JOURNAL OF BACTERIOLOGY, June 1992, p. 3629-36360021-9193/92/113629-08$02.00/0Copyright ©) 1992, American Society for Microbiology

Vol. 174, No. 11

Carboxylation of Phenylphosphate by Phenol Carboxylase, anEnzyme System of Anaerobic Phenol Metabolism

ACHIM LACK AND GEORG FUCHS*Angewandte Mikrobiologie, University of Ulm, P.O. Box 4066, W-7900 Ulm, Gennany

Received 26 December 1991/Accepted 2 March 1992

Several lines of evidence indicate that the first step in the anaerobic metabolism of phenol is phenolcarboxylation to 4-hydroxybenzoate; this reaction is considered a biological Kolbe-Schmitt carboxylation. Aphenol carboxylase system was characterized by using a denitrifying Pseudomonas strain, K 172, whichcatalyzes an isotope exchange between '4CO2 and the carboxyl group of 4-hydroxybenzoate. The enzymaticisotope exchange activity (100 nmol min-' mg-' of protein) requires Mn2' and K+. We show that this systemalso catalyzes the carboxylation of phenylphosphate (the phosphoric acid monophenyl ester) to 4-hydroxyben-zoate and phosphate. The specific activity of phenylphosphate carboxylation at the optimal pH of 6.5 is 12 nmolof CO2 fixed min-' mg-' of protein. Phenylphosphate cannot be replaced by Mg2' -ATP and phenol. Thecarboxylase activity requires Mn2' but, in contrast to the isotope exchange activity, does not require K+. Theapparent Km values are 1.5 mM dissolved CO2 and 0.2 mM phenylphosphate. Several convenient assays forphenylphosphate carboxylation are described. The isotope exchange reaction and the net carboxylationreaction are catalyzed by the same oxygen-sensitive enzyme, which has a half-life in an air-saturated solutionof less than 1 min. Both activities cochromatographed with a protein with a Mr of 280,000, and both activitieswere induced only after anaerobic growth on phenol. The carboxylation of phenylphosphate suggests thatphenylphosphate itself is the physiological CO2 acceptor molecular of this novel CO2 fixation reaction.Alternatively, phenylphosphate could simulate the unknown natural precursor. It is suggested that theformation of an enzyme-bound phenolate anion from the activated phenolic compound is the rate-determiningstep in the carboxylation reaction.

Phenol is metabolized by microorganisms via two totallyunrelated strategies. Aerobically, phenol is hydroxylated tocatechol and the aromatic ring is cleaved by dioxygenases(10). Anaerobically, phenol appears to be metabolized viabenzoyl-CoA (5, 11, 18, 26, 31). The initial steps in anaerobicphenol catabolism for a denitrifying Pseudomonas strain(Fig. 1) and for sulfate-reducing bacteria (1) have beenstudied.

Different experiments indicated that phenol is carboxy-lated to 4-hydroxybenzoate. 4-Hydroxybenzoate is thenactivated to 4-hydroxybenzoyl-CoA, which subsequently isreductively dehydroxylated to benzoyl-CoA. Benzoyl-CoAis reduced to the coenzyme A (CoA) thioester of an alicycliccompound (16). This reaction sequence involves at least fourcharacteristic enzymes, phenol carboxylase, 4-hydroxyben-zoate-CoA ligase (AMP forming), 4-hydroxybenzoyl-CoAreductase (dehydroxylating), and benzoyl-CoA reductase(aromatic ring reducing). Evidence supporting this pathwayhas been discussed previously (5, 30, 31) as follows. (i)Anaerobic growth on phenol is CO2 dependent, whereasgrowth on 4-hydroxybenzoate is not. (ii) Cells grown onphenol incorporate substantial amounts of 14C02 into cellmaterial, four times more than do cells grown on 4-hydrox-ybenzoate. (iii) Cells grown on phenol plus nitrate catalyzean isotope exchange between 14C02 and 4-hydroxybenzoate;this is not the case with cells grown on 4-hydroxybenzoateplus nitrate. (iv) All activities required were demonstrated,and their regulation strongly supports the postulated func-tion in anaerobic phenol metabolism.

Subsequently, a phenol-induced enzyme that catalyzes theisotope exchange reaction between "4CO2 and the carboxyl

* Corresponding author.

group of 4-hydroxybenzoate observed in vivo was charac-terized in vitro (18). This reaction requires Mn2' and K+.The actual substrate is CO2 rather than bicarbonate, and theenzyme is not inhibited by avidin. The carboxylase, tenta-tively named phenol carboxylase, did not exchange[14C]phenol with the aromatic ring moiety of 4-hydroxyben-zoate. So far, a net carboxylation of phenol to 4-hydroxy-benzoate has not been observed in cell extracts.The properties of the isotope exchange reaction suggest

that in the course of the reaction a strongly enzyme-boundphenolate anion is formed by the decarboxylation of 4-hy-droxybenzoate (pK. at 25°C, 4.67 and 9.37 for COOH andOH groups, respectively) (6), which subsequently becomescarboxylated again. This exchange reaction is considered areversible partial reaction of the overall net carboxylationcatalyzed by the same enzyme. This implies that the sameenzyme-bound phenolate anion is an intermediate of the netcarboxylation reaction. However, the carboxylation of phe-nol would be mechanistically unfavorable at physiologicalconcentrations of the free phenolate anion (pK. at 25°C,10.0) (6) and CO2 (pKYa at 25°C, 6.3) (32), which are theactual substrates of the chemical carboxylation of phenol(Kolbe-Schmitt carboxylation) (12). This theoretical argu-ment and the lack of phenol exchange indicated that areactive phenol derivative is the intermediate in phenolcarboxylation in this organism, delivering a strongly en-zyme-bound phenolate anion. This would require the activa-tion of phenol or of the enzyme. The question, what may bethe actual phenolate source for this reaction, was addressedin this investigation.We demonstrate that phenylphosphate, the phosphoric

acid ester of phenol, is carboxylated by phenol carboxylase.Several convenient enzyme assays which facilitate the puri-

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3630 LACK AND FUCHS

SCoA

coo c=oX-11 ATP + 2 [H

2(- O HSCoA2p [

OH "'~~ 0 - ,-" OHAMP +

OHH20

OH o~~~~~~~ OH~~~~ pp1 O

phenolateanion

4-hydroxy-benzoate

4-hydroxy-benzoyl-CoA

SCoA

benzoyl-CoA

SCoA/C =O

cyclohex-1 -enecarboxyl-CoA

FIG. 1. Intermediates and enzymes involved in the initial steps of anaerobic phenol metabolism in the denitrifying Pseudomonas strain K172. T and c0, Phenol carboxylase system; 0, 4-hydroxybenzoate-CoA ligase (AMP forming); (0), 4-hydroxybenzoyl-CoA reductase(dehydroxylating); e, benzoyl-CoA reductase (aromatic ring reducing).

fication and characterization of this novel carboxylase sys-

tem are described.

MATERIALS AND METHODS

Materials and reagents. Chemicals and biochemicals were

obtained from Fluka (Neu-Ulm, Germany), BoehringerGmbH (Mannheim, Germany), and Serva (Heidelberg,Germany). ['4C]Na2CO3 (specific radioactivity, 1.85 GBqmmol-1) was purchased from Amersham Buchler (Braun-schweig, Germany). All chemicals were of the highest purityavailable. Disodium phenylphosphate (99%) (Fluka) was freeof contaminating 4-hydroxybenzoate. This was verified byseparating a 250 mM phenylphosphate solution by high-pressure liquid chromatography (HPLC) (see below) andmaking a photometric determination at 254 nm (4-hydroxy-benzoate content, <10 ,uM). Carbon dioxide (99.7%), 02-

free nitrogen gas (99.999%), and N2-H2 (95%:5%, vol/vol)gas mixtures were from Linde (H6llriegelskreuth, Germany)or Nussle (Ulm-Soflingen, Germany). Pseudomonas strainK 172 was described by Tschech and Fuchs (30).Growth conditions. Pseudomonas K 172 was cultivated

anaerobically at 28°C in a defined mineral medium (30) byusing 1-liter glass flasks or 50-liter stainless steel tanks.Phenol and CO2 or 4-hydroxybenzoate served as the carbonsources, and nitrate served as the electron acceptor. Thisstrain is unable to grow aerobically with phenol, but it doesgrow with 4-hydroxybenzoate. Growth was monitored bydetermining the apparent increase in the A.78 (1-cm lightpath), corrected for the absorbance of the medium. The dryweight content was 0.38 g of cells per liter of culture at an

A578 of 1. The culture was inoculated with 2% of an expo-nentially growing preculture. Cells were grown on 4-hydroxy-benzoate (5 mM) and nitrate (20 mM) in a batch culture.Cells grown on phenol and nitrate were continuously fedwith an anaerobic stock solution of 0.5 M phenol and 2 Mnitrate. The initial phenol, CO2, and nitrate concentrationswere 1, 20, and 4 mM, respectively. The substrates were

supplied via a motor-driven large syringe (Perfusor; Braun,Melsungen, Germany) through Viton tubing, and the solu-tion was filter sterilized. The applied rate of substrate supply(7.4 x 10 6 mol of phenol min'- per liter of culture at a celldensity of A578 = 1, four times more nitrate) was sufficientfor exponential growth with a generation time of 14 h. Thistechnique maintained the phenol concentration below 0.5mM and the nitrate concentration below 2 mM; nitrite wasnot detectable. The rate of substrate supply, based on a

molar growth yield of 42 g of dry cell matter formed per mol

of phenol consumed and a generation time of 14 h, was

calculated. In accordance with the preset exponential supplyof the limiting substrate, the cells grew exponentially with a

generation time of 14 h up to an A57., of 1. Then, the cellswere harvested by centrifugation with a separator (Westfa-lia, Oelde, Germany). The cell paste was stored frozen inliquid nitrogen until use.

Simultaneous adaptation experiments. Cells were culti-vated anaerobically on phenol or 4-hydroxybenzoate in1-liter glass flasks. At an A7.8 of 1, the cells were harvestedanaerobically by centrifugation at 10,500 x g for 10 min(4°C). The cells were washed twice in anaerobic mineral saltsmedium (30) free of CO2 and aromatic substrate and were

then suspended in the same medium at a final density ofA578of 10. The assay was performed in stoppered glass vials with10 ml of bacterial suspension of phenol-grown or 4-hydroxy-benzoate-grown cells. After 10 min of preincubation at 30°C,phenol (1 mM) and nitrate (5 mM) or 4-hydroxy-benzoate (1 mM) and nitrate (5 mM) were added. Parallelexperiments in which NaHCO3 (20 mM) was included were

carried out. Samples (500 ,ul) were withdrawn after 6, 12, 30,45, 60, 120, and 180 min of incubation at 30°C, cooled, andrapidly centrifuged aerobically in an Eppendorf centrifuge.The cell-free supernatant was analyzed by scanning the UVspectrum from 250 to 300 nm at pH 14.

Cell extracts. All steps were carried out under anaerobicconditions. Cells (3 g, wet weight) were suspended in 3 ml ofanoxic 50 mM imidazole-HCl (pH 7) solution containing 2mM dithioerythritol and 0.05 mg of DNase I. Cells werebroken by a French pressure cell (American InstrumentsCompany, Silver Spring, Md.) at 137 MPa. Cell debris wasremoved by centrifugation at 100,000 x g for 60 min or at39,000 x g for 20 min (4°C). For some experiments, thesupernatant (cell extract) was fractionated by gel filtration.

Protein determination. The protein content was deter-mined by the method of Markwell et al. (19), with crystallinebovine serum albumin as the standard.

Phenol carboxylase assays. The amount of phenol carbox-ylase activity was determined at 30°C under strictly anaero-

bic conditions by two types of assays. In the first type ofassay, the incorporation of 14C from 4C02 into the carboxylgroup of 4-hydroxybenzoate by an isotope exchange reac-

tion was determined. In brief, the method is as follows.Method 1: isotope exchange reaction. The assay mixture (1

ml; 4.8 ml of nitrogen gas, head space) contained 100 mMimidazole-HCl (pH 7), 1 mM MnCl2, 2 mM 4-hydroxyben-zoate, 20 mM KCl, 25 ,umol of 'CO2' ('CO2' is the totalamount of dissolved CO2, HCO3-, and CO2 gas) (50 ,ul of 0.5

phenol

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CARBOXYLATION OF PHENYLPHOSPHATE BY PHENOL CARBOXYLASE 3631

M NaHCO3 added per assay), and 50 ,u of cell extract (2 mgof protein). The complete assay was preincubated for 10 minin a shaking water bath before [14C]bicarbonate (15 kBq;final specific radioactivity, 600 Bq pumolVl of 'CO2') wasadded with a syringe to start the isotope exchange reaction.Samples (300 PA) were taken at different times and pipettedinto Eppendorf tubes containing 30 PlA of 3 M perchloric acid.The precipitate was centrifuged; from the clear supernatant,150 ,u was transferred into scintillation vials containing 150,ul of 1 M KHCO3. Formic acid (100 ,ul, 10 M) was added,and the solution was gassed with CO2 for 30 min in order toremove 14Co2. Then, 4 ml of scintillation cocktail wasadded, and the amount of 14C incorporated was determinedby liquid scintillation counting using external standardiza-tion. The molar amount of CO2 exchanged was calculatedfrom the amount of fixed 14C and the initial specific radio-activity of [14C]'C02' (18).Method 2. In the second type of assay, the carboxylation

of phenylphosphate to 4-hydroxybenzoate and phosphatewas determined by five different methods (a through e).Method 2a. Fixation of "4C into acid-stable products. The

phenylphosphate-dependent fixation of 14C from 14CO2 intoacid-stable products was studied essentially as describedabove for method 1. 4-Hydroxybenzoate was omitted, andthe reaction was started by adding 2 mM phenylphosphate asthe aromatic substrate. Because of unspecific phosphatasesin the extract, phenylphosphate was hydrolyzed to a signif-icant degree (0.25 mM in 30 min).Method 2b. Spectrophotometric assay. The phenylphos-

phate- and C02-dependent formation of 4-hydroxybenzoatewas determined spectrophotometrically by the determina-tion of the absorption of the sample at an alkaline pH (pH 14)at two wavelengths (235 nm and 280 nm) (Fig. 2). Samples(50 RI) were diluted 20-fold with 950 ,u of 1 M KOH. Thephenolate anion has an absorption maximum at 235 nm (r235= 9,400 M-1 cm-') (6); the 4-hydroxybenzoate dianion hasan absorption maximum at 280 nm (6280 = 16,300 M-1 cm-1)(6). Phenylphosphate has an absorption maximum at 266 nm;the molar absorption coefficient was estimated to be thefollowing: E266 = 580 M-1 cm-1. The respective molarabsorption coefficients at other wavelengths were estimatedfrom the UV spectra as follows: for phenol at 280 nm, r280 =2,190 M-1 cm-,; for 4-hydroxybenzoate at 235 nm, E235 =2,500 M--cm-1. The contribution of phenylphosphate to theA235 (r235 = 100 M-1 cm-') and A280 (F280 = 6 M-1 cm-')was neglected; this is justified since the respective valueswere <5% of the absorption contributed by either phenol or4-hydroxybenzoate. The concentrations of 4-hydroxyben-zoate and phenol were calculated from the absorption at 235and 280 nm according to two equations derived from Lam-bert-Beer's law (17). The amount of phenylphosphate in theassay samples which was not carboxylated or hydrolyzedwas calculated as the difference between the amount ofphenylphosphate added minus the amount converted intophenol and 4-hydroxybenzoate.Method 2c. HPLC analysis. The phenylphosphate- and

C02-dependent formation of 4-hydroxybenzoate and theformation of phenol due to hydrolysis were determined byHPLC and detection at 254 nm. The method was calibratedby internal standardization. The HPLC conditions includedthe following: a ,u-Bondapak reversed-phase C-18 column(Waters, Millipore Corp., Milford, Mass.); solvent, 150 mMpotassium phosphate buffer, pH 3.5, plus 0.1% (vol/vol)butanol at a flow rate of 1 ml min-'; and sample, 50 ul.Retention times were as follows (minutes): 18.7, phenol; 24,4-hydroxybenzoate; 27,3-hydroxybenzoate; 28.7,2-hydroxy-

1 .0

0.8

cXam-o30

-o

0.6

0.4

0.2

0.0

1 .0

cXa,

o0)n

0.8

0.6

0.4

0.2

0.0

240 260 280 300 320

Wavelength (nm)

240 260 280 300 320

Wavelength (nm)FIG. 2. Spectrophotometric analyses of reaction products of

phenylphosphate carboxylation. (A) Spectra of reference com-pounds at pH 14. 1, phenylphosphate (0.1 mM); 2, phenol (0.05mM); 3, 4-hydroxybenzoate (0.05 mM); 4, phenylphosphate (0.1mM) plus phenol (0.05 mM) plus 4-hydroxybenzoate (0.05 mM). (B)Spectra of deproteinized supernatant of the phenylphosphate car-boxylase assay at pH 14 after different periods of incubation. Fordescriptions of the preparations, see methods 1 and 2b in Materialsand Methods.

benzoate; and 54.4, benzoate. Phenylphosphate was elutedin the void volume.Method 2d. Phosphate assay. The phenylphosphate- and

C02-dependent release of Pi was determined. Samples (50RI) were diluted with 950 RI of distilled water. An aliquot of100 ,ul was added to 750 [lI of phosphate reagent (13); after 3min, 150 pI of 34% (wt/vol) citric acid was added, and after60 min of incubation, the A630 was measured. Calibrationwas by the Pi standard.Method 2e. Determination of 14C-labeled 4-hydroxyben-

zoate. The phenylphosphate- and "4C02-dependent fixationof 14C into 4-hydroxybenzoate was determined as describedfor method 2c. Fractions (1 ml) were collected, and 14C inthe 4-hydroxybenzoate fraction was determined by liquidscintillation counting using external standardization.

Sepharose CL-6B gel filtration. Cell extract (4.2 ml of100,000 x g supernatant) was chromatographed under an-aerobic conditions at 4°C on a Sepharose CL-6B column(Pharmacia, Freiburg, Germany; diameter, 3 cm; volume,

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0-

2E

a

on

0._

.0

E0

1.0

0.8

0.6

0.4

0.2

0 II I I

0 1 0 20 30

Incubation time (min)FIG. 3. Study of cellular regulation and of the C02 requirement

of anaerobic phenol metabolism. The simultaneous adaptationmethod was applied to thick suspensions (A578 = 10) of Pseudomo-nas K 172 cells. 0, phenol consumption in the presence of 'CO2' bycells pregrown on phenol; 0, phenol consumption by cells pregrownon phenol when CO2 was omitted; V, 4-hydroxybenzoate consump-

tion in the presence of 'CO2' by cells pregrown on phenol; V,

4-hydroxybenzoate consumption by cells pregrown on phenol when'CO2' was omitted; *, phenol consumption in the presence of 'CO2'by cells pregrown on 4-hydroxybenzoate. Note that these cellsimmediately used 4-hydroxybenzoate (not shown). In all experi-ments, the aromatic substrate was 1 mM and nitrate was 5 mM.'CO2', if present, was added as 20 pLmol of bicarbonate per ml ofsuspension. For details, see Materials and Methods.

607 ml). Imidazole-HCl buffer (pH 7; 100 mM) containing 2mM dithioerythritol was used at a flow rate of 40 ml h-1.Fractions (6.6 ml) were collected anaerobically. Phenolcarboxylase activity in 1-ml aliquots of the individual frac-tions was assayed by methods 1 and 2a. The fractionscontaining carboxylase activity were combined and used fordeterminations of the requirements of the enzyme in theisotope exchange reaction and in the phenylphosphate car-

boxylation reaction. The column was calibrated with thefollowing molecular mass (daltons) marker proteins: thyreo-globulin, 669,000; ferritin, 440,000; catalase, 232,000; andaldolase, 155,000.

RESULTS

Experiments with whole cells. The dependence of anaero-bic phenol metabolism on CO2 and the regulation of thephenol carboxylating enzyme system were studied withthick suspensions of whole cells of Pseudomonas strain K172 in mineral salts medium containing nitrate as the electronacceptor. Meaningful short lag phases in the substrate con-

sumption can only be observed with concentrated cell sus-

pensions, which normally consume the substrate within a

few minutes.Cells grown on phenol instantaneously metabolized phe-

nol provided that bicarbonate was added. When 'CO2' was

omitted, phenol degradation started only after a 10-min lagphase (Fig. 3). Phenol-grown cells were simultaneouslyadapted to metabolizing 4-hydroxybenzoate. The specific4-hydroxybenzoate consumption was similar in cells grownon phenol or 4-hydroxybenzoate (not shown in Fig. 3).4-Hydroxybenzoate metabolism, as opposed to phenol me-

tabolism, was unaffected by CO2. The absolute substrate

8000

0 L 600

co

.0 co0 0WI L 40010

200

00 50 100 150 200

Time (min)FIG. 4. Enzymatic carboxylation of phenylphosphate to 4-hy-

droxybenzoate catalyzed by a cell extract of Pseudomonas K 172.Cells were grown anaerobically with nitrate. *, extract (2 mg ofprotein) from cells grown on phenol; El, same as the above extractbut minus phenylphosphate; *, extract (2 mg of protein) from cellsgrown on 4-hydroxybenzoate.

consumption rates in different anaerobic preparations variedby up to a factor of two, but the relative differences in thesubstrate consumption rates were reproducible.

Cells grown on 4-hydroxybenzoate immediately metabo-lized 4-hydroxybenzoate, whereas the consumption of phe-nol started only after a lag phase of 120 min. These dataprovide additional evidence that phenol metabolism involvescarboxylation, that 4-hydroxybenzoate is a likely intermedi-ate of phenol metabolism, and that the phenol carboxylatingenzyme system is induced by phenol.

Carboxylation of phenylphosphate by extracts of cells grownon phenol. Extracts of cells anaerobically grown on phenolplus nitrate catalyzed a K+- and Mn2+-dependent 14C02-4-hydroxybenzoate isotope exchange reaction (100 nmolmin-' mg-'), but phenol was not carboxylated even whenMg2+-ATP was included (18). When 4-hydroxybenzoate wasomitted, virtually no 14CO2 was fixed into acid-stable prod-ucts (<0.1 nmol min-' mg-' of protein). However, when4-hydroxybenzoate was substituted by phenylphosphate inthe standard isotope exchange assay, a net fixation of 14Cfrom 14C02 into acid-stable products occurred (Fig. 4).

Phenylphosphate carboxylation was linearly dependent onthe amount of protein added (0.1 to 1.4 mg of protein ml-1)and proceeded linearly with time only for a few minutes (Fig.4). Afterwards, the reaction rate continuously decreased butwas detectable for several hours. When the reaction rate hadslowed down, the addition of phenylphosphate did not resultin an activity increase; this demonstrates that the substratewas not limiting. However, when cell extract was addedagain, the initial rate was obtained and then decreased againafter a few minutes. This indicates that the decrease inenzyme activity was not due to a product accumulation butwas most likely due to inactivation of the enzyme. Theaddition of boiled cell extract (95°C, 10 min) to the completeassay had no effect.

This reaction was strictly dependent on phenylphosphateand was extremely oxygen sensitive. In air-saturated solu-tion, the half-life of the activity was 30 s. Unfortunately, thepresence of a reducing compound such as dithioerythritol (2mM), Ti(III)citrate (1 mM) (34), or sodium dithionite (0.1mM) inhibited phenylphosphate-dependent 14C02 fixation.In addition, up to 0.1 mM [14C]formate was formed underreducing conditions, possibly catalyzed by a formate-accep-tor oxidoreductase.The phenylphosphate carboxylating enzyme activity was

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CARBOXYLATION OF PHENYLPHOSPHATE BY PHENOL CARBOXYLASE 3633

recovered almost exclusively in the 100,000 x g supernatant,it had a bell-shaped pH dependence with an optimum at pH6.5, and it had a specific activity of 12 nmol min-1 mg- ofprotein. Half-maximal activity was obtained at pHs 5.5 and7.8.

Requirements of the reaction. The carboxylation of phe-nylphosphate with the soluble protein fraction of phenol-grown cells was studied. Cell extract (supernatant centri-fuged at 100,000 x g) was fractionated by anaerobic gelfiltration on Sephadex CL-6B, and phenylphosphate carbox-ylating fractions were combined. The carboxylation of phe-nylphosphate catalyzed by this protein fraction was strictlydependent on CO2, phenylphosphate, and Mn2+, but not onK+. A strict K+ and Mn2' dependence has been reported forthe isotope exchange reaction (18) and was confirmed. Thesame results were obtained after the precipitation of phenolcarboxylase by ammonium sulfate (60% saturation).The dependence of the phenylphosphate carboxylating

enzyme activity on the individual substrates and on thecocatalyst Mn2+ was determined at nearly saturating con-centrations of all other reactants. The reaction followedMichaelis-Menten kinetics. The apparent Km values were0.2 mM phenylphosphate, 1.5 mM dissolved CO2 (not in-cluding dissolved bicarbonate), and 2 mM Mn2+. The appar-ent Km value for Mn2+ may be much lower, since phe-nylphosphate (2 mM) complexes some Mn2' and thereforecompeted for cocatalyst.

Analysis of the products of phenylphosphate carboxylationand independent assays of phenylphosphate carboxylase activ-ity. The products of phenylphosphate carboxylation activityshould be either 2-hydroxybenzoate (salicylate; ortho car-boxylation) or 4-hydroxybenzoate (para carboxylation). Theproducts of phenylphosphate carboxylation formed during300 min of incubation were monitored by five differentmethods (for details of methods 2a through e, see Materialsand Methods) (Fig. 5) as follows. (i) The substrate andpossible products of phenylphosphate metabolism were an-alyzed by HPLC and detected by UV absorption at 254 nm(method 2c). There was only one acid-stable product ofphenylphosphate carboxylation activity, which cochromato-graphed with authentic 4-hydroxybenzoate and whose spec-trum at pHs of 7 and 14 was identical with that of 4-hydroxy-benzoate (see Fig. 2 for spectra at pH 14). (ii) In a similarassay, CO2 was 14C labeled and the amount of 14C in theindividual HPLC fractions was determined (method 2e). 14Cwas found only in the 4-hydroxybenzoate fraction. Theamount of 14C incorporated was identical with the amount of4-hydroxybenzoate formed, as determined by the spectro-photometric uantitation of method 2c. (iii) The phenylphos-phate- and 1 CO2-dependent fixation of radioactivity intoacid-stable products was analyzed without purification of thereaction products (method 2a). The results were identicalwith those obtained by measuring 14C in the HPLC fractionof 4-hydroxybenzoate (method 2e). This shows that no otherlabeled products were formed. (iv) 4-Hydroxybenzoate andphenol formation were monitored by recording the spectra ofdiluted aliquots at pH 14, as described for method 2b. Theresults obtained by this simple method were completelyconsistent with the more-cumbersome HPLC determinationof substrates and products or with the 14C methods. (v) Thephenylphosphate- and C02-dependent release of Pi couldalso serve as a measure for phenylphosphate carboxylation;however, because of the high background of phosphateformation due to unspecific phenylphosphate hydrolysis,this method is not suitable for cell extracts.

Determination of reaction stoichiometry using cell extract.

2.0

0EU)

0

0~

a-

1.5

1.0

0.5

0.0

4.0

0E

3.5 50

3.0 .c0.0

02.5 L

2.0 0-

Time (min)

U)

U4i

00U-)

D0 X

00

U)

10 0.'L-

0 Oc

600

500

400

300

200

100

00 50 100 150 200 250 300

Time (min)FIG. 5. Time course of ['4C]4-hydroxybenzoate and phosphate

formation from phenylphosphate plus 14C02 by cell extracts ofPseudomonas K 172. (A) O, phenylphosphate; V, 4-hydroxyben-zoate determined spectrophotometrically; V, phenol determinedspectrophotometrically; El, 4-hydroxybenzoate determined byHPLC; *, Pi. For details, see methods 2b, -c, and -d in Materialsand Methods. (B) 0, '4C in acid-stable products; El, 14C in the4-hydroxybenzoate fraction of HPLC. For descriptions of the radio-active assay and HPLC separation, see methods 2a and -c inMaterials and Methods. Note that besides phenylphosphate carbox-ylation, an unspecific phenylphosphate hydrolysis to phenol andphosphate occurred. The amount of phenylphosphate was calcu-lated from the sum of 4-hydroxybenzoate and phenol formed.

The stoichiometry of the reaction was determined by HPLCanalysis of the aromatic compounds and colorimetric phos-phate determination (methods 2c and d) (Fig. 5). After 300min of incubation of 4 ,umol of phenylphosphate, 1.2 ,umol of4-hydroxybenzoate and 1 ,umol of phenol were formed; 2.2,umol of phenylphosphate was consumed. Approximately 2,umol of phosphate was formed from phenylphosphate. Thisstoichiometry excludes the possibility that some contami-nant in the commercial phenylphosphate was carboxylatedby phenol carboxylase. If CO2 was omitted, 4-hydroxyben-zoate was not formed and less phosphate was released,suggesting that phenylphosphate hydrolysis was unspecific.

Molecular mass determination of native phenylphosphatecarboxylase and of the enzyme catalyzing the radioisotopeexchange reaction. When cell extract was fractionated on

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3634 LACK AND FUCHS

1.2

1.0

0.80CO4< 0.6

0.4

0.2

0.0

50 --

0.6b

20.4 CdE

I ~~~~~~~~x0.2-

0.0 30 Ei05 Da 106l

20 --

10

E10 N

0 100 200 300 400 500 600 700

Elution volume (ml)FIG. 6. Cochromatography of the phenylphosphate carboxylating enzyme activity (0) with the '4CO2-4-hydroxybenzoate isotope

exchange enzyme activity (0) during gel filtration with Sepharose CL-6B. The insert shows the determination of the molecular mass of thenative phenol carboxylase. The molecular mass standards in the calibration curve were thyreoglobulin (669 kDa) (a), ferritin (440 kDa) (b),catalase (232 kDa) (c), and aldolase (155 kDa) (d).

Sepharose CL-6B, both the phenylphosphate carboxylatingenzyme activity and the 14C02-4-hydroxybenzoate isotopeexchange activity cochromatographed in a symmetrical peak(Fig. 6). The ratio of these two activities was constant in theactive fractions and was approximately 1:3. The peak frac-tion corresponded to a molecular mass of 280,000 Da. Gelfiltration resulted in an 80% loss of both activities. Thecochromatography of these two activities is a strong argu-

ment for the identity of the enzyme catalyzing both activi-ties.

Expression of phenylphosphate carboxylating activity. An-other argument in support of one enzyme is the similarregulation. Extract from cells grown on 4-hydroxybenzoatecatalyzed neither the 14C02-4-hydroxybenzoate isotope ex-

change reaction nor the phenylphosphate carboxylation re-

action. Cells grown on either substrate catalyzed phe-nylphosphate hydrolysis. This reaction is ascribed to an

unspecific phosphatase, e.g., alkaline phosphatase (q28),which had a specific activity of 7 nmol min-1 mg- ofprotein, independent of the growth substrate of the cells.

DISCUSSION

We have demonstrated that phenylphosphate is carboxy-lated to 4-hydroxybenzoate plus phosphate by a solubleenzyme system. This enzyme is involved in the anaerobicmetabolism of phenol and catalyzes also the 14C02-4-hy-droxybenzoate isotope exchange reaction. The argumentsthat a single enzyme catalyzes these two activities are (i)both enzyme activities are induced by phenol, (ii) bothenzyme activities cochromatograph, (iii) the enzyme activi-ties are similarly oxygen sensitive, and (iv) they requireMn2+ as a cocatalyst. It has yet to be shown whetherphenylphosphate is a physiological intermediate of anaero-bic phenol metabolism in growing cells and, if so, how it isformed from phenol. The experiments with whole cellssupport the proposal that phenol metabolism requires CO2,

that the active enzyme system is only present in cells grownon phenol, and that the formation of sufficient amounts ofactive enzyme requires 2 h.The whole system converting phenol to 4-hydroxyben-

zoate appears to be complex. Comparison of the proteinpattern of cells grown on phenol or 4-hydroxybenzoaterevealed five additional proteins in the range of 65 to 13 kDain phenol-grown cells (18). The molecular mass of 280,000Da of native phenylphosphate carboxylase suggests that theenzyme is composed of subunits. Purification is impeded bythe extreme oxygen sensitivity of the protein and the nega-tive effect of reducing agents in the assay. The inactivationof a carboxylase by oxygen is uncommon and indicates thatthe reaction mechanism of the enzyme may be unusual.The formation of phenylphosphate from phenol requires

an energy-rich phosphate donor, e.g., a nucleotide triphos-phate, pyrophosphate, or phosphoenolpyruvate. There arenumerous examples of nucleotide triphosphate-dependentphosphorylation of phenolic OH groups, e.g., by tyrosinekinases. Furthermore, a phosphate ester can be formed fromglycosidic OH groups in a phosphotransferase reaction withphosphoenolpyruvate as the energy-rich phosphate eno-lester. The best-studied example is the sugar phosphotrans-ferase system of certain anaerobic or facultative bacteria(20). Such a system would represent an efficient mechanismof phenol transport via phosphate group translocation.Why does phosphorylation of phenol activate the sub-

strate for carboxylation? In any enzyme-catalyzed reactionin which a carbon-carbon bond is formed, there must be acarbanion (or a carbanion equivalent) to serve as the attack-ing nucleophile at some other carbon atom that is electro-philic, e.g., CO2 (33). Therefore, a stabilized carbanion mustbe formed, in the case of phenol carboxylation, this carban-ion is the phenolate anion (pKa = 10.0). Since at a neutral pHthere is only a small amount of phenolate anion and thecarboxylation direction is endergonic, an anionic leavinggroup would greatly help to form the carbanion and to drive

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CARBOXYLATION OF PHENYLPHOSPHATE BY PHENOL CARBOXYLASE 3635

14 CO0ecoo

1 4

OH P <3

02H

H2O~~~~~

0a0

LH COO COO

OH

- P= O

o08FIG. 7. Unifying scheme of the two catalytic properties of the

phenol carboxylase system, (i) the reversible isotope exchangereaction between '4CO2 and the carboxyl group of 4-hydroxyben-zoate (K+ and Mn2' dependent) and (ii) the exergonic carboxylationof phenylphosphate to 4-hydroxybenzoate and phosphate (Mn2+dependent). The common intermediate is suggested to be an en-zyme-bound nucleophilic phenolate anion. The electrophilic addi-tion of CO2 rather than bicarbonate is freely reversible.

the reaction to the product side. Otherwise, 4-hydroxyben-zoate decarboxylation would be favored because of thearomatic ring, which functions as a built-in electron sink tostabilize the incipient carbanion formed on the loss of CO2.A leaving group with a sufficiently large free energy changeof hydrolysis would be phosphate in phenylphosphate. Thephenolic OH group is far more acidic (pKa = 10) than analiphatic OH group (pKa of ethanol = 18). Therefore, thefree energy of hydrolysis of phenylphosphate is more similarto an energy-rich phosphate-anhydride bond than to a low-energy phosphate-ester bond.A unifying scheme describing phenylphosphate carboxy-

lation and 14CO2-4-hydroxybenzoate isotope exchange via acommon, strongly enzyme-bound phenolate anion is shownin Fig. 7. The phenolate anion must be protected from water,which could explain why it is not freely exchangeable withphenol. It may be formed either by reversible decarboxyla-tion of 4-hydroxybenzoate or by hydrolysis of phenylphos-phate. Hence, the enzyme may have a dual function, (i) tocatalyze a base-catalyzed exergonic cleavage of the phenol-phosphate ester bond, and (ii) to stabilize the phenolateanion, keeping off water while allowing CO2 to attack. Onefeature is difficult to explain. The isotope exchange reactionis dependent on K+, in contrast to the phenylphosphatecarboxylation reaction. Both reactions, however, requireMn2+. This seems to suggest that K+ is required to decar-boxylate 4-hydroxybenzoate but not to carboxylate theintermediate.Phenol carboxylation is considered a biological Kolbe-

Schmitt reaction. Not only phenol but also o-cresol wasshown to be metabolized anaerobically by nitrate-reducingbacteria via para carboxylation (3, 25). Interestingly, alsom-cresol has been reported to become para carboxylated(24). Substrates besides phenol which are suitable for aparaor ortho carboxylation would be para-hydroquinone, a-

napththol, 3-aminophenol, and phenol with alkyl substitu-ents in one or both ortho positions (12).

Indirect evidence for thepara carboxylation of phenol hasbeen obtained with mixed cultures of various anaerobicbacteria (1, 2, 8, 9, 14, 15, 35; for a different type of phenolmetabolism, see reference 22). However, in most casesstudied, it remained unclear whether 4-hydroxybenzoate is afree intermediate or whether benzoate is formed directly. Anenzyme system which catalyzes the H/D exchange betweenD20 and the proton at C-4 but not at C-2 of phenol wasdescribed previously (7). This exchange would also requirethe formation of a phenolate ion, the abstraction of a proton,and the addition of a deuterium specifically at C-4, the site ofcarboxylation. In this case, apparently phenylphosphate isnot involved.Two similarly fascinating carboxylation reactions have

been recently documented, the carboxylation of aniline to4-aminobenzoate (27) and the carboxylation of acetone to3-oxobutyrate (4, 23), although no enzymatic properties areknown. It will be interesting to test whether the phosphateamide of aniline and the enolphosphate of acetone, respec-tively, is being used as a substrate. The equilibrium constantof the acetone carboxylation,

CH3-CO-CH3 (aq) + CO2 (g) -- CH3-CO-CH2-COO (aq) + H+ (aq)where aq is aqueous and g is gas, is near 10-4 (Go' = 22kJ/mol) (29). At pH 7, an acetone concentration of 1 mM,and 1 atm of CO2 (1 atm = 101.29 kPa), the resulting3-oxobutyrate concentration would be near 0.1 ,uM. Thisindicates that the activation of the substrate is important formechanistic and thermodynamic reasons. In the case ofanaerobic phenol metabolism, the product 4-hydroxyben-zoate is removed by an active CoA ligase (21, 31, 36).

ACKNOWLEDGMENTS

This investigation was financially supported by the DeutscheForschungsgemeinschaft and the Fonds der Chemischen Industrie.

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