriacetic acid, 0.2 g; 4-hydroxyphenylaceticacid, 0.5 g, brought to pH 7 with NaOH; and 4 ml ofthe stock solution of salts specified by Rosenbergerand Elsden (23). Procedures for obtaining washed-cellsuspensions and cell extracts were described previ-ously (10).

Analytical methods. Compounds were chromato-graphed by using aluminum sheets precoated withsilica gel F-254 (EM Laboratories, Inc., Elmsford,N.Y.). The compositions of solvents used for 2,4-dini-trophenylhydrazones, expressed as ratios by volume,were as follows: solvent A (22), ethyl formate-pro-pionic acid-light petroleum ether (bp 60 to 80 C;30:15.4:70); solvent B, benzene-dioxane-formic acid(6:3:1). The respective R, values for the derivatives ofpyruvic acid, succinic semialdehyde, and 4-hydroxy-2-ketopimelic acid were: in A, 0.21, 0.42, and0.03; and in B, 0.56, 0.55, and 0.38. For amino acidswe used: solvent C, n-butanol-acetic acid-water(4:1:1); and solvent D, 2-butanone-pyridine-water-acetic acid (70:15:15:2). The R, values for authenticsamples of L-alanine, 4-aminobutyric acid, and 2-aminopimelic acid were, respectively: in C, 0.35, 0.40,and 0.45; and in D, 0.22, 0.18, and 0.22. 2-Amino-4-

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SPARNINS, CHAPMAN, AND DAGLEY

CH2-COOH CH2-COOH

H

OH OH

CH2-COOH

CH2

ICOOH

CH2-COOHNADP

CH2f

CHO

CH2-COOH

02 -CHO

a NCOOH

OH

II

CH2- COOH

CH2

eCH2- CO-CO

C H3-CO -COOHv

H2-COOH

NAD-

COOH

b COOH

OH

T,C02/{c

I CH2-COOH

H20

d COOH)OH OH

17

FIG. 1. Reaction sequence for the metabolism of 4-hydroxyphenylacetic acid and homoprotocatechuic acidby Acinetobacter sp. and Pseudomonas putida.

hydroxypimelic acid, obtained by reduction of the2,4-dinitrophenylhydrazone of the corresponding syn-thetic keto acid, showed an R, of 0.25 in solvent C.Gas chromatography, used to obtain informationconcerning the structure of compound HI, was

performed with a glass column (152 by 0.32 cm)packed with 3% OV 17 on Supelcoport (Supelco Inc.,Bellefonte, Pa.). The instrument used was an LKB9000A gas chromatograph-mass spectrometer, andseparations were achieved by linear temperature pro-gramming from 150 C at 5 degrees per min. Massspectra were obtained at 70 eV. When compound Ill,isolated from incubation mixtures, was treated withTri-Sil (Pierce Chemical Co., Rockford, Ill.) in anhy-drous tetrahydrofuran, the derivative formed gave a

single symmetrical peak with a retention time of 9.5min, having a parent ion of 504 m/e. The relativeintensities of the ions of the mass spectrum were alsoconsistent with the chemical structure assigned,namely tetra trimethylsilyl 5-carboxymethyl-2-hydroxymuconic acid, indicating that the enol func-tion in III was derivatized in addition to the threecarboxyl groups. Pyruvate and succinic semialdehydewere determined spectroscopically at 340 nm, theformer by monitoring the oxidation of reduced NAD(NADH) when lactate dehydrogenase was added, andthe latter by observing the reduction of NAD phos-phate (NADP) which was catalyzed by succinicsemialdehyde dehydrogenase present in extracts ofcells grown with 4-hydroxyphenylacetic acid.

Materials. Homogentisic acid, 3,4-dihydroxy-phenylacetic acid, dl-2-aminopimelic acid, dl-3,4-dihydroxymandelic acid, and dl-4-hydroxymandelicacid were from Sigma Chemical Co.; 4-aminobutyricacid, 3- and 4-hydroxyphenylacetic acids were fromAldrich Chemical Co. Succinic semialdehyde was

synthesized either by treating glutamic acid with so-

dium hypochlorite (17) or as described by Jakoby (15).2,3,4-Trihydroxyphenylacetic acid was synthesizedby the following method. 2,3,4-Trimethoxyphenylpyru-vic acid was prepared from 2,3,4-trimethoxybenzalde-hyde (Aldrich Chemical Co.) via a-acetamino-2,3,4-

trimethoxycinnamic acid by a procedure similar tothat for phenylpyruvic acid (14), and was then oxi-dized directly, without isolation, by means of hydro-gen peroxide (27) to give 2,3,4-trimethoxyphenyl-acetic acid (mp 102 to 103 C; literature [13] mp 102C). This compound was then demethoxylated by re-

fluxing with pyridine hydrochloride, as described byBlakley and Simpson (6), to give 2,3,4-trihydroxy-phenylacetic acid (mp 163 C). 2-Hydroxymuconic acid(4-oxalocrotonic acid) was the kind gift of L. M. Hen-derson. 4-Hydroxy-2-ketopimelic acid was synthe-sized by P.-T. Leung (21).

Assays of enzymatic activity. The buffer used inall reactions was 0.1 M K+-Na+ phosphate at the pHspecified. Individual enzymatic activities were deter-mined as follows.

Homoprotocatechuate 2,3-dioxygenase (enzyme a

of Fig. 1) was assayed by measuring the consumptionof 0, at 30 C by means of an oxygen electrode (9). Forextracts of Acinetobacter (see Table 4), the reactionmixture contained, in a total volume of 2.7 ml ofphosphate buffer (pH 7.5), 0.2 ml of 0.04 M ascorbicacid, 0.01 ml of 0.04 M FeSO4, and 1.5 to 6.3 mg of cellextract protein. After preincubation for 2.5 min, thereaction was started by adding 0.01 ml of 0.01 Mhomoprotocatechuic acid. In experiments when ex-

tracts of the two strains of P. putida were used, it wasnot necessary to activate this enzyme by addingascorbic acid and FeSO,, and these compounds weretherefore omitted from the reaction mixture.The dehydrogenase for compound II (enzyme b of

Fig. 1) was assayed by measuring the decrease inabsorbancy at 380 nm which occurred as II was oxi-dized. Cuvettes contained, in 1.0 ml of phosphatebuffer (pH 7.0), 11 nmol of II, 27 nmol of NAD, and0.1 to 0.4 mg of cell extract protein. The rate ofdisappearance of substrate was calculated from e=30,000 for compound II at pH 7.0.

Succinic semialdehyde dehyrogenase (enzyme f ofFig. 1) was assayed from the increase in absorbancy at340 nm as NADP was reduced. Reaction mixturescontained, in 1.2 ml of phosphate buffer (pH 7.5), 84

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nmol of succinic semialdehyde, 5 ,umol of MgCl,, 0.2pmol of NADP, and 0.6 to 0.8 mg of cell extractprotein.

4-Hydroxy-2-ketopimelate aldolase (enzyme e ofFig. 1) gives rise to succinic semialdehyde and pyru-vate, each of which can be determined in separateassays. In the first assay, the aldolase was coupledwith lactate dehydrogenase (type II crystalline, 865U/mg of protein; Sigma Chemical Co.), and thedecrease in absorbancy at 340 nm, due to oxidation ofNADH, was monitored during the reaction. Thisprocedure was suitable for Acinetobacter and Pseudo-monas T since NAD does not serve as coenzyme forthe succinic semialdehyde dehydrogenase that is alsopresent in cell extracts of these organisms. Reactionmixtures contained in 1.2 ml of phosphate buffer (pH7.5), 10 Mmol of MgCl,, 0.15 pmol of 4-hydroxy-2-ketopimelate, 0.2 pmol of NADH, 3.2 U of lactatedehydrogenase, and 0.1 to 0.8 mg of cell extractprotein. In the second assay, succinic semialdehydewas formed by the aldolase and then oxidized by thespecific NADP-dependent dehydrogenase containedin extracts. Increase in absorbancy at 340 nm wasmeasured for a reaction mixture similar to that usedin the first assay, except that lactate dehydrogenasewas omitted and NADP was added instead of NADH.The value e = 6,200 at 340 nm was used to calculateNAD(P)H formed or oxidized in these spectrophoto-metric assays.

Fractionation with ammonium sulfate. Cell ex-tracts were cooled in an ice bath, and powdered(NH,),SO, was added slowly with stirring to give therequired percentage of saturation. The precipitatewas removed by centrifugation and discarded, and asecond calculated amount of (NH,)S04 was added tothe supernatant solution. The precipitate was col-lected on the centrifuge and dissolved in a suitablevolume of phosphate buffer, pH 7.0.

RESULTSEnzyme-catalyzed formation of reaction in-

termediates: (i) isolation of compound II.Fresh, crude cell extracts of the three organismsrapidly oxidized homoprotocatechuic acid (I,Fig. 1), with an uptake of 1 mol of 03 per mol ofsubstrate, to give a yellow compound havingAmeX at 320 nm in acid, 380 nm in alkali, andwith absorption maxima at both these wave-lengths at pH 7.0. These are the spectral proper-ties reported previously (1, 4) for 5-carboxy-methyl-2-hydroxymuconic semialdehyde (II).For use as a substrate in later experiments,samples of compound II were conveniently pre-pared by using an extract of Pseudomonas U inwhich dehydrogenase b (Fig. 1) had been de-stroyed by being maintained at 57 C for 3 min,followed by removal of the precipitate by cen-trifugation. This treatment scarcely affectedthe activity of dioxygenase a. In each of a seriesof Warburg vessels containing 3 ml of phosphate

buffer (pH 7.5), 5 ,umol of homoprotocatechuatewas shaken with 0.5 ml of heat-treated extract(5 mg of protein) until uptake of 03 ceased.Flask contents were then pooled, deproteinizedwith 10% metaphosphoric acid, and extractedthree times with equal volumes of ether, and thesolution after drying with sodium sulfate wasevaporated to give yellow microcrystals of com-pound II.

(ii) Isolation of compound III. When acrude extract of Pseudomonas U was held at52 C for 2 min, the activity of enzyme b wasdiminished, but that of enzyme c was abolished.Accordingly, these extracts in the presence ofNAD converted homoprotocatechuate into com-pound III, having Xmex at 300 nm, which wasisolated as follows. Reaction mixtures contain-ing 100 umol of homoprotocatechuate, 135 j,molof NAD, and 5 ml of heat-treated extract (50 mgof protein) in 200 ml of phosphate buffer (pH7.5) were incubated for 1 h at 37 C until alltraces of the yellow intermediate compound IIhad disappeared. The mixtures were brought topH 1.5 by addition of HCl, the precipitate wasremoved by centrifugation, and the solution wasextracted four times with equal volumes of ethylacetate. When the extract was dried with so-dium sulfate and the ethyl acetate evaporated,a pale yellow solid remained that gave theultraviolet absorption spectrum shown in Fig.2a. The spectral characteristics, which are com-pared with those of 2-hydroxymuconic acid inTable 1, are consistent with the assignment ofstructure Ill, namely, 5-carboxymethyl-2-hy-droxymuconic acid. About 1.8 mg of the com-pound was treated with trimethylsilane, and themass spectrum showed that four trimethylsilane

(a) (b)0.4 0.4

0.3- 30.La

z

0.2- 0.2 au, acid base U

250 300 350 400 250 300 350

WAVE LENGTH (nm)FIG. 2. Ultraviolet absorption spectra of (a) 14 gM

5-carboxymethyl-2-hydroxymuconic acid, compoundIII; (b) 2-hydroxyhepta-2,4-diene-1,7-dioic acid, com-pound IV, approximate concentration 20 ,M.

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TABLE 1. Comparison of spectroscopic properties ofmetabolites with those of related compounds

Absorbance maxima (nm)Compound

Acid pH 7 Base

2-Hydroxymuconic acid 303a 295a 350aCompound III 304 300 345

2-Oxopent-4-enoic acid 270b 265b 305bCompound IV 278 274 307

a From Adachi et al (1).bFrom Bayly and Dagley (2).

groups were attached to the parent compound,indicating that the hydroxyl and three carboxylgroups were silylated as expected for III. Solu-tions that showed the ultraviolet absorption ofthis compound were also obtained by less con-venient procedures with the other two organ-isms. Fractionation of crude extracts of Acineto-bacter resulted in loss of dioxygenase activitywhich could be restored only by incubation withascorbic acid and FeSO,. However, withoutthese additions a fraction precipitating between20 and 40% saturation with ammonium sulfaterapidly oxidized compound II in the presence ofan equimolar amount of NAD to give solutionsexhibiting the spectra of Fig. 2a. A compoundwith these characteristics was also formed fromhomoprotocatechuate by extracts of Pseudomo-nas T when inhibited with sodium ethylenedi-aminetetraacetic acid. Extracts were first dia-lyzed for 95 min against phosphate buffer (pH7.0) containing 0.02 M ethylenediaminetetra-acetic acid, a procedure which diminished theactivity of dehydrogenase b besides inactivatingenzyme c. Cuvettes at 25 C contained, in 1 ml ofphosphate buffer (pH 7.5), 20 mM ethylenedi-aminetetraacetic acid, 0.1 mg of NAD, 0.1 umolof homoprotocatechuate, and 0.02 ml of dialyzedextract. After 2 h, compound II, which formedinitially, had disappeared and was replaced byIII. This compound was not metabolized furtheruntil an addition was made of 0.025 ml of 1.0 MMgCl2, when the peak at 300 nm rapidly disap-peared.Formation of compound IV. The last experi-

ment was repeated with the same concentra-tions of reactants and inhibitor, but in this casethe extract of Pseudomonas T was not dialyzedagainst ethylenediaminetetraacetic acid beforeuse. Under these conditions there was a transi-tory accumulation of compound III, but thefinal product had Xmax at 274 nm (Fig. 2b). Theaddition of MgCl2 again abolished the light

absorption peak. Table 1 shows that theseabsorption characteristics are consistent withthose expected for a dienoic hydroxyacid IVarising from III by decarboxylation. This wasconfirmed by forming a 2,4-dinitrophenylhydra-zone of the product, followed by reduction togive a saturated amino acid according to theprocedures of Bayly and Dagley (2). Theseprocedures required larger amounts of material,and reaction mixtures at pH 7.5 and 25 Ccontained 400 ml of 0.1 M phosphate buffer, 100ml of 0.1 M ethylenediaminetetraacetic acid, 40mg of NAD, and 0.43 g of cell extract protein.The reaction was started by the addition of 200,gmol of homoprotocatechuate after incubationfor 6 min and was stopped by addition of 39 mlof 6% perchloric acid 35 min later when theyellow color due to the presence of compound IIhad disappeared. The acidic dinitrophenylhy-drazone was formed from the deproteinizedsolution, extracted through ethyl acetate andsodium carbonate solution, reduced with Pdand H2 (2), and chromatographed with solventsC and D. Single spots were given with Rf valuesthe same as those for authentic 2-aminopimelicacid. When reaction mixtures were maintainedovernight at 4 C after incubating for 35 min at25 C, a second acidic dinitrophenylhydrazonecould be detected having the same chromato-graphic properties as the derivative of 4-hydroxy-2-ketopimelic acid (V). In the case ofAcinetobacter, an extract fraction collected at50 to 80% saturation with ammonium sulfatewas used to demonstrate the conversion ofcompound III into IV by following changes inlight absorption.Pyruvate and succinic semialdehyde. Ex-

tracts of the three organisms containedNAD(P)-dependent succinic semialdehyde de-hydrogenases; formation of pyruvate from ho-moprotocatechuate or its reaction intermedi-ates was determined in several experiments bymeans of lactate dehydrogenase. Succinic semi-aldehyde and pyruvate were identified as theproducts formed when 1.74 jimol of compound IIand 0.05 mg of NAD were incubated at 25 C for20 min with a 20 to 80% ammonium sulfatefraction of Acinetobacter extract. Chromatog-raphy of dinitrophenylhydrazones from the de-proteinized reaction mixture showed pyruvateand succinic semialdehyde, and when thesederivatives were reduced (2) amino acids weregiven with the R, values of authentic alanineand 4-aminobutyric acid, respectively. A similarexperiment was performed with compound V,except that NAD was omitted from the incu-

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bation mixture; succinic semialdehyde and pyr-

uvate were again identified as reaction prod-ucts.

Kinetics of formation of reaction interme-diates. The successive formation of compoundsin the sequence of Fig. 1 was shown by measure-

ments near their respective light absorptionmaxima, taken at intervals of time during thereaction (Fig. 3). When dilute extracts of Pseu-domonas T were used (60 lAg of protein per ml ofreaction mixture), rates of reaction were suffi-ciently slow to permit such measurements to bemade. The concentration of compound II, whichwas formed first from homoprotocatechuate,decreased immediately when NAD was addedat 6 min; and the amounts of compounds III andIV that accumulated were maximal at 15 and 40min, respectively.Stoichiometry of formation of pyruvate,

succinc semialdehyde, and carbon dioxide,and reduction of NAD. Different amounts of

TIME, minFIG. 3. Kinetics of formation of reaction intermedi-

ates from homoprotocatechuate. Absorbances weremeasured at the following wavelengths: compound II,380 nm (-); compound III, 300 nm (.....); com-

pound IV, 274 nm (- - -). The reaction mixture in I mlof phosphate buffer (pH 7.5) contained 50 nmol ofhomoprotocatechuate, and 27 nmol ofNAD was addedafter 6 min. At the point indicated by the arrow, 10Agmol of MgCI, was added.

homoprotocatechuate were incubated with adialyzed extract of Pseudomonas T for 2 h incuvettes at 25 C, at which time formation ofpyruvate was determined with lactate dehydro-genase. From 0.01, 0.02, 0.05 and 0.10 gmol ofhomoprotocatechuate there were formed, re-spectively, 0.01, 0.021, 0.052 and 0.096 umol ofpyruvate. Extracts of Pseudomonas T were alsoused to show the formation of 1 mol of pyruvateper 1 mol of compound II. Similar experimentswere performed with a 20 to 80% ammoniumsulfate fraction of Acinetobacter, at whichyields of succinic semialdehyde were also deter-mined. The average of three experiments (Table2) gave 0.92 and 0.97 jsmol of succinic semialde-hyde and pyruvate, respectively, from 1.00 umolof compound H. The carbon dioxide formedfrom homoprotocatechuate by an extract frac-tion of Acinetobacter was determined by War-burg respirometry. Reaction mixtures contained20 umol of ascorbic acid and 5 jsmol of FeSO4,which were added to activate the homo-protocatechuate dioxygenase present in 6 mg ofextract protein (in a total volume of 3 ml ofphosphate buffer, pH 7). When acid was tippedat 45 min to release CO2 from the buffer, thefollowing quantities were determined. From 3.0ismol of homoprotocatechuate, 3.1 Amol of CO2was evolved and 4.8 ,umol of O, was taken up.The consumption of 0, above that required byFig. 1 (1.6 Mmol instead of 1.0 ,mol) is ac-counted for by appreciable NADH oxidase ac-tivity at the extract concentrations required forrespirometry. Measurements made with an ex-tract of Pseudomonas T gave almost identicalresults.

Succinic semialdehyde dehydrogenase in Ac-inetobacter and Pseudomonas T requiredNADP, whereas NADP- and NAD-dependentactivities were present in Pseudomonas U. Acrude extract of this organism accumulated0.045 jsmol of pyruvate from 0.05 gmol ofhomoprotocatechuate and formed 0.094 ,umol of

TABLE 2. Stoichiometry of conversion of compound IIto pyruvate and succinic semialdehyde by an extract

of Acinetobactera

Compound II Pyruvate (nmol) Succinic semi-mo (nmol) aldehyde (nmol)

11.6 9.6 9.517.6 15.8 15.735 37 34

a Fraction precipitating at 20 to 80% saturationwith ammonium sulfate. Reactions reached comple-tion in 1.2 ml of phosphate buffer containing 10 Amolof MgCl2 and 0.4 mg of cell extract protein.

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NADH, whereas a heat-treated extract (52 C for2 min) formed 0.051 gmol of NADH from thesame amount of substrate and accumulatedcompound III.

Specificity of enzymes. In accordance withobservations made with the crystalline enzyme(26), we found that homoprotocatechuate 2,3-oxygenase in extracts of Pseudomonas T exhib-ited narrow substrate specificity, with little orno activity against catechol and 4-methylcate-chol and low activity against 3,4-dihydroxy-phenylpropionic acid. Racemic 3,4-dihydroxy-mandelic acid was oxidized to give a compoundsimilar to II but with Xmax at 310 nm for acid and373 nm for the base, and the amounts of 02consumed indicated that both enantiomerswere attacked. The enzyme did not attack2,3,4-trihydroxyphenylacetic acid, and thiscompound inhibited oxidation of homo-protocatechuate. This behavior contrasts withthat of protocatechuate 4,5-oxygenase, whichoxidizes 3,4,5-trihydroxybenzoic acid (gallicacid) as readily as protocatechuate (29). Mu-conic semialdehydes formed by meta-cleavageof other catechols were not oxidized by extractsof the three organisms. These compounds wereprepared in cuvettes by incubating suitableconcentrations of catechol or 3- and 4-methyl-catechols with heat-treated extracts of Pseudo-monas U grown with phenol (2); heating greatlyreduced but did not entirely abolish ring-fissionproduct dehydrogenases. Compound II was alsoprepared from homoprotocatechuate in situ byusing an extract of 4-hydroxyphenylacetate-grown Pseudomonas T that had been stored inthe frozen state for 8 weeks and lacked dehydro-genase activity. A 20 to 72% ammonium sulfatefraction of 4-hydroxyphenylacetate-grown Ac-inetobacter was active only against compoundII; NADP did not replace NAD in this reaction(Fig. 4). This same fraction rapidly metabolizedcompound III upon addition of 10 mM MgCl2but had no detectable activity against 2-hydroxymuconic acid, an intermediate in themeta-fission pathway of catechol (24, 25). Ittherefore appears that the substrate specificityof enzyme c (Fig. 1) is also narrow. The proper-ties of enzyme e will be reported later (21).Induction of enzymes. Washed-cell suspen-

sions of the three organisms grown at theexpense of various aromatic carbon sourcesother than 4-hydroxyphenylacetate failed tooxidize homoprotocatechuate at significantrates. This is shown in Table 3 for Acinetobac-ter, where growth with 3-hydroxyphenylacetateconferred ability to readily oxidize homogenti-sate, whereas homoprotocatechuate was oxi-

0.6 7

Enzyme

E 04 NAD

0

0.2 T

0

0 2 3 4TIME (min)

FIG. 4. Oxidation of meta-fission products by anextract of Acinetobacter grown with 4-hydroxy-phenylacetic acid. Ring-fission products were gener-ated in situ from: (a) 3-methylcatechol; (b) catechol;(c) 4-methylcatechol; and (d) homoprotocatechuate.In addition to the dioxygenase used in this reaction,the extract contained traces of ring-fission producthydrolase for (a) and dehydrogenases for (b) and (c).The disappearance of products was monitored fromthe decrease in absorbancy at 380 nm. At the timesindicated by the first arrow on each curve, 27 nmol ofNAD was added to cuvettes (1.2 ml of phosphatebuffer, pH 7.5). The extract of Acinetobacter (170,ugof protein) was added at the second arrow.

dized at a much slower rate, probably due tononspecific induction. Specific activities of en-zymes were also assayed for extracts of Acineto-bacter grown with phenylacetate and 4-hydroxy-phenylacetate, respectively. Enzymes a, b, ande (Fig. 1) were effectively absent for the firstgrowth substrate, and a fivefold increase in suc-

cinic semialdehyde dehydrogenase resultedafter growth with the second (Table 4).

DISCUSSIONThe proposed metabolic pathway (Fig. 1)

includes two compounds, III and IV, that havenot been described previously. Adachi et al. (1)showed that II was formed when homo-protocatechuate (I) was oxidized by crystalline3,4-dihydroxyphenylacetic acid 2,3-oxygenase;furthermore, Senoh et al. (26) proposed com-

pound III as the next intermediate in thereaction sequence, although no supporting datawere provided. We have isolated this compound

t 4 0

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from incubation mixtures and have shown thatits ultraviolet and mass spectral properties arethose expected for 5-carboxymethyl-2-hydroxy-muconic acid (III). It is formed by reactions ofsimilar type to those involved when nonfluores-cent pseudomonads degrade 4-hydroxybenzoateby meta-fission of protocatechuate, a propertyof those organisms of proved significance intaxonomic studies (28). Oxidation of the meta-fission product of protocatechuate by NAD(P)proceeds much faster than hydrolytic fission(11) and is probably the only route of physiolog-ical importance; however, in this system thebenzene nucleus is cleaved between C4 and C5of protocatechuate (9) whereas homoprotocate-chuate is cleaved between C2 and C3 (1).Furthermore, when 4-carboxy-2-hydroxymu-conic acid is formed from protocatechuate (11)or gallate (29), it is immediately hydrated, andcarbon dioxide is then released from the oxal-oacetate that arises when the hydration productundergoes aldol fission (30). By contrast, in Fig.1 the sequence of hydration and decarboxyla-tion is reversed: compound Im is decarboxylatedto give compound IV, which is then hydrated.Our evidence for this sequence rests mainlyupon the structure assigned to intermediates IV

TABiE 3. Rates of oxidation of aromatic acids bywashed cells of Acinetobacter

Oxygen uptakea

Aromatic acid oxidized 4-Hydroxy- 3-Hydroxy-phenyl- phenyl-

acetic acidb acetic acidb

4-Hydroxyphenylacetic ...... 11.5 <0.1Homoprotocatechuic ........ 10.6 1.03-Hydroxyphenylacetic ...... <0.1 8.6Homogentisic ............... <0.1 7.42-Hydroxyphenylacetic ...... <0.1 <0.1Phenylacetic ............... <0.1 <0.1

a Expressed as microliters of 0° per minute, mea-sured after subtraction of endogenous respiration(0.25 Mliters of 02 per min). Warburg cups contained,in 3 ml of phosphate buffer (pH 7.0), 4 mg (dryweight) of cells, 140 gsg of chloramphenicol, and 3Amol of an aromatic acid.

° Compound on which cells were grown.

and V. 4-Hydroxy-2-ketopimelic acid (V) wassynthesized by Leung et al. (21), and we haveshown that this compound is cleaved enzymati-cally to give the same products as those ob-tained by enzymatic degradation of homo-protocatechuate, namely, succinic semialde-hyde and pyruvate. The dinitrophenylhydra-zone of compound IV was reduced to give2-aminopimelic acid, indicating a dicarboxylicketoacid of seven carbon atoms which, in itsenol form, had the spectral properties of thedienoic hydroxyacid, 2-hydroxyhepta-2,4-diene-1,7-dioic acid.The catabolism of homoprotocatechuate (I)

bears a closer resemblance to the NAD-depend-ent degradation of 4-methylcatechol (25) thanto the meta-fission pathway for protocatechu-ate. Thus, P. putida cleaves 4-methylcatecholby meta-fission of the nucleus between C2 andC3; in that reaction sequence, decarboxylationof 5-methyl-2-hydroxymuconic acid precedesthe hydration step that furnishes the substratefor aldol cleavage, namely, L-(S)-4-hydroxy-2-oxohexanoate (8). However, the enzymes cata-lyzing the reactions of Fig. 1 exhibit rathernarrow substrate specificities and fail to me-tabolize intermediates in the degradation ofcatechol and methylcatechols. The various me-ta-fission pathways employ aldolases, hydra-tases, and hydrolases. These enzymes appar-ently catalyze their reactions by similar mech-anisms, each depending upon the presence inthe substrate molecule of an enolizable ketogroup and also, in some instances, a suitablyplaced ethylenic bond (8). Furthermore, most ofthe chemical structures which these enzymesmust bind are seldom, if ever, encountered asmetabolites outside their particular catabolicpathways. It has therefore been suggested thatthe enzymes of such a sequence might possess acommon ancestral gene (16).Compounds HI and IV accumulated in reac-

tion mixtures containing ethylenediaminetet-raacetic acid and were metabolized upon addi-tion of Mg2+ ions. On the basis of mechanismspreviously proposed for reactions of this type(8), stimulation of enzymes d and e (Fig. 1) bydivalent cations is to be expected. A similar

TABLE 4. Induction of enzymes for homoprotocatechuate degradation by Acinetobacter

Sp act (nmol per min per mg of protein)

Growth substrate Homopro- Dehydrogenase for 4-Hydroxy-2- Succinictocatechuate compoundae ketopimelate semialdehyde

2,3-dioxygenase aldolase dehydrogenase

Phenylacetic acid.<0.1 <0.1 0.7 6.54-Hydroxyphenylacetic acid.25 26 17 35

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SPARNINS, CHAPMAN, AND DAGLEY

reaction mechanism for the decarboxylationcatalyzed by enzyme c (Fig. 1) is shown in Fig. 5(sequence a). For an analogous reaction of4-oxalocrotonate, Sala-Trepat and Evans (24)proposed that the enol form of the substrateundergoes tautomerization before decarboxyla-tion. Their proposal was based on the observa-tion that a spectral change occurs when oxalo-crotonate is dissolved in buffer at pH 7.5, theXmax at 295 nm shifting to X at 237 nm. Thechange was found to be enzyme-catalyzed andwas accompanied by increased carbonyl reac-tivity and decreased enol reactivity. Upon thisview, the decarboxylation of compound IIIwould be formulated as shown in Fig. 5 (se-quence b). We consider that sequence a is moreprobable since: (i) the conversion of enol intoketo form, which is observed to be spontaneousand is therefore energetically favored, involvessignificant loss of conjugation in b, but not in a;and (ii) the isomer of III, in sequence a, pos-sesses a chemical structure more susceptible todecarboxylation than the keto tautomer in se-quence b. We have observed an enzyme-cat-alyzed spectral change for compound III, simi-

lar to that described for 4-oxalocrotonate. Ac-cording to sequence a, this enzyme should bedescribed as an isomerase rather than a tau-tomerase; but the question as to whether it is anecessary component of the overall reactionsequence must await purification of the decar-boxylase for III. It should be noted that no datarelating to the stereochemistry of compound IVare available; furthermore, participation of theisomer of III as an intermediate in sequence awould permit changes in configuration to occursince C4 and C5 are no longer joined by a doublebond.

ACKNOWLEDGMENTSWe are grateful for the facilities for mass spectrometry

provided and maintained by the Minnesota AgriculturalExperiment Station and for the skilled technical assistance ofTom Krick. We also thank Terry Sayther for help insynthesizing succinic semialdehyde.

This investigation was supported by Public Health Servicegrant ES Al 00678 from the National Institute of Environ-mental Health Sciences.

LITERATURE CITED1. Adachi, K., Y. Takeda, S. Senoh, and H. Kita. 1964.

Metabolism of p-hydroxyphenylacetic acid in Pseudo-

SEQUENCE a

COOH

H+ CH2- Coo-

Q:.cooH

117 , enol

COOH

M,3 isomer

COOH

CH2C02

Ncoo-0

H

Tv, enol

SEQUENCE b

COOH

CH2

-COO- >

N.A COO-C8ooH

H

, enol

COOH

CH2

-> COO-oocoo -

0

Co2>0

Ti, keto

COOH

CH2

<,COO-o

Iv, ketoFIG. 5. Alternative mechanisms for conversion of compound III into compound IV. Sequence a involves an

isomerization, sequence b involves a tautomerization of III.

166 J. BACTERIOL.

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