Journal of Bacteriology, July 2004, p . 4556-4567, Vol . 186, No . 14

Phenylphosphate Carboxylase: a New C-C Lyase Involved in Anaerobic Phenol Metabolism in Thauera aromatica

Karola Schühle and Georg Fuchs^*

Mikrobiologie, Institut für Biologie II, Universität Freiburg, D-79104 Freiburg, Germany

Received 27 February 2004/ Accepted 19 April 2004

 
The anaerobic metabolism of phenol in the beta-proteobacterium Thauera aromatica proceeds via carboxylation to 4-hydroxybenzoate and is initiated by the ATP-dependent conversion of phenol to phenylphosphate . The subsequent para carboxylation of phenylphosphate to 4-hydroxybenzoate is catalyzed by phenylphosphate carboxylase, which was purified and studied . This enzyme consists of four proteins with molecular masses of 54, 53, 18, and 10 kDa, whose genes are located adjacent to each other in the phenol gene cluster which codes for phenol-induced proteins . Three of the subunits (54, 53, and 10 kDa) were sufficient to catalyze the exchange of ¹⁴CO₂ and the carboxyl group of 4-hydroxybenzoate but not phenylphosphate carboxylation . Phenylphosphate carboxylation was restored when the 18-kDa subunit was added . The following reaction model is proposed . The ¹⁴CO₂ exchange reaction catalyzed by the three subunits of the core enzyme requires the fully reversible release of CO₂ from 4-hydroxybenzoate with formation of a tightly enzyme-bound phenolate intermediate . Carboxylation of phenylphosphate requires in addition the 18-kDa subunit, which is thought to form the same enzyme-bound energized phenolate intermediate from phenylphosphate with virtually irreversible release of phosphate . The 54- and 53-kDa subunits show similarity to UbiD of Escherichia coli, which catalyzes the decarboxylation of a 4-hydroxybenzoate derivative in ubiquinone (ubi) biosynthesis . They also show similarity to components of various decarboxylases acting on aromatic carboxylic acids, such as 4-hydroxybenzoate or vanillate, whereas the 10-kDa subunit is unique . The 18-kDa subunit belongs to a hydratase/phosphatase protein family . Phenylphosphate carboxylase is a member of a new family of carboxylases/decarboxylases that act on phenolic compounds, use CO₂ as a substrate, do not contain biotin or thiamine diphosphate, require K⁺ and a divalent metal cation (Mg²⁺or Mn²⁺) for activity, and are strongly inhibited by oxygen .

 
The anaerobic metabolism of phenol has been studied to some extent in the beta-proteobacterium Thauera aromatica . The two initial steps of the pathway consist of the phosphorylation of phenol to phenylphosphate and the carboxylation of phenylphosphate to 4-hydroxybenzoate (36-38) (Fig. 1) . Both enzyme activities are induced in cells grown anoxically on phenol and nitrate and not in cells grown on 4-hydroxybenzoate, the product of this process . 4-Hydroxybenzoate is also an intermediate in the metabolism of p-cresol (53)

 
Further metabolism of 4-hydroxybenzoate proceeds via benzoyl coenzyme A (benzoyl-CoA) in two steps (Fig . 1) . A specific CoA ligase forms 4-hydroxybenzoyl-CoA (7, 25), which is reductively dehydroxylated to benzoyl-CoA by a molybdo-flavo-iron-sulfur protein, 4-hydroxybenzoyl-CoA reductase (13, 15, 26) . The electron donor is a 2-[4Fe/4S] ferredoxin which is reduced by 2-oxoglutarate-ferredoxin oxidoreductase (21) . Benzoyl-CoA is a common intermediate in the metabolism of many aromatic compounds . It is reductively dearomatized (11, 12), and further metabolism results in the production of three acetyl-CoA molecules and one CO₂ molecule (note that one CO₂ molecule was originally introduced into phenol) (reviewed in reference 31) .

Phenol-induced cells contain an enzyme activity, E₁, that catalyzes the Mg-ATP-dependent conversion of phenol to phenylphosphate, Mg-AMP, and inorganic phosphate (36, 37; S . Schmeling, A . Narmandakh, O . Schmitt, K . Schühle, and G . Fuchs, unpublished results) . The use of ATP makes this endergonic carboxylation process unidirectional even in the presence of ambient concentrations of phenol and CO₂; at the same time the electron-withdrawing phosphoryl group makes phenylphosphate a poor substrate for a conventional electrophilic attack by CO₂ .

Hence, the subsequent phenylphosphate carboxylase, E₂ (36), which is also a phenol-induced enzyme, is expected to exhibit special features . It requires divalent metal ions (Mg²⁺ or Mn²⁺) and catalyzes the carboxylation of phenylphosphate to 4-hydroxybenzoate (equation 1) (referred to as the net carboxylation reaction) . Simultaneously, an enzyme activity catalyzing an exchange of free ¹⁴CO₂ and the carboxyl group of 4-hydroxybenzoate (equation 2) is induced (referred to as the CO₂ exchange reaction) . This reaction has been proposed to be a partial reaction of the phenylphosphate carboxylase-catalyzed reaction (36) . Free [¹⁴C]phenol did not exchange with the phenol moiety of phenylphosphate (38) . This suggests that the E₂ enzyme follows a ping-pong mechanism .
 

The postulated mechanism involves an enzyme E₂-phenolate intermediate (equations 3 and 4), which is also formed in a presumably exergonic reaction from phenylphosphate (equation 5), followed by the reversible carboxylation reaction (equation 6) . The actual substrate is CO₂ rather than bicarbonate (as is the case for biotin-dependent carboxylases), and the carboxylating enzyme is not inhibited by avidin, a potent inhibitor of biotin-dependent carboxylases . Both of these results suggest that biotin is not involved in carboxylation .

Two-dimensional gel electrophoresis resulted in identification of a number of phenol-induced proteins, some of which were N terminally sequenced . A DNA segment which included the open reading frames (ORFs) coding for six of the phenol-induced proteins identified was cloned and sequenced (Fig . 2) (16; K . Schühle, H . Schägger, and G . Fuchs, unpublished results) . All but two of these ORFs are transcribed in the same direction and are thought to be involved in the anaerobic metabolism of phenol or related phenolic compounds . Another gene, orf11, which is transcribed in the opposite direction, was found upstream of the gene cluster . This gene codes for a protein similar to a regulatory protein (DmpR) of Pseudomonas putida which controls transcription of the genes of aerobic phenol metabolism . This may suggest a similar role for the orf11 gene product (ORF11) in the regulation of anaerobic phenol metabolism in T . aromatica .

 
The products of the orf1 and orf2 genes show similarities to parts of phosphoenolpyruvate synthetase (46), and these genes are thought to code for enzyme E₁, phenylphosphate synthase . ORF4, ORF6, and ORF7 showed similarity to the ubiD gene product of Escherichia coli, 3-octaprenyl-4-hydroxybenzoate carboxylyase; UbiD catalyzes the decarboxylation of an isoprenylated derivative of 4-hydroxybenzoate in ubiquinone biosynthesis . ORF8 is similar to the ubiX gene product, an isoenzyme of UbiD . It has been proposed that (some of) these proteins are involved in the carboxylation of phenylphosphate; this proposal was based on analogy of the 3-octaprenyl-4-hydroxybenzoate decarboxylase and phenylphosphate carboxylase reactions (16) . Also, a 54-kDa 4-hydroxybenzoate decarboxylase from Clostridium hydroxybenzoicum (35) and subunits of various proven or putative vanillic acid decarboxylases from E . coli, Bacillus subtilis, and Streptomyces sp . show similarity to UbiD and UbiX (18, 20, 40), yet these enzymes are likely to function as decarboxylases, yielding phenolic compounds .

It was the aim of this work to purify and study enzyme E₂, phenylphosphate carboxylase, and to identify the genes coding for its subunits . Furthermore, we investigated whether phenylphosphate carboxylation and the ¹⁴CO₂ exchange reaction are catalyzed by the same enzyme and, if so, whether the ¹⁴CO₂ exchange reaction is a partial reaction catalyzed by a fragment of the holoenzyme .

 
Materials. Chemicals were purchased from Bio-Rad (Munich, Germany), Biomol (Hamburg, Germany), Fluka (Neu-Ulm, Germany), Merck (Darmstadt, Germany), Roth (Karlsruhe, Germany), Serva (Heidelberg, Germany), or Sigma-Aldrich (Deisenhofen, Germany) . Biochemicals were obtained from Roche Diagnostics (Mannheim, Germany) . The scintillation cocktail and acrylamide stock solution were purchased from Roth . Na₂¹⁴CO₃ (2 GBq mmol^–1), 4-[¹⁴C]hydroxybenzoate (1.85 GBq mmol^–1), and [U-¹⁴C]phenol (2.7 GBq mmol^–1) were purchased from American Radiolabeled Chemicals Inc./Biotrend Chemikalien GmbH (Cologne, Germany) . H₂¹⁸O was obtained from Campro (Emmerich, Germany) . Gases were purchased from Sauerstoffwerke Friedrichshafen (Friedrichshafen, Germany) . Enzymes were obtained from MBI Fermentas (St . Leon-Rot, Germany) and Amersham Biosciences (Freiburg, Germany) . Positively charged nylon membranes for phage hybridization were obtained from Schleicher and Schuell (Dassel, Germany) . Materials and equipment for protein purification were obtained from Amersham Biosciences .

Growth conditions, cell harvesting, and storage. T . aromatica type strain K 172 (= DSMZ 6984) (1, 60) was cultured anaerobically on mineral salt medium at 30°C in a 200-liter fermentor . Cultivation was started with 0.5 mM phenol, 10 mM KHCO₃, and 2 mM NaNO₃ or with 5 mM 4-hydroxybenzoate-Na⁺ and 15 mM NaNO₃ as carbon and energy sources (13, 60) . After growth had started, the fermentor was operated in a fed-batch mode (22) . Growth was measured by monitoring the optical density at 578 nm with a 1-cm light path . An optical density at 578 nm of 1.0 corresponded to a concentration of approximately 0.3 g (dry weight) of cells per liter . The harvested cells were immediately frozen and stored in liquid nitrogen . E . coli strains XL1blue MRF' {(mcrA)183 (mcrCB-hsdSMR-mrr)173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac F' proAB lacI^qZM15 Tn10 (Tet^r)} and XLOLR (mcrA)183 (mcrCB-hsdSMR-mrr)173 endA1 thi-1 recA1 gyrA96 relA1 lac [F' proAB lacI^qZM15 Tn10 (Tet^r)] Su^{– R}} (Stratagene, Heidelberg, Germany) used in screening and E . coli SURE {e14^– (McrA^–) (mcrCB-hsdSMR-mrr)171 endA1 supE44 thi-1 gyrA96 relA1 lac recB recJ sbcC umuC::Tn5 (Kan^r) uvrC F' proAB lacI^qZM15 Tn10 (Tet^r)} (Stratagene, Heidelberg, Germany) and TUNER(DE3)pLacI [F^– ompT hsdS_B (r_B^– m_B^–) gal dcm lacY1 (DE3) pLacI(Cm^r)] (Novagene, Darmstadt, Germany) used in overexpression experiments were grown at 37°C in Luria-Bertani medium (54) . Anaerobic cultivation of E . coli was performed in TGYEP medium (6) at 20°C . Antibiotics were added to E . coli cultures at the following final concentrations: ampicillin, 50 to 100 µg/ml; chloramphenicol, 34 µg/ml; kanamycin, 50 µg/ml; and tetracycline, 20 µg/ml .

Preparation of cell extract. All cell extract preparation steps were performed under strictly anaerobic conditions . Frozen cells (5 g) were suspended in 5 ml of a solution containing 10% glycerol, 0.5 mM dithioerythritol (DTE), and traces of DNase I, disrupted by a passage through a French press (137 MPa), and ultracentrifuged (1 h at 100,000 x g and 4°C) . The supernatant containing the soluble protein fraction (approximately 80 mg of protein/ml) was used immediately or was stored at –20°C for later experiments . The soluble protein fraction (cell extract) contained all ¹⁴CO₂-4-hydroxybenzoate isotope exchange activity and the net phenylphosphate carboxylation activity .

Enzymatic tests. All enzymatic tests were conducted at 30°C under strictly anaerobic conditions . The assays were performed with 1-ml assay mixtures in stoppered vials (or cuvettes) having an approximately 250-µl gas phase . The radioactive assays for ¹⁴CO₂-4-hydroxybenzoate isotope exchange and the net carboxylation of phenylphosphate with ¹⁴CO₂ were performed as described elsewhere (36, 38) . The standard assay mixture contained enzyme (0.3 to 1 µg of protein), 20 mM 2-(N-morpholino)ethanesulfonate (MES)-K⁺ buffer, 10% (vol/vol) glycerol, 50 mM ß-mercaptoethanol, 20 mM KCl, and 2 mM MgCl₂ . The assay mixture for the net carboxylation of phenylphosphate also contained 2 mM phenylphosphate-Na⁺ and 20 mM NaH¹⁴CO₃ (0.2 Bq nmol^–1), and the CO₂ exchange assay mixture also contained 2 mM 4-hydroxybenzoate-Na⁺ and 40 mM NaH¹⁴CO₃ (0.1 Bq nmol^–1) . Each reaction was started by adding ¹⁴C-labeled bicarbonate . The reaction was stopped after 5 min by adding a 350-µl sample to 35 µl of 3 M perchloric acid .

The amount of radioactivity in an acid-stabile product (4-hydroxybenzoate) was analyzed by liquid scintillation counting . The amount of labeled 4-hydroxybenzoate formed was calculated from the amount of fixed radioactivity by taking into account the known specific radioactivity of the total bicarbonate added to the assay mixture . The net carboxylation of phenylphosphate to 4-hydroxybenzoate could also be measured indirectly by measuring phosphate release (34, 36) . To reduce background values because of the nonspecific reaction of the phosphate detection reagent, the concentration of phenylphosphate added to the assay mixture was reduced to 0.5 mM, and only 15 to 30 µg of protein was added . The formation of 4-hydroxybenzoate could also be measured directly by a spectrophotometric assay as described previously (36) .

[¹⁴C]phenol-4-hydroxybenzoate isotope exchange, [¹⁴C]phenol-phenylphosphate isotope exchange, and test for net carboxylation of phenol. We tested whether the enzyme system also catalyzed an exchange reaction between [¹⁴C]phenol and the ring positions of 4-hydroxybenzoate . The assay and sample preparation procedures described above for the ¹⁴CO₂-4-hydroxybenzoate isotope exchange assay were used, but 1 mM [U-¹⁴C]phenol (15 Bq nmol^–1) was used instead of ¹⁴C-labeled bicarbonate . To test for [¹⁴C]phenol-phenylphosphate isotope exchange activity, the standard assay mixture described above for the assay for the net carboxylation of phenylphosphate was used, but ¹⁴CO₂ was omitted and 1 mM [¹⁴C]phenol (15 Bq nmol^–1) was added instead . The net carboxylation of phenol was tested by using the radioactive standard assay for the net carboxylation of phenylphosphate described above, but phenylphosphate was replaced by 1 mM [U-¹⁴C]phenol (15 Bq nmol^–1) and unlabeled bicarbonate was used . Stopped samples (350 µl plus 35 µl of 3 M perchloric acid) were neutralized by adding 20 µl of 5 M KOH and centrifuged, and 10-µl portions of the supernatants were analyzed by thin-layer chromatography (TLC) . The reference standards used were [U-¹⁴C]phenol (15 Bq nmol^–1; R_f, 0.7) and 4-[ring-¹⁴C]hydroxybenzoate (8 Bq nmol^–1; R_f, 0.55) . TLC was carried out on aluminum plates (20 by 20 cm) with a 0.2-mm silica gel (Kieselgel 60F₂₅₄; Merck) by using ethanol-methylene chloride-water-NH₃ (8:1:1:0.1, vol/vol/vol/vol) as the solvent . Radioactive areas on the TLC plates were detected by audioradiography with phosphorimager plates (Fujix BAS-IP MP 2040S; Fuji, Tokyo, Japan) and were analyzed with Molecular Imager FX (Bio-Rad, Hercules, Calif.) .

Isotope exchange of H₂¹⁸O into 4-hydroxybenzoate. The CO₂ exchange reaction into 4-hydroxybenzoate and the phenylphosphate carboxylation reaction were studied in the presence of unlabeled bicarbonate in an assay mixture in which 56% of the water was ¹⁸O labeled . After 180 min of incubation at 20°C, 4-hydroxybenzoate was isolated by high-performance liquid chromatography (Grom-Sil 120 ODS HE 5 µm column; 125 by 4 mm; Grom, Herrenberg-Kayh, Germany) at a rate of 1 ml/min by using 0.1% (vol/vol) aqueous trifluoroacetic acid and 3% methanol in a 35-min 3 to 50% methanol linear gradient . 4-Hydroxybenzoate was analyzed by mass spectrometry by using electron ionization (220°C, 70 eV, 1 mA, 3 kV) .

Protein determination. The protein content was determined by the method of Bradford (14) by using bovine serum albumin as the standard .

Purification of phenylphosphate carboxylase. For the purification procedure we started with 5 g (wet weight) of phenol-grown cells . Purification was performed at 15°C under strictly anaerobic conditions in an anaerobic glove box, and 50 mM ß-mercaptoethanol was added to all buffers .

(i) DEAE-Sepharose. Cell extract (6 ml of a 100,000-x-g supernatant, 550 mg of protein) was applied at a flow rate of 2 ml/min to a DEAE-Sepharose column (fast flow; volume, 15 ml; Amersham Biosciences) which had been equilibrated with 20 mM MES-KOH (pH 6.8) containing 10% (vol/vol) glycerol (buffer A) . The column was washed with 3 bed volumes of buffer A and 3 bed volumes of buffer A containing 5 mM (NH₄)₂SO₄ . Phenylphosphate carboxylase was eluted with 5 bed volumes of buffer A containing 75 mM (NH₄)₂SO₄ .

(ii) . Butyl TSK-Sepharose. Solid (NH₄)₂SO₄ was added to the phenylphosphate carboxylase pool (80 ml) to a final concentration of 1.5 M, and this was followed by centrifugation . The supernatant was applied to a Butyl TSK-Sepharose column (bed volume, 25 ml; flow rate, 3 ml/min) which had been equilibrated with buffer A containing 1.5 M (NH₄)₂SO₄ The column was washed with 3 bed volumes of buffer A containing 1.5 M (NH₄)₂SO₄ and 3 bed volumes of buffer A containing 1.2 M (NH₄)₂SO₄ . Phenylphosphate carboxylase was eluted with 3 to 4 bed volumes of buffer A containing 0.6 M (NH₄)₂SO₄ .

(iii) Ammonium sulfate precipitation. The phenylphosphate carboxylase fraction (80 ml) was concentrated by precipitation with ammonium sulfate (60%, wt/vol) . The pellet was dissolved in 5 ml of buffer A .

(iv) Gel filtration. The phenylphosphate carboxylase pool (5 ml) was applied in two runs to a Superdex 200 gel filtration column (bed volume, 330 ml; diameter, 2.6 mm; flow rate, 1.5 ml/min) equilibrated with buffer A containing 65 mM (NH₄)₂SO₄ . Active fractions were pooled and concentrated by precipitation with ammonium sulfate (60%) . The molecular mass of the native enzyme was estimated by using a 20-ml Superdex 200 gel filtration column as described above for the Superdex 200 gel filtration column, but the flow rate was 0.2 ml min^–1 . The elution volumes of the following standard molecular mass marker proteins (2 mg/200 µl) were determined under the same conditions: ferritin (450 kDa), catalase (220 kDa), alcohol dehydrogenase (150 kDa), bovine serum albumin (67 kDa), and ovalbumin (45 kDa) .

Purification of ORF5. ORF5 was purified from 5 g of T . aromatica cells by four chromatography steps at 15°C under strictly anaerobic conditions, and 50 mM ß-mercaptoethanol was added to all buffers .

(i) DEAE-Sepharose. Five milliliters of cell extract (100,000-x-g supernatant, 450 mg of protein) was applied to a DEAE-Sepharose column (fast flow; volume, 15 ml; flow rate, 2 ml/min; Amersham Biosciences) which had been equilibrated with buffer A . The column was washed with 3 bed volumes of buffer A and 3 bed volumes of buffer A containing 20 mM (NH₄)₂SO₄ . ORF5 was eluted with 3 bed volumes of buffer A containing 35 mM (NH₄)₂SO₄ .

(ii) Butyl TSK-Sepharose. Solid (NH₄)₂SO₄ was added to the ORF5 pool (50 ml) at a final concentration of 1.5 M, and this was followed by centrifugation . The supernatant was applied to a Butyl TSK-Sepharose column (bed volume, 25 ml; flow rate, 3 ml/min) which had been equilibrated with buffer A containing 1.5 M (NH₄)₂SO₄, and the column was washed with 3 bed volumes of buffer A containing 1.5 M (NH₄)₂SO₄ and 3 bed volumes of buffer A containing 1.3 M (NH₄)₂SO₄ . ORF5 was eluted with 3 bed volumes of buffer A containing 1 M (NH₄)₂SO₄ .

(iii) Ammonium sulfate precipitation. The ORF5 pool (40 ml) was concentrated by 60% ammonium sulfate precipitation . The pellet was dissolved in 2 ml of buffer A and desalted with a PD-10 desalting column (Amersham Biosciences) .

(iv) . Hydroxyapatite. Protein was applied to a hydroxyapatite column (bed volume, 10 ml; flow rate, 1 ml/min; Bio-Rad) which had been equilibrated with buffer A containing 5 mM MgCl₂ (buffer H) . The column was washed with 3 bed volumes of buffer H and 3 bed volumes of buffer H containing 150 mM potassium phosphate buffer (pH 6.8) . ORF5 was eluted with 2 bed volumes of buffer H containing 220 mM potassium phosphate buffer . The ORF5 pool (15 ml) was concentrated by 60% ammonium sulfate precipitation, and the pellet was dissolved in 0.7 ml of buffer A .

(v) Gel filtration. The precipitated ORF5 pool was applied in two runs to a Superdex 200 gel filtration column (bed volume, 25 ml; flow rate, 0.25 ml/min) which had been equilibrated with buffer A containing 65 mM (NH₄)₂SO₄ . Active fractions were pooled and concentrated by 60% ammonium sulfate precipitation .

Modified chromatographic steps used to obtain subfractions of phenylphosphate carboxylase. Phenylphosphate carboxylase consists of four subunits, , ß, , and , encoded by orf6, orf4, orf12, and orf5, respectively . The subunits were separated on a DEAE-Sepharose column by using modified elution conditions . The DEAE-Sepharose column (fast flow; volume, 15 ml; flow rate, 2 ml/min; Amersham Biosciences) was equilibrated with buffer A, and 5 ml of cell extract (100,000-x-g supernatant, 450 mg of protein) was applied . Subunits and eluted with 3 column volumes of buffer A containing 20 mM (NH₄)₂SO₄; subunit (and minor parts of subunits and and ß) eluted with buffer A containing 35 mM (NH₄)₂SO₄; and most of the ß subunit eluted with buffer A containing 75 mM (NH₄)₂SO₄ . Subunit was separated from the , ß, and subunits either on a Butyl TSK-Sepharose column or by gel filtration . On the Butyl TSK-Sepharose column (bed volume, 25 ml; flow rate, 3 ml/min), which was equilibrated with buffer A containing 1.5 M (NH₄)₂SO₄, the subunit eluted with buffer A containing 1 M (NH₄)₂SO₄, while subunits , ß, and eluted with buffer A containing 0.6 M (NH₄)₂SO₄ . During gel filtration, the subunit eluted at 60 ± 10 kDa, and the ß subunits eluted at 360 ± 30 kDa .

Metal analysis. Purified core enzyme (2.2 mg ml^–1) and purified 18-kDa subunit (1.4 mg ml^–1) were analyzed for metals and selenium by inductively coupled plasma emission spectrometry (ICP-OES) by R . Auxier, Chemical Analysis Laboratory, University of Georgia, Athens, Ga . The proteins were dissolved in buffer A from which glycerol was omitted and were hydrolyzed in 1.1 M HCl at 95°C for 36 h .

UV-visible spectroscopy. The UV-visible spectra (250 to 650 nm) of the phenylphosphate carboxylase core enzyme (ORF4, ORF6, and ORF12) and ORF5 were recorded anaerobically by using gas-tight sealed quartz cuvettes and a nitrogen headspace (reduced state) . Purified protein fractions (concentrated gel filtration pools; 0.35 and 0.25 mg/ml, respectively; dissolved in buffer A containing 65 mM ammonium sulfate and 50 mM ß-mercaptoethanol) were measured against the same solvent . The spectra of the oxidized enzyme were recorded under aerobic conditions with enzyme preparations that were dissolved in buffer without ß-mercaptoethanol; these spectra did not differ from the reduced spectra .

Reconstitution experiments. Subfractions of phenylphosphate carboxylase obtained by using modified chromatographic steps were used in reconstitution experiments with native and overexpressed protein to determine their possible roles in phenylphosphate carboxylation . The separated subunits were pooled in various combinations and tested for the ability to catalyze the net carboxylation of phenylphosphate and the ¹⁴CO₂-4-hydroxybenzoate exchange .

Study of kinetic properties. The radioactive assays and enriched protein (Butyl TSK-Sepharose fraction) were used to study kinetic properties . All buffers contained 10% glycerol and 50 mM ß-mercaptoethanol . The pH optimum of the net carboxylation reaction was determined in 20 mM MES-K⁺ buffer (pH 5.5 to 6.8) or morpholinepropanesulfonic acid (MOPS)-K⁺ buffer (pH 6.5 to 8.0) . The pH optimum of the CO₂ exchange reaction was determined in 50 mM K⁺-phosphate buffer (pH 5.5 to 8) . The dependence on mono- and divalent cations (K⁺, Na⁺, NH₄⁺, Mg²⁺, Mn²⁺, and Ca²⁺) was determined in 20 mM imidazole-Cl^– (pH 6.8) after the enzyme fraction was desalted by two passes over a PD-10 desalting column with 20 mM imidazole-Cl^– (pH 6.8) as the solvent . Phenylphosphate-Na⁺ was passed over a Dowex-50 WX8 column (proton form; Serva) . CO₂ gas (0.5 ml in the CO₂ exchange assay and 0.25 ml in the phenylphosphate carboxylation assay) was added to a 0.5-ml assay mixture (headspace volume, 0.75 ml) instead of sodium bicarbonate; the assay mixture was equilibrated by 1 h of shaking at room temperature . The apparent K_m values were determined at various concentrations of the substrate and saturating concentrations of the cosubstrates and cocatalysts (2 mM phenol, 2 mM 4-hydroxybenzoate, 2 mM Me²⁺ (divalent metal cations), 25 mM K⁺, 25 mM Na⁺, or 25 mM NH₄⁺) .

Inactivation experiments. To study the inhibition of phenylphosphate carboxylase by oxygen and dithionite, various concentrations of air (0 to 10% of the gas space, corresponding after equilibration at 30°C to 0 to 115 µM dissolved O₂) and sodium dithionite (0 to 300 µM) were added to the standard radioactive assay mixtures without ß-mercaptoethanol, and the residual activity was determined after 1, 3, 10, and 30 min of preincubation with inhibitor before the reaction was started by adding ¹⁴C-labeled bicarbonate . Approximately 6 µM enriched enzyme (1.5 mg of the DEAE-Sepharose pool) was used . As a control in the dithionite inactivation experiment, 0.3 mM NaHSO₃ instead of dithionite was added . To study the reactivation of the enzyme activity after treatment with oxygen, the assay mixtures were made anaerobic again by adding 50 mM ß-mercaptoethanol and applying four cycles consisting of alternating 2 min of gassing with N₂ and 2 min of degassing with a vacuum pump .

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Polyacrylamide gels (normally 13.5% acrylamide) were prepared as described previously (39) . The proteins were stained as described by Zehr et al . (64) . Usually, 20 µg of extract protein and enriched protein fractions and 5 to 8 µg of pure enzyme were applied .

Cloning, transformation, amplification, and purification of nucleic acids. Standard protocols were used for DNA cloning, transformation, amplification, and purification (54). -ZAP Express gene libraries of Sau3A- and EcoRI-digested genomic DNA from T . aromatica were prepared as described in the ZAP Express cloning kit instruction manual (Stratagene, Amsterdam, The Netherlands) . PCR products used as probes in the screening analysis were labeled with digoxigenin-11-dUTP by PCR . Probes were detected by using anti-digoxigenin-AP, nitroblue tetrazolium chloride, and X-Phosphate (5-chloro-4-bromo-3-indolyl-phosphate toluidine salt) (Biomol) .

Overexpression of genes. The following primers were used to PCR amplify genes: for orf4, orf4for (ATG GAC CTG CGC TAC TTC AT) and orf4rev (CCG AGA TCT GTC ATG CGG CTA AG); for orf5, orf5for (ATG GAA CAG GCG AAG AAC ATC) and orf5rev (CCA TGT AGA TCT CCT TGT CGA TGG); and for orfs6 to orf12, orf6/12for (ATG GGA AAG ATT TCA GCA CC) and orf6/12rev (CCA GAT CTT TAG CGG GTT GAG TG) . Fragments were cloned in the pET-Blue1 expression vector (Novagene) and were expressed in E . coli TUNER (DE3)pLacI (TGYEP medium, 16 h at 20°C under anaerobic conditions) .

Computer analysis. The nucleotide and amino acid sequences were analyzed by using the PC/gene software package (Genofit) and the Open Reading Frame Finder (ORF Finder; http://www.ncbi.nlm.nih.gov/gorf/gorf.html) . Similar sequences were identified by performing a BLAST search with the TBLASTN algorithm provided by the National Center for Biotechnology Information (Bethesda, Md.) .

Nucleotide sequence accession number. All sequence data have been deposited in EMBL Nucleotide Sequence Database under accession number AJ272115 .

 
Purification of phenylphosphate carboxylase. Extracts of phenol-grown cells catalyzed the ¹⁴CO₂-4-hydroxybenzoate isotope exchange reaction and the carboxylation of phenylphosphate at specific rates of 100 to 350 and 10 to 32 nmol min^–1 mg^–1, respectively (30°C, pH 6.8) . The specific activities varied with the cell batches due to the extreme oxygen sensitivity of the activities . These activities had a half-life of 30 s in air . Therefore, the enzyme had to be purified under strictly anaerobic conditions, and at the same time it was completely inactivated by low concentrations (0.1 mM) of dithionite . Furthermore, its subunits dissociated, and active enzyme had to be reconstituted from four subunits by recombination of separate enzyme subfractions . We succeeded in purifying the enzyme on a small scale from 5 g of phenol-grown cells and in identifying its subunits and coding genes (see below) (Table 1 and Fig . 3) .

 
The last chromatographic step, gel filtration, resulted in a separate protein peak at 360 ± 30 kDa . This protein fraction was inactive in phenylphosphate carboxylation; however, it still catalyzed the CO₂ exchange reaction (11% recovery) (Table 1) . The purified incomplete enzyme consisted of three subunits having apparent molecular masses of 59, 57, and 10 kDa (for the deduced molecular masses, see below) . Protein staining indicated that approximately equal molar amounts of the 59- and 57-kDa subunits were present; the amount of the 10-kDa protein could not be estimated exactly .

Reconstitution of phenylphosphate carboxylation activity by an 18-kDa protein. We noticed that loss of phenylphosphate carboxylation activity was consistently associated with loss of a 18-kDa phenol-induced protein in the course of the chromatography procedure . In contrast, the CO₂ exchange activity was almost unaffected . The lost protein was likely to be a subunit of the enzyme . Therefore, the 18-kDa protein was purified separately (Fig . 3B) . The protein had a native molecular mass of 58 kDa as determined by Superdex 200 gel filtration . This suggested that the purified 18-kDa subunit formed a homotrimer . Adding this protein fraction to the 360-kDa gel filtration protein fraction at a molar ratio of 3:1 (0.1 mg of the 18-kDa protein and 0.25 mg of core enzyme) resulted in reconstitution of the phenylphosphate carboxylation activity (Table 1), whereas the ¹⁴CO₂ isotope exchange reaction was stimulated only slightly, if at all . Hence, the ¹⁴CO₂ isotope exchange reaction is a partial reaction catalyzed by a fragment of the holoenzyme consisting of three subunits having apparent molecular masses of 59, 57, and 10 kDa, whereas the net carboxylation of phenylphosphate requires in addition the 18-kDa subunit .

UV-visible spectra and metal content. The UV-visible spectra of the enzyme subfractions isolated under reducing conditions showed protein absorption maxima at around 280 nm and no other significant absorption in the range from 320 to 650 nm, indicating that no commonly known cofactor absorbing in this range of wavelengths was present . The purified enzyme subfractions were analyzed for metals and selenium . The core enzyme preparation (subunits , ß, and ) contained (per mole) less than 0.1 mol of Mg, Mn, Fe, Co, Ni, Cu, Zn, Mo, V, or Se but 0.55 mol of Al . The subcomplex contained less than 0.1 mol of Mn, Fe, Co, Ni, Cu, Zn, Mo, V, or Se per mol but 0.41 mol of Mg per mol and 0.13 mol of Al per mol .

Test for exchange of H₂¹⁸O into 4-hydroxybenzoate and phenylphosphate. We tested whether the oxygen atom of the phenolic hydroxyl group remains on the aromatic ring when the carboxyl group becomes incorporated . It was expected that the oxygen atoms of the carboxyl group would exchange to some degree with oxygen from water . The CO₂ exchange reaction and the phenylphosphate carboxylation reaction were performed in the presence of 56% H₂¹⁸O, and the 4-hydroxybenzoate that formed after prolonged incubation with cell extract was analyzed by mass spectroscopy . The mass peak at 138 Da corresponding to the unlabeled 4-hydroxybenzoate contributed 50%, a mass peak at 140 Da corresponding to a singly labeled product contributed 40%, and a 142-Da mass peak corresponding to a double-labeled product contributed 10% . No peak at 144 Da corresponding to a triple-labeled product was observed . Furthermore, the phenolic mass peak at 93 Da did not have a satellite mass peak at 95 Da .

These results indicated that one or two of the oxygen atoms of CO₂ had been incorporated from ¹⁸O-labeled water via free carbonic acid into the carboxyl group of 4-hydroxybenzoate, but no oxygen from water was incorporated into the phenolic hydroxyl group .

Test for net carboxylation of phenol and for [U-¹⁴C]phenol exchange into 4-hydroxybenzoate and phenylphosphate. Under the conditions optimized for net carboxylation of phenylphosphate, we tested whether the enzyme system also catalyzed a net carboxylation of phenol to 4-hydroxybenzoate . No such activity was detected . We also tested whether the enzyme catalyzed an isotope exchange between [¹⁴C]phenol and the ring positions of 4-hydroxybenzoate and phenylphosphate . Neither of these activities was detected . These findings support the hypothesis that an enzyme-bound phenolate intermediate is formed by the irreversible release of the phosphoryl group of phenylphosphate in the course of the net carboxylation reaction or, alternatively, is formed by decarboxylation of 4-hydroxybenzoate in the course of the CO₂ exchange reaction .

Requirements and kinetics of the reaction. The net carboxylation of phenylphosphate to 4-hydroxybenzoate was strictly dependent on CO₂, phenylphosphate, Mg²⁺ (or Mn²⁺[about 40% activity]), and K⁺ . The reaction was linearly dependent on time in the range from 0 to 10 min and on the amount of protein added in the range from 0 to 0.9 mg . The reaction followed Michaelis-Menten kinetics with the following apparent K_m values: for phenylphosphate, 0.09 ± 0.01 mM; for Mg²⁺, 0.1 ± 0.015 mM; for Mn²⁺, 0.09 ± 0.01 mM; for K⁺, 14 ± 2.5 mM; and for dissolved CO₂, 1.5 ± 0.5 mM . The CO₂ exchange activity was strictly dependent on CO₂, 4-hydroxybenzoate, Mg²⁺ (or Mn²⁺ [about 90% activity]), and K⁺ . No CO₂ exchange activity was measured with 3-hydroxybenzoate (38) or 2-hydroxybenzoate . The apparent K_m values were as follows: for 4-hydroxybenzoate, 4.5 ± 0.5 mM; for Mg²⁺, 0.2 ± 0.025 mM; for Mn²⁺, 0.1 ± 0.02 mM; for K⁺, 2.6 ± 0.45 mM; and for dissolved CO₂, 1 ± 0.3 mM . For both reactions, Mg²⁺ could be replaced to some extent by Mn²⁺ or Ca²⁺, and K⁺ could be replaced to some extent by NH₄⁺ or Na⁺ (Table 2) . The optimal pH for both activities was 6.8 . Half-maximal activity was observed at pH 6.0 and 7.8, respectively .

 
Inactivation by oxygen and dithionite. The inactivation of the enzyme by molecular oxygen was studied . The enzyme was inactivated in a stoichiometric way, and the half-life was less than 1 min . The phenylphosphate carboxylation and the CO₂ exchange activities were equally affected . A preparation containing 6 µM enzyme which still contained some contaminant proteins was completely inactivated by 60 µM dissolved molecular oxygen (Fig . 4A) . The enzyme activity could be completely restored by subsequently making the assay mixture anaerobic again and by adding 50 mM mercaptoethanol as a reducing agent (data not shown) .

 
The phenylphosphate carboxylation and the CO₂ exchange activities also were both inactivated by trace concentrations of dithionite in a stoichiometric way . A similar enzyme preparation containing 6 µM enzyme was completely inactivated by 100 µM dithionite, and the half-life was less than 1 min (Fig . 4B) . Both the net carboxylation and isotope exchange activities were affected in the same way . In an aqueous solution dithionite forms sulfite ions . Therefore, the effect of 300 µM sulfite on the net carboxylation and isotope exchange activities was studied, and the treatment was found to have no inhibitory effect or only a small inhibitory effect (10%) . Therefore, sulfite ions are not the inactivating molecular species, but the SO₂ radical anion may be . Other reducing agents, like 50 mM ß-mercaptoethanol, 1 mM dithioerythritol, or 0.5 mM titanium(III) citrate (in the presence of excess Mg²⁺), did not inhibit the enzyme activity .

Reinvestigation of the phenol gene cluster and explanation for unexpected results in T7 polymerase experiments. Cloning and sequencing of the genes coding for phenol-induced proteins in T . aromatica resulted in the discovery of a large cluster of ORFs (Fig. 2) . Six of these ORFs (orf1, orf4, orf5, orf6, orf8, and orf13) could be assigned to phenol-induced proteins whose N-terminal amino acid sequences have been determined (16; Schühle et al., unpublished) . Purification of phenylphosphate carboxylase (see above) showed that the enzyme consists of four subunits . N-terminal sequencing revealed that the , ß, and subunits were encoded by orf6, orf4, and orf5, respectively (see below) . The deduced molecular masses for ORF6 (54 kDa), ORF4 (53 kDa), and ORF5 (18 kDa) were close to the experimentally determined molecular masses for these subunits (59, 57, and 18 kDa, respectively) . However, the ORF coding for the fourth subunit (the subunit) was not found in the known gene cluster . Resequencing of the intergenic region between orf6 and orf7 provided new insights . The missing gene coding for the 10-kDa subunit of phenylphosphate carboxylase was identified as orf12 located between orf6 and orf7 (Fig . 2) . orf12 was overlooked before simply because of its very small size and the lack of similarity of ORF12 to other proteins . Furthermore, a frameshift in the former intergenic DNA sequence was found, which changed the transcription start of the following orf7; the corrected sequence of orf7 predicts a larger size for ORF7 (the size determined previously was 38 kDa, and the protein was seemingly N terminally truncated compared to UbiD; the corrected size was 48 kDa, and the protein shows full sequence similarity with UbiD) .

The previous T7 polymerase transcription experiments (16) gave odd results (for genes see Fig . 2) . When the DNA region between orf5 and orf7 was transcribed, instead of three fragments having the expected sizes, 18 kDa (ORF5), 54 kDa (ORF6), and 38 kDa (formerly ORF7), an 18-kDa fragment (ORF5; observed at 19 kDa), a 54-kDa fragment (ORF6; observed at 60 kDa), a 10-kDa fragment (unknown), and a 48-kDa fragment (unknown) were observed . These T7 polymerase experimental data turned out to be correct and can now be reinterpreted in terms of the new data . The 10-kDa protein represents the overlooked ORF12 protein, and the 48-kDa protein represents ORF7 with the corrected size (48 kDa rather than 38 kDa, as deduced previously) . The T7 polymerase experiments therefore provided evidence that orf1 to orf8 and orf12 are cotranscribed; orf9, orf10, and orf13 to orf15 have not been tested . However, the range of phenol-induced proteins is larger, and the genes comprises at least orf1 to orf10, orf12, and orf13; orf11 probably acts as the regulator gene .

Identification of the genes coding for the four subunits. We N terminally sequenced the subunits of the purified 360- ± 30-kDa enzyme fraction consisting of three proteins and the separate 18-kDa protein and identified the coding genes . The four subunits having apparent molecular masses of 59, 57, 18, and 10 kDa, which make up active phenylphosphate carboxylase, are encoded by orf4, orf5, orf6, and orf12, respectively, in the phenol gene cluster (Fig . 2) . The experimentally determined molecular masses of the subunits match the predicted sizes (53, 18, 54, and 10 kDa) well . Below the molecular masses predicted from the genes are used for the subunits . ORF4, ORF6, and ORF12 were present in the gel filtration fraction which catalyzed the ¹⁴CO₂ isotope exchange reaction . ORF5 represented the 18-kDa protein that reconstituted phenylphosphate carboxylation but had no significant effect on the CO₂ exchange reaction .

The requirement for these four proteins for the complete phenylphosphate carboxylation reaction was suggested by additional findings . Modified DEAE exchange chromatography separated the enzyme into two subfractions, one of which contained ORF6 and ORF12 and one of which contained ORF4 and ORF5; these subfractions were inactive in phenylphosphate carboxylation . The subfraction containing only ORF6 and ORF12 showed slight isotope exchange activity . Recombination resulted in an active enzyme that catalyzed both reactions . A modified Butyl TSK-Sepharose chromatography step separated the enzyme into two subfractions, one of which contained ORF5 and one of which contained ORF4, ORF6, and ORF12; the fraction containing ORF4, ORF6, and ORF12 was virtually inactive in phenylphosphate carboxylation but was active in the CO₂ exchange reaction, and recombination with the ORF5 fraction resulted in an active phenylphosphate carboxylation enzyme . Gel filtration resulted in the loss of ORF5; the resulting enzyme preparation was active in the CO₂ exchange reaction and virtually inactive in phenylphosphate carboxylation . Addition of the fraction containing ORF5 reconstituted the phenylphosphate carboxylation activity .

Overexpression of orf4, orf5, orf6, and orf12. The results of the purification and complementation studies strongly suggested that phenylphosphate carboxylase consists of a three-subunit core enzyme which catalyzes the ¹⁴CO₂ exchange reaction and a subunit which complements phenylphosphate carboxylation activity . However, the possibility of participation of minor contaminant T . aromatica proteins cannot be eliminated by these results . Therefore, we cloned and overexpressed orf4, orf5, orf6, and orf12 separately in pETBlue-1 in E . coli (Fig . 5) .

 
Whereas orf4 and orf5 could be expressed individually in soluble form, ORF12 formed inclusion bodies when orf12 was expressed separately . Overexpression was possible when both orf12 and orf6 were present in the same DNA fragment and expressed simultaneously . This phenomenon cannot be explained at this time and should be studied further . A possible explanation may involve the very basic deduced pI of ORF12 (pI 9.8) . Overexpressed ORF5 was active in reconstituting the phenylphosphate carboxylase activity when it was added to the ORF4-ORF6-ORF12 fraction . This indicates that in the presence of ORF4, ORF5, and ORF12, ORF5 alone is sufficient to reconstitute active phenylphosphate carboxylase . Overexpressed ORF6 and ORF12 alone showed slight ¹⁴CO₂-4-hydroxybenzoate isotope exchange activity but no net phenylphosphate carboxylation activity . The expressed proteins were also active in reconstituting native ORF4 and ORF5 . Overexpressed ORF4 was apparently inactive; therefore, it has not been possible to reconstitute net phenylphosphate carboxylation activity from all four overexpressed subunits . It seems possible that for correct expression overproduced ORF4 requires the presence of (some of) the other phenylphosphate carboxylase subunits . However, we failed to overexpress the genes of all of the subunits in one DNA clone .

 
General properties of phenylphosphate carboxylase. Phenylphosphate carboxylase (E.C . 4.1.1.-) represents a new type of carbon-carbon lyase which eliminates or adds a carboxy group . The recommended name is phenylphosphate carboxylase, and the systematic name is orthophosphate:4-hydroxybenzoate carboxylyase (phosphorylating) . The reaction catalyzed is Phenylphosphate + CO₂ + H₂O 4-Hydroxybenzoate + Orthophosphate . This reaction is strongly in favor of the carboxylation direction . The enzyme also catalyzes the exchange of free CO₂ with the carboxyl group of 4-hydroxybenzoate . The active CO₂ species is CO₂ rather than bicarbonate, and mono- and divalent cations are required for activity . The protein appears to require four subunits; three subunits of these subunits (54, 53, and 10 kDa) are present in the enzyme fraction that catalyzes the CO₂ exchange reaction (core enzyme) . An 18-kDa protein is required in addition for the net carboxylation of phenylphosphate and for phosphate release . The enzyme system may be useful for biotechnical phenol carboxylation (3) . An enzymatic reaction similar to the phenylphosphate carboxylase reaction is involved during methanopterin biosynthesis in the formation of 4-(ß-D-ribofuranosyl)aminobenzene 5'-phosphate from 4-aminobenzoate and 5-phosphoribosyl-1-pyrophosphate, during which CO₂ and pyrophosphate are released (50) .

Molecular composition of the enzyme. The enzyme could not be isolated as a single protein complex, and two different subfractions had to be recombined . If the three subunits of the core complex (54, 53, and 10 kDa) were present in equal molar amounts, the native molecular mass (360 ± 30 kDa) suggests a (ß)₃ composition (calculated molecular mass, 351 kDa) . The ORF5 protein (18 kDa), as isolated, may be a homotrimer with a molecular mass of 54 kDa (experimentally determined molecular mass, 58 kDa) . So far, no indication of the presence of a cofactor has been obtained .

Similarities of phenol-induced ORF-encoded proteins to other proteins and possible role of the second part of the phenol gene cluster (orf7 to orf15). Phenylphosphate carboxylase of T . aromatica is composed of ORF4, ORF5, ORF6, and ORF12 . The products of orf4, orf6, and orf7 of the phenol gene cluster showed 48 to 59% similarity to UbiD (55.2 kDa), and the product of orf8 showed 87% similarity to UbiX (20.7 kDa) of E . coli . ORF4 and ORF6 showed 29% amino acid sequence identity and 46% sequence similarity to each other . UbiD of E . coli catalyzes the third reaction in ubiquinone biosynthesis, the decarboxylation of 3-octaprenyl-4-hydroxybenzoate to 3-octaprenylphenol . The ubiX gene encodes an isoenzyme of UbiD that catalyzes the same reaction (reviewed in references 44 and 45) . Of the four decarboxylase-like ORF-encoded proteins of T . aromatica, two (ORF4 and ORF6) were present in phenylphosphate carboxylase . The other two (de)carboxylase-like proteins, ORF7 and ORF8, although phenol induced, were missing, and they are therefore likely to play a role in the carboxylation or decarboxylation of other phenolic compounds . T . aromatica can grow under anoxic conditions on a variety of different aromatic compounds, but not all potential phenolic candidates have been tested as growth substrates . Hence, the 5' part of the gene cluster comprising orf1, orf 2, orf3, orf4, orf5, orf6, and orf12 appears to be directly involved in phenol phosphorylation (orf1, orf2, and orf3) (Schmeling et al., unpublished) and the carboxylation of phenylphosphate (orf4, orf5, orf6, and orf12) . The 3' part of the gene cluster comprising orf7, orf8, orf9, and the following ORFs may play a role in the metabolism (decarboxylation or carboxylation) of other phenolic compounds, perhaps in conjunction with, for example, orf5 (Fig . 2) .

So far, only limited biochemical data are available for UbiD and UbiX . The holoenzyme UbiD of E . coli has a molecular mass of 340 kDa, and its activity is dependent on the presence of Mn²⁺ and an unidentified heat-stable factor with a molecular mass of <10 kDa (40, 44, 45) . Moreover, the activity was increased by adding membrane preparations or phospholipids, indicating that the enzyme, as expected, normally functions in association with the membrane . Like the activity of E . coli UbiD, the phenylphosphate carboxylase and CO₂ exchange activities were both dependent on Mg²⁺ or Mn²⁺ . Unlike 3-octaprenyl-4-hydroxybenzoate decarboxylase, apparently no low-molecular-mass factor is required for phenylphosphate carboxylase enzyme activity, and the enzyme is soluble . Interestingly, phenylphosphate carboxylase contains both UbiD-like proteins (ORF4 and ORF6) in equal molar amounts .

Role of ORF5 and similarity to other proteins. ORF5 seems to be responsible for binding of phenylphosphate and dephosphorylation of this molecule . At the same time this subunit must ensure trapping of the released phenolate in a reactive form bound to the core carboxylase enzyme composed of ORF4, ORF6, and ORF12 . Otherwise, phenylphosphate would simply hydrolyze, and the energy of the relatively energy-rich phenol ester would be dissipated as heat; also, [¹⁴C]phenol exchange into 4-hydroxybenzoate would be observed . ORF5 indeed shows similarity to a hydrolase family of proteins, including phosphatases, and contains the typical domain of these proteins (COG1778, conserved domain database, National Center for Biotechnology Information) . The postulated interaction of the proteins during catalysis raises some interesting questions which cannot be answered at the present state of knowledge .

Catalytic properties and partial reactions of the catalytic cycle. The CO₂ exchange reaction indicates that phenylphosphate carboxylase follows a ping-pong mechanism, with a phenolate anion intermediate bound to the core enzyme (Fig . 6) (note that for sake of simplicity the phenolate anion intermediate shown in Fig. 6 represents only the quinone form, which is one of numerous possible resonance structures) . It will be interesting to study the nature and type of binding of the phenolate . The binding must be tight since exchange of ¹⁴C-labeled phenol into 4-hydroxybenzoate was not observed . Also, no exchange of oxygen from water into the phenolic hydroxyl group was observed, excluding, for example, the possibility of formation of a Schiff base with the phenolate unit . Furthermore, the same bound phenolate must also be formed from phenylphosphate, which requires interaction of ORF5 and the core enzyme . The ¹⁴CO₂ exchange reaction with 4-hydroxybenzoate is 10 times faster than phenylphosphate carboxylation to 4-hydroxybenzoate and is freely reversible (bidirectional); hence, this partial step of the catalytic cycle must be readily reversible .

 
In contrast, the formation of the enzyme-bound phenolate from phenylphosphate is probably unidirectional (exergonic) since dephosphorylation of phenylphosphate is exergonic . The electrophilic CO₂ addition requires that the electron pair of the P-O bond stays with the oxygen atom since the leaving group is a phosphoryl or metaphosphate-like unit . Whether this unit is directly transferred to water or is intermediately accepted by an amino acid residue cannot be determined . The rate-limiting step in phenylphosphate carboxylation appears to be the formation of the enzyme-bound phenolate from phenylphosphate by ORF5 . The equilibrium constant of phenol carboxylation, [4-hydroxybenzoic acid] x [phenol]^–1 [CO_2]^–1, is estimated to be 0.2 M^–1 . Since carboxylation of phenol at ambient concentrations of phenol (in the range of <1 mM) and CO₂ (in the millimolar range when CO₂ is dissolved) is endergonic (30) and not freely reversible, the enzyme-bound phenolate must be activated compared to free phenol .

Postulated mechanism of phenylphosphate carboxylase. Based on the considerations described above, we postulate the following mechanism for phenylphosphate carboxylation (Fig . 6) . The ß core enzyme, which shows similarity to known decarboxylases, catalyzes the reversible carboxylation of an enzyme-bound phenolate intermediate . This phenolate intermediate can be formed by the reversible decarboxylation of 4-hydroxybenzoate, which can be measured as a partial reaction of phenylphosphate carboxylation (¹⁴CO₂-4-hydroxybenzoate isotopic exchange) . Alternatively, it can be formed during the irreversible, exergonic dephosphorylation of phenylphosphate (the activated phenol) by the subcomplex, which indeed shows similarity to known phosphatases (see above) . Only in the presence of the phosphatase subcomplex is the net carboxylation of phenylphosphate to 4-hydroxybenzoate catalyzed .

Possible roles of K⁺ and Mg²⁺. Phenylphosphate carboxylase requires mono- and divalent cations for activity . The specificity was not high, but, taking into account the cellular conditions, K⁺ and Mg²⁺ are likely to represent the physiological cocatalysts . Me²⁺ may act as a Lewis acid to increase the electrophilic character of CO₂ . In addition, an interaction with the phenolic hydroxyl groups is conceivable . K⁺ is known to support the chemical para carboxylation in Kolbe-Schmitt-type carboxylations of phenolic compounds (3, 38) . K⁺ may stabilize carbon dioxide coordinated to transition metals (30) . Na⁺, which can substitute to some extent for K⁺ (Table 2), is known to support the chemical ortho carboxylation of phenol to 2-hydroxybenzoate . However, phenylphosphate carboxylase did not show any CO₂ exchange activity with 2-hydroxybenzoate, and no aromatic acid-CoA ligase acting on this substrate was found in cells anoxically grown on phenol . Therefore, the carboxylation of phenylphosphate seems to take place exclusively in the para position .

Inactivation by oxygen and dithionite. Another property, which is unexpected for a carboxylase, is inactivation with molecular oxygen which is reversible . The most intriguing feature, however, is the inactivation by dithionite, which may be due to the action of the SO₂ radical anion . The possibility of an inhibitory effect of sulfite can be excluded . This may indicate that a redox-sensitive component of the system, which needs to be in an intermediate redox state, is crucial for activity and that oxidizing or reducing agents change the redox state and thereby lead to inactivation . The possibility of even a radical form cannot be excluded since molecular oxygen, as well as the SO₂ radical, may act as a radical trapping agent .

Related (de)carboxylating systems in bacteria acting on aromatic compounds. A sequence comparison with known decarboxylases also supports the proposed role of ORF5 . It appears that all arylic acid decarboxylases acting on 4-hydroxybenzoate and its derivatives (protocatechuate 3,4-dihydroxybenzoate, vanillate 4-hydroxy-3-methoxybenzoate) consist of three different subunits or require three genes to form an active enzyme . These decarboxylases seem to contain two proteins of the UbiD/UbiX type and an additional small subunit which show some similarity to each other (Fig. 7) but not to ORF12, the small subunit of phenylphosphate carboxylase . The physiological role of these enzymes is probably decarboxylation of 4-hydroxybenzoate (derivative) rather than carboxylation of phenol (derivative) . In line with our interpretation, the decarboxylating enzymes lack a subunit which corresponds to ORF5 .

 
The similarity of proteins of the UbiD/UbiX type is shown in Fig. 8 . In Streptomyces sp . strain D7 the vdcBCD gene cluster codes for a vanillic acid (4-hydroxy-3-methoxybenzoate) decarboxylase; the product is 2-methoxyphenol (guaiacol) (20) . An E-X-P motif was found in the decarboxylases, which may play a role in substrate binding or catalysis of (hydroxy-)arylic acid decarboxylation or carboxylation of the corresponding phenolic compounds . VdcD is a small protein which is also required and does not show similarity to any known protein . There are homologues of the VdcBCD proteins in various (vanillic acid) decarboxylases from Lactobacillus plantarum (17), B . subtilis (18), Bacillus pumilus (63), Novosphingobium aromaticivorans (52), and E . coli (10), and all of them have levels of sequence identity greater than 50% for the C subunit and 30% for the D subunit (D . Y . Lyon and J . Wiegel, Abstr . 102nd Gen . Meet . Am . Soc . Microbiol., abstr . Q-72, 2002) .

 
The genes for 4-hydroxybenzoate decarboxylase (54 kDa) in C . hydroxybenzoicum can also be interpreted in terms of the vdcBCD system of Streptomyces sp . Initially, a 1,440-bp ORF (vdcC-like) was identified as the only gene required for 4-hydroxybenzoate decarboxylase (32, 35) . However, an additional 270-bp ORF (vdcD-like) downstream of this ORF was recently found to be necessary for activity (Lyon and Wiegel, Abstr . 102nd Gen . Meet . Am . Soc . Microbiol.) . A 3,4-dihydroxybenzoate decarboxylase with a 54-kDa monomeric subunit and similar catalytic properties from C . hydroxybenzoicum has also been characterized (33), but no genetic data are available . The vanillic acid decarboxylase from Streptomyces sp . requires vdcB when it is expressed in Streptomyces lividans 1326 (20) . However, only vdcCD were needed for activity when the genes were cloned in pUC18 and expressed in E . coli JM109, and the activity of the enzyme was contingent on expression of vdcC and vdcD together . This situation seems to be similar to the requirement for coexpression of orf6 and orf12 in T . aromatica . VdcD may form inclusion bodies when it is expressed separately from VdcC .

New related database entries from genome projects. Screening of databases revealed unknown ORFs which clearly are similar to ORFs of the phenol gene cluster of T . aromatica (16) . Notably, Geobacter metallireducens (Fig . 2) has a gene cluster containing homologues of orf11, orf1, orf2, orf3, orf4, orf8, orf15, and orf16 . A HAD phosphatase gene is also located in this cluster, but it shows little similarity to the ORF5 phosphatase gene and is oriented in the opposite direction; a better orf5 homologue is located separately from this cluster . G . metallireducens is an iron(III)-reducing anaerobic respirer belonging to the delta subclass of the Proteobacteria that reportedly grows on various aromatic substrates, including phenol (43) . In addition, in various genome sequences, genes coding for hypothetical proteins with levels of similarity greater than 50% to ORF4, ORF6, and ORF7 from T . aromatica, 4-hydroxybenzoate decarboxylase from C . hydroxybenzoicum, and UbiD from E . coli were found .

(De)carboxylases acting on aromatic compounds that are not related to phenylphosphate carboxylase or that have not been studied in greater detail. A completely different 4-hydroxybenzoate decarboxylase from an anaerobic coculture showed similarity to pyruvate-flavodoxin oxidoreductase (41, 42) . Furthermore, a very interesting 4-hydroxyphenylacetate decarboxylase which belongs to the glycine radical enzyme family was found in Clostridium difficile (56) . Other carboxylases have not been studied in more detail, although anaerobic metabolism of phenol or chlorophenols (4, 9, 19, 41, 57-62, 65-67) and other phenolic or anilinic compounds is widespread . o-Cresol (2-methylphenol) (8, 9, 53), m-cresol (3-methylphenol) (49, 51), hydroquinone (1,4-dihydroxybenzene) (28, 29), catechol (1,2-dihydroxybenzene) (27), and aniline (aminophenol) (55) are metabolized by pure cultures of denitrifying and sulfate-reducing bacteria and consortia of fermenting bacteria, and the process involves carboxylation of the aromatic ring para or ortho to the hydroxy or amino substituent . Growth on these substrates is therefore dependent on the presence of CO₂ as the cosubstrate for the initial attack (60) . Consortia of fermenting bacteria convert phenol to benzoate (41) and decarboxylate 4-hydroxybenzoate to phenol (66) . They also catalyze an isotope exchange between D₂O and the proton at C-4 of the aromatic ring of phenol (24) . Aerobic ortho carboxylation of aniline to 2-aminobenzoic acid has been reported for Rhodococcus erythropolis (2) . In sulfate-reducing consortia the initial reaction in the anaerobic metabolism of naphthalene may be carboxylation specifically at C-2 to form 2-naphthalene carboxylic acid . Anaerobic phenanthrene transformation leads to phenanthrene carboxylic acid, and the carboxylation site is unknown (67) .

Other recently discovered energy-dependent carboxylases that differ from the phenol carboxylase system. Some unusual carboxylation reactions concern the aliphatic side chain of aromatic compounds . Acetophenone, an intermediate of the anaerobic ethylbenzene catabolic pathway (5, 48), is carboxylated to benzoylacetate (3-oxophenylpropionate) . This energy-driven carboxylation is thought to be analogous to carboxylations found in aerobic and anaerobic degradation of aliphatic ketones, such as acetone (23, 47) . In these cases a phosphorylated enzyme or a phosphoenol substrate intermediate has to be assumed .

 
This work was supported by the Deutsche Forschungsgemeinschaft, Bonn, Germany, by the Fonds der Chemischen Industrie, Frankfurt, Germany, and by cooperation with E . I . DuPont de Nemours, USA .

We thank Johann Heider, Freiburg, Germany, for helpful suggestions and critical reading of the manuscript, and we thank Jürgen Wörth, Freiburg, Germany, for performing mass spectrometry .

 
* Corresponding author . Mailing address: Mikrobiologie, Institut Biologie II, Schaenzlestr . 1, D-79104 Freiburg, Germany . Phone: 49-761-2032649 . Fax: 49-761-2032626 . E-mail: georg.fuchs@biologie.uni-freiburg.de .

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