Metabolism of Phenol and Resorcinol in Trichosporon cutaneum

JOURNAL OF BACTERIOLOGY, Jan. 1979, p. 13-21 0021-9193/79/01-0013/09$02.00/0 Vol. 137, No. 1 Metabolism of Phenol and Resorcinol in Trichosporon cutaneum ANDRAS GAAL AND HALINA Y. NEUJAHR* Department of Biochemistry and Biochemical Technology, The Royal Institute of Technology, S 100 44 Stockholm, Sweden Received for publication 2 October 1978 Trichosporon cutaneum was grown with or resorcinol as the carbon source. The formation of 8i-ketoadipate from, catechol, and resorcinol was shown by a manometric method using antipyrine and also by its isolation and crystallization. Metabolism of begins with o-hydroxylation. This is followed by ortho-ring fission, lactonization to muconolactone, and delactonization to f,-ketoadipate. No meta-ring fission could be demonstrated. Metabolism of resorcinol begins with o-hydroxylation to 1,2,4-benzenetriol, which undergoes ortho-ring fission yielding maleylacetate. Isolating this product leads to its decarboxylation and isomerization to trans-acetylacrylic acid. Maleylacetate is reduced by crude extracts to,8-ketoadipate with either reduced nicotinamide adenine dinucleotide or reduced nicotinamide adenine dinucleotide phosphate as a cosubstrate. The enzyme catalyzing this reaction was separated from catechol 1,2- oxygenase, hydroxylase, and muconate-lactonizing enzyme on a diethylaminoethyl-sephadex A50 column. As a result it was purified some 50-fold, as was the muconate-lactonizng enzyme. Methyl-, fluoro-, and chloros are converted to a varying extent by crude extracts and by purified enzymes. None of these derivatives is converted to maleylacetate,,8-ketoadipate, or their derivatives. Cells grown on resorcinol contain enzymes that participate in the degradation of and vice versa. Most of the information on metabolic pathways of aromatic degradation comes from studies on bacterial soil isolates or selected and classified strains of bacteria (for selected review see refs. 4, 27). The few early reports on degradation of ic compounds by yeast also concern selected and classified strains (10, 12, 14, 32). A few yeast strains were included in the extensive investigation of aromatic degradation by fungi (1). In all these cases, the utilization of s by yeasts was limited and slow. We reported previously from this laboratory that strains of Trichosporon cutaneum and Candida tropicalis, isolated from soil, can metabolize and a number of monosubstituted s at a comparatively high rate (20, 21, 29; H. Y. Neujahr and J. M. Varga, Fed. Eur. Biochem. Soc. Proc. Meet. Abstr. 5, no. 624). Two enzymes initiating the metabolic sequence of degradation were purified to homogeneity and studied in detail. These two enzymes are hydroxylase (EC 1.14.13.X) (16-18) and catechol 1,2-oxygenase (EC 1.13.11.1) (30, 31). They catalyze the first and second steps of degradation, respectively. These enzymes have rather broad substrate specificity. It was therefore earlier proposed (21) that all the monosubstituted s are metabolized in T. cutaneum by the f,-ketoadipate pathway. This paper provides evidence that and resorcinol are indeed metabolized via the,6-ketoadipate pathway. A new enzyme, maleylacetate reductase, is described. This enzyme participates in the conversion of resorcinol to f8-ketoadipate. A number of other monosubstituted s are metabolized through part of the pathway only. Preliminary results of this investigation have been reported (H. Y. Neujahr and A. Gaal, Proc. 6th Intern. Symp. Yeast, Montpellier, France, abstr. no. S-VI-16). MATERIALS AND METHODS Chemicals and equipment. All chemicals were commercial products, of reagent grade whenever available. Inorganic salta, ethylenediaminetetraacetic acid, 2-mercaptoethanol,, resorcinol, and catechols were products of Merck AG (Darmstadt, Germany). Levulinic acid, cresols, fluoros, and m- and p- chloro were from Fluka AG (Switzerland); o- chloro was purchased from Aldrich-Europe (Belgium). 2-(N-morpholino)ethanesulfonic acid, all enzymes, proteins, dithiothreitol, and 8i-ketoadipic 13

14 GAAL AND NEUJAHR J. BACTERIOL. acid came from Sigma (St. Louis, Mo.). Acetylacrylic acid was from Polysciences Inc., (Warrington, Pa.). Sephadex gels and Sephadex ion exchangers came from Pharmacia (Uppsala, Sweden). Hydroxyapatite was from BioRad Laboratories (Richmond, Calif.). Chromatographic equipment for enzyme purification was from Pharmacia and LKB-Produkter (Bromma, Sweden). Spectrophotometry was carried out on a Perkin-Elmer recording spectrophotometer, model 124, or a Zeiss spectrophotometer PMQ II. Manometry was done in a Warburg apparatus, model Braun VL 166. Dissolved oxygen was measured with a Clark oxygen electrode YSI 4004. Organism and culture. Cultures (200 to 1,000 liters) of T. cutaneum were grown in large fermentors as described earlier (20) using or resorcinol as the carbon source. Cell paste was stored frozen until disrupted. Crude cell-free preparations. Cell paste (50 to 200 g) was suspended in 0.05 M K-phosphate buffer (ph 7.6) containing 1i-' M mercaptoethanol, 10-4 M ethylenediaminetetraacetic acid, and 10-6 M flavin adenine dinucleotide. The thick suspension was mixed with an equal volume of glass beads (B. Braun, Melsungen Apparatebau, Germany) and disrupted in a rotary disintegrator manufactured at this institute (11). Smaller quantities (0.5 to 5.0 g of cell paste) were disrupted in a similar manner using a centrifuge tube and a glass propeller (22). Supernatants from centrifugations at 50,000 x g or 105,000 x g were used as the source of all the enzymes concerned. Enzyme assays. All enzymes were assayed spectrophotometrically at ambient temperature. One enzyme unit is defined as the amount of enzyme that catalyzes the disappearance of 1 umol of substrate or cosubstrate per min, or the formation of 1,umol of product per min. Phenol hydroxylase was determined by following the disappearance of its cosubstrate, reduced nicotinamide adenine dinucleotide phosphate (NADPH), i.e., recording the change in absorbance at 340 nm (-AA34o/min), as described earlier (18). Catechol 1,2-oxygenase was assayed by following the appearance of its product, cis,cis-muconic acid (+AA260/min) under conditions reported earlier (30). Catechol 2,3-oxygenase was measured according to Sala-Trepat and Evans (24), but using 0.033 M tris(hydroxymethyl)aminomethane (Tris)-SO4 instead of phosphate buffer. cis,cis-muconate-lactonizing enzyme was determined by following the disappearance of its substrate (-AA2w/min). The assay was carried out in 1.0-cm cells. The final volume of 1.0 ml contained 0.033 M 2-(N-morpholino)ethanesulfonic acid buffer adjusted to ph 6.6 with NaOH, 0.1 t,mol of cis,cis-muconate, and 0.5 to 200 jig of enzyme protein. In our system, 1 enzyme unit is equivalent to a decrease of 17.2 optical density units at 260 nm per minute. Delactonizing activity was measured by following the disappearance of (+)-muconolactone (-AAmo/min), essentially as described by Sistrom and Stanier (26). The assay was done in 1.0-cm cells. The final volume of 1.0 ml contained 0.033 M Tris-SO4 (ph 7.6), 1.0,mol of racemic muconolactone, and 50 to 200,ug of enzyme protein. The reverse reaction to cis,cismuconate (by the action of the lactonizing enzyme) was neglected. One enzyme unit is assumed to represent a decrease of about 1.5 optical density units per min. Enzymic reduction of maleylacetate was measured by following the decrease of Am%0, using NADPH as the cosubstrate. The reaction was done in 1.0-cm cells. The final volume of 1.0 ml contained 0.05 M Tris-SO4 (ph 7.6), 0.2,umol of maleylacetate, 0.2 ymol of NADPH, and 50 to 200 itg of enzyme protein. Under these conditions, 1 enzyme unit corresponds to an absorbance decrease of 6.1 optical units per min. Purification of enzymes. Phenol hydroxylase was purified to electrophoretic homogeneity, essentially as described earlier (17). Catechol 1,2-oxygenase, cis,cismuconate-lactonizing enzyme, and the maleylacetatereducing enzyme were purified about 50 times. These three enzymes could be recovered during the purification of hydroxylase. Separation of all four enzymes on the same diethylaminoethyl-sephadex column is shown in Fig. 1. The most active fractions of each peak were pooled and used as sources of the respective enzymes. Chemical syntheses. cis,cis-muconic acid was prepared by oxidizing with peracetic acid according to Elvidge et al. (7). It was recrystallized from ethanol to give white crystals of mp 192 to 193 C (uncorrected). Racemic y-carboxymethyl-a0-butenolide (muconolactone) was prepared from pure cis,cismuconic acid as described by Elvidge et al. (7) to give colorless prisms of mp 110 to 112 C (uncorrected). Enzymic syntheses. Maleylacetate was prepared enzymically using 300 U of purified catechol 1,2-oxygenase in 50 ml of 0.05 M K-phosphate buffer (ph 6.9). Crystalline 1,2,4-benzenetriol (recrystallized from ether-petroleum after filtering its ethereal solution through activated carbon) was added in about 3-mg portions over a period of 2 h (a total of 133 mg). The mixture was stirred magnetically in a 1-liter beaker. Oxygen concentration was monitored with a Clark oxygen electrode. The ph of the mixture was held between 6.5 and 6.9 by adding 1.0 M NaOH. The concentration of maleylacetate was measured enzymically using partially purified maleylacetate reductase in the spectrophotometric assay, with NADPH as cosubstrate. When the reaction with 1,2,4-benzenetriol was complete, the ph was adjusted to 7.5 and the mixture was filtered through a Diaflo PM 10 membrane. The filtrate consisted of 50 ml of 15 mm maleylacetate. It remained stable for several days when stored at 4 C. Manometric procedures. Oxygen uptake measurements were done by conventional procedures (28). B8-Ketoadipic acid was determined by catalytic decarboxylation at ph 4.2 using 4-amino-antipyrine as a catalyst, according to Sistrom and Stanier (25). Analytical procedures. Phenol concentration in culture media was determined by its UV absorbance at 270 nm, after filtering through a Millipore Millex disposable filter unit. Protein in crude extracts was determined by the biuret method (9). When low protein readings were expected, the Folin method of Lowry et al. (13) was used. Bovine serum albumin was used as a reference. f?-ketoadipic acid was detected by the Rothera reaction (23) and also determined manometrically. Thin-layer chromatography was carried out in solvent system I (benzene-ethyl alcohol-acetic acid, 80:14:7). Silica plates from Schleicher and Schiill

VOL. 137, 1979 METABOLISM OF PHENOLS IN YEAST 15 15-~~~~~~~~~~~~~~~~~~~w REz 250 500 750 1000 Effluent, ml FIG. 1. Separating the enzymes involved in metabolism of and resorcinol, using a diethylaminoethyl- Sephadex column. Symbols: (RE) maleylacetate reductase; (LA) cis,cis-muconate lactonase; (HY) hydroxylase; (CO) catechol 1,2-oxygenase; (---) (NH4)2SO4 concentration. The column (2.5 by 35 cm) was equilibrated with 0.02 M Tris-SO4 (ph 7.6) containing 1 mm mercaptoethanol, 0.1 mm ethylenediaminetetraacetic acid, 2 pm flavin adenine dinucleotide, and (NH4)2SO4 as indicated. The material applied to the column was from -grown cells. Crude extracts were pretreated by precipitation with protamine sulfate followed by precipitation with ammonium sulfate and by gel filtration on Sephadex G-50. Resorcinol-grown cells yielded a corresponding pattern; however, the amount ofmaleylacetate reductase was higher (see Table 4). were used, F1510/LS254 for preparative work and F1500/LS254 for analyses. Detection was achieved by using a UV lamp. Isolation of f8-ketoadipate after incubating crude extracts with catechol, resorcinol, 1,2,4- benzenetriol, or maleylacetate. Crude extracts of -induced cells were used in the incubations with catechol. However, extracts of resorcinol-induced cells were used in the incubations with resorcinol, 1,2,4- benzenetriol, or maleylacetate. The usual ratio in this series of experiments was about 30 g of cell paste to 100 ml of buffer. Supernatants contained 9 to 11 mg of protein per ml. Such cell extracts contained glucose-6- phosphate dehydrogenase activity, about 0.3 U/mg of protein. Additional glucose-6-phosphate dehydrogenase was supplied in some incubations. The substrates were gradually added in small portions during a period of 2 to 3 h. When required, oxygen was supplied by vigorous shaking. The ph was held between 6.5 and 7.0 by intermittent additions of 1.0 M NaOH. Incubations took place at room temperature.,b-ketoadipate was isolated essentially as described by Darrah and Cain (5). However, direct crystallization of the product after ether extraction and evaporation could be done only in the case of extracts from -grown cells after incubation with catechol. In all other cases, the residue failed to crystallize. Samples of the residue were separated by thin-layer chromatography in solvent system I. The substance that co-chromatographed with authentic fb-ketoadipate was extracted and crystallized from ethyl acetate-n-hexane. Other incubation conditions varied somewhat, according to the nature of the primary substrate. All recoveries are calculated on a molar basis. Incubation with catechol. A 20-mmol quantity of catechol was added in portions of 0.5 mmol every 5 to 10 min to 450 ml of extract of -grown cells. After extraction with diethyl ether as described by Darrah and Cain (5), the extracted solids were crystallized from ethyl acetate-light petroleum (boiling point range 60 to 80 C) to give 1.6 g of white crystals of mp 116 to 118 C. This corresponds to an overall recovery of 50%. Incubation with resorcinol. Portions of 0.4 mmol of resorcinol mixed with 0.8 mmol of glucose-6-phosphate were added at 20-min intervals to 300 ml of extract of resorcinol-grown cells, supplemented with 0.05 mmol of nicotinamide adenine dinucleotide phosphate (NADP). About 5 mmol of resorcinol was consumed during 3 h. Overall recovery of 8l-ketoadipate, mp 116 to 118 C, was about 25% of the consumed resorcinol. Incubation with 1,2,4-benzenetriol. The incubation mixture consisted of 300 ml of extract of resorcinol-grown cells, supplemented by 500 U of glucose- 6-phosphate dehydrogenase and 0.05 mmol of NADP. It was held in a 1-liter Erlenmeyer flask on a rotary shaker. Another mixture was prepared by dissolving 7 mmol of 1,2,4-benzenetriol and 8 mmol of glucosq-6- phosphate in 80 ml of oxygen-free 0.05 M K-phosphate (ph 6.5) in a flask stoppered with a rubber membrane cap. A peristaltic pump slowly transferred the latter mixture anaerobically into the incubation mixture. Incubation proceeded for 2.5 h. Oxygen concentration was monitored continuously by means of a Clark oxygen electrode. The concentration of 1,2,4-benzenetriol was determined enzymically in small samples, using purified catechol 1,2-oxygenase. Overall recovery of f8-ketoadipate was about 10% of the consumed 1,2,4- benzenetriol.

16 GAAL AND NEUJAHR Incubation with maleylacetate. A total of 0.70 mmol of maleylacetate (47 ml of the 15 mm solution prepared as described above) was added in 0.8-ml portions to 20 ml of extract of resorcinol-grown cells, supplemented by 100 U of glucose-6-phosphate dehydrogenase, 0.01 mmol of NADP, and 0.8 mmol of glucose-6-phosphate. The incubation was allowed to proceed for 2 h. Concentration of maleylacetate was determined enzymically with purified maleylacetate reductaae. Overall recovery of /8-ketoadipate was about 42% of the consumed maleylacetate. An identical incubation was then done, omitting the NADPH-generating system. After extracting and isolating just as with /8-ketoadipate, a sample of the residue was separated by thin-layer chromatography, using solvent system I. The substance that co-chromatographed with authentic trans-acetylacrylic acid was extracted with ether and crystazlied from ether-petroleum. A total of 10 mg was isolated, with an overall recovery of 48%. RESULTS Formation of 8-ketoacilds by crude extracts. Table 1 shows the formation of,-ketoacids by crude extracts of -grown cells as determined by catalytic decarboxylation. Oxygen consumption with or resorcinol was 2 mol of 02 per mol of substrate. This is consistent with a hydroxylation step followed by a ringcleaving step. With catechol, only 1 mol of 02 was consumed per mol of substrate. Omitting the NADPH-generating system depressed this value slightly. Thus, practically all the oxygen taken up in the presence of catechol was utilized for ring cleavage, whereas hydroxylation of catechol by crude extracts was iificant. The reaction products of, resorcinol, and catechol gave a strongly positive Rothera reaction. Any one of these three s gave rise to the formation of 1 mol of C02 per mol of substrate upon catalytic decarboxylation of its reaction product. This is conotent with the formation of 1 mol of,6-ketoacid from 1 mol of, catechol or resorcinol. The substance isolated after incubating with catechol had mp 112 to 115 C (unconected). The mp was undepressed by admixing authentic fl-ketoadipic acid. The product co-chromatographed with authentic,8-ketoadipate in solvent system I - (Rf 0.29). The phenylhydrazone derivative of the product had mp 136 to 1370C (uncorrected). This value was undepresed by admiing the phenylhydrazone derivative of authentic levulinic acid. Similar results were obtained with the products isolated from incubating with resorcinol, 1,2,4-benzenetriol, or maleylacetate. No phenylhydrazone derivatives were prepared in these cases. Conversion of methyl- and halogen-sub- J. BAcTzIOL. TABLE 1. Formnation of,b-ketoadipate by crude extracts of-grown cells in the presence of various? 02 uptake C02 evolve Substrate Sbtae Quantity (jlmol) Rothera test pumol (,umol/ of pjmolio substrate) sate) substtej Phenol 1.5 + 2.05 0.95 7.5 + 2.02 0.96 Catechol 3.0 + 1.06 0.96 7.5 + 1.08 0.94 7.5b + 0.97 0.94 Resorcinol 3.0 + 2.04 0.93 7.5 + 2.02 0.95 o-cresol 3.0-0.14 0.13 m-cresol 3.0-0.32 0.18 p-cresol 3.0-0.62 0.48 o-fluoro- 3.0-3.09 0.07 m-fluoro- 3.0-3.22 0.08 p.fluoro- 3.0-3.18 0.07 a Each Warburg flask contained (total volume of 3 ml) 150 pol of 2-(N- norpholino)ethanesulfonic acid buffer (ph 6.9), 2.5 U of glucose 6.phosphate dehydrogenase, 10 pmol of glucone 6-phosphate, 0.1 pmol of NADP, and the 50,000 x g supernatant (7.1 mg of protein). Phenolic substate was added from the side arm. The center well contained a piece of filter paper saked in 0.2 ml of 20% KOH. Temperature was 30 C, and the gas phase was air. After 15 min of equilibration, the manometer was closed and the substrate was tipped in. At termination of 02 uptake, the reaction mixture was quantitatively transerred to a clean Warburg flask containig 0.4 ml of 0.1 M 4-aminoantipyrine in its side arm. Glacial acetic acid (0.1 ml) was added to terminate the oxidation reaction. After 2 min of equilibration, the manometer ws closed and the catalyst was tipped in. The amount of p-ketoadipate is calculated from the amount of CO2 evolved. Results are corrected for changes in gas preure in the absence of substate. With cresols and fluoros, the oxidative stage of the reaction was interrupted after 3.5 h, despite a continued low 02 uptake. b NADPH-generating system omitted. stituted s by crude extracts. Table 1 shows that all three cresols were poor substrates for oxidation by crude extracts. The efficiency of oxidation increasd in the order of o-, m-, andpsubstitution. So did the proportion of CO2 evolved. All three fluoros were very good substrates for oxidation. The stoichiometry of oxygen consumption indicated 3 mol of 02 per mol of fluoro. This is one mol of 02 more than would be consitent with the introduction ofone hydroxyl group, followed by ring cleavage. The evolution of CO2 upon decarboxylation of the reaction products of fluoros was insignificant. This indicates that fluoros are not completely metabolized by the enzymes of the,-ketoadipate pathway. None of the oxidation products of methyl- or halogen-substituted s gave a positive Rothera reaction. To obtain information about the fate of these derivatives during conversion by crude extracts, we used purified enzymes of the,-ketoadipate

VOL. 137, 1979 pathway (Table 2). The rate of hydroxylation of cresols was low. So was the rate of ring cleavage of the resulting methyl catechols. Formation of either a- or,b-methyl derivatives of cis,cis-muconate is indicated by UV spectra of the reaction products (15). Judging from the decrease of A260, the lactonizing enzyme had no activity towards a-methyl muconate, whereas,8-methyl muconate was lactonized. p-fluoro was stoichiometrically converted to a lactone, judging from A2mo decrease. We did not investigate whether dehalogenation occurred at this stage or not. o- Fluoro was not stoichiometrically con- TABLE 2. Relative enzyme activities towards derivatives and their degradation productsa Relative enzyme activity Relative extent of P15mary Phenol Catechol cts,cis-mu- conversion substrate conate-lac- of muconahec genase tonizing en- ate derivazyme tiveb Phenol 100 100 100 100 o-cresol 7 2d 0 0 m-cresol 10 p-cresol 16 21' 25 100 o-fluoro- 30 5 7 10 m-fluoro- 72 16 10 55 p-fluoro- 90 58 20 100 o-chloro- 10 <1 m-chloro- 26 <1 p-chloro- 29 <1 a Enzymes were purified from cells grown on. Phenol hydroxylase was incubated with monosubstituted s in 4.0-ml final volume containing 100 Amol of Tris-SO4 buffer (ph 7.6), 1 U of hydroxylase, 0.8 pimol of ic substrate, 0.1 pmol of NADP or NADPH, 1.0 pmol of glucose- 6-phosphate, and 1.2 U of glucose-6-phosphate dehydrogenase. Incubation was done at room temperature until all substrate had reacted (10 to 60 min). The mixture was then diluted twice with 0.05 M Tris-Cl buffer to ph 8.3. About 3 U of catechol 1,2-oxygenase were added. Absorbance at 260 nm was recorded. At the termination of the reaction, the mixture was diluted twice with 0.05 M 2-(N-morpholino)ethanesulfonic acid buffer to ph 6.6. About 3 U of ciu,ci-muconate lactonase were then added, and the reaction was followed by recording absorbance at 260 nm. b Approximate values asesed from absorbance decrease. From ref. 17. d 3-Methylcatechol was used as the substrate since it is the presumed hydroxylation product of o-methyl (17). The ring cleavage product has the UW absorption spectrum of a- methyl-ciu,cis-muconate (15). '4-Methylcatechol was used as the substrate since it is the hydroxylation product of either m- or p-methyl (20). The ring cleavage product has the UV absorption spectrum of fl-methyl-cis,cis-muconate (17). METABOLISM OF PHENOLS IN YEAST 17 verted to a lactone. Although the initial rate of lactonization was high, the reaction came to an early end. Hydroxylation of m-fluoro seemed to yield 3- and 4-fluorocatechol in equimolar proportion. This was indicated by the extent of the lactonization reaction. The ringcleaving activity towards chlorocatechols was less than 1% of the activity towards catechol. In all the experiments described, practically identical results were achieved if purified enzymes of cells grown on resorcinol instead of were used. Evidence for the formation of maleylacetate and for the enzymic reduction of maleylacetate to 8-ketoadipate. The hydroxylation product ofresorcinol is 1,2,4-benzenetriol (17). The nonenzymic oxidation of 1,2,4-benzenetriol is shown in Fig. 2. When purified catechol 1,2-oxygenase was added to 1,2,4-benzenetriol, a different spectrum was observed (Fig. 3). The peak at 260 nm was absent, but a new peak appeared at 243 mm. When the mixture was acidified to ph 3.0, the peak at 243 nm disappeared, reappearing on neutralization. These spectral characteristics are consistent with the formation of maleylacetate as observed by Larway and Evans (P. Larway and W. C. Evans, Biochem. J. 95:52P) and also by Chapman and Ribbons (3). Attempts to isolate maleylacetate (described in Materials and Methods) led to its decarboxylation and isomerization tof,-trans-acetylacrylic acid. The isolated product co-chromatographed with authentic trans-acetylacrylic acid (Rf = 0.51) in solvent system I. It had mp 123 to 125 C, undepressed by adding the authentic acid. Both the isolated and the authentic product had a UV absorption peak at 220 nm in neutral and acidic solutions. Table 3 shows that crude extracts of resorcinol-induced cells catalyzed the reduction of maleylacetate by stoichiometric amounts of either NADPH or NADH. Furthermore, with a NADPH-generating system, the reduction of maleylacetate was indicated by disappearance of its peak at 243 nm. From these results we conclude that /1-ketoadipate is a metabolite of both and resorcinol. However, the 81-ketoadipate pathway of resorcinol degradation differs from that of degradation. The two pathways are indicated in Fig. 4. Enzymes of the and resorcinol degradation pathways in celis induced by or resorcinol. Table 4 shows that both types of cells contained all the relevant enzymes (cf. Fig. 3). However, the level of maleylacetate reductase was higher in resorcinol-grown cells, whereas the levels of the lactonizing and delac-

18 GAAL AND NEUJAHR J. BACTERIOL. Wavelength, nm FIG. 2. Spectral changes during nonenzymic oxidation of 1,2,4-benzenetriol. Scans were repeated as indicated (minutes). Cuvette contained, in a final volume of 3.0 ml at 25 C, 90 pmol of K-phosphate (ph 6.5) and 0.5 pmol of 1,2,4-benzenetriol. 8'V 6.0 0 L4 FIG. 3. Spectral changes during enzymic oxidation of 1,2,4-benzenetriol bypurified catechol 1,2-oxygenase. Scans were repeated as indicated (minutes) using a control lacking 1,2,4-benzenetriol. Conditions were as described in the kgend to Fig. 2. Reaction was inuitiated by adding about 0.2 U of catechol 1,2-oxygenase. Dotted line shows spectrum of enzymic oxidation product after acidifying to ph 3.0. tonizing enzymes were higher in -grown cells. DISCUSSION Metabolism of phenol Our results show that and catechol are metabolized to,bketoadipate in T. cutaneum, following ring cleavage of ortho type. Meta-cleavage could not be demonstrated in extracts of cells grown on or resorcinol. Purified hydroxylase has considerable affinity for catechol (19). The product of this hydroxylation reaction is pyrogallol (17). However, Table 1 indicates that hydroxylation of catechol by crude extracts is insignificant. Hydroxylation of catechol thus lacks physiological imnportance. The reason is probably the much lower activity towards catechol of the hydroxylating enzyme as compared to the activity of the ring-cleaving enzyme. It is worth noting that crude extracts oxidized pyro-

VOL. 137, 1979 TABLE 3. Enzymic reduction of makylacetatea Maleylacetate NADPH or NADH oxidized (UMOl) (IAmol) 0.00 0.03 0.20 0.24 0.50 0.55 a Assay cuvettes contained (3.0-ml final volume) 0.1 mmol of K-phosphate (ph 6.7); 0.6 pmol of NADPH or NADH; 100 fd of crude extract (0.5 mg of protein); about 0.5 U of purified catechol 1.2-oxygenase and 1,2,4-benzenetriol as indicated. The reaction was started by the addition of 1,2,4-benzenetriol. After incubation for 3 min, crude extract and NADPH (NADH) were added, and the reaction was followed spectrophotometrically at 340 nm. The amount of maleylacetate was estimated from the amount of added 1,2,4-benzenetriol, assuming a complete conversion by catechol 1,2-oxygenase. Maleylacetate disappeared (decrease of Am3 peak) when incubated with a NADPH-generating system consisting of 0.02 umol of NADP or NADPH, 1.0,umol of glucose-6-phosphate, and 0.2 U of glucose-6-phosphate dehydrogenase. a ; b c OH Phenol 0O OH ff r::[~c5 4f r'ocaur,z Catechot cis,cis-muconate (-lucono1actone C>=O dffi~ ~ ~ ~ d HO a; HO,( b 'fz*, CH O4H 2 "X,~o MWeylseta f-kdoadipate 0-Ketoadipate anotaione FIG. 4. Metabolic sequences for and resorcinol catabolism in T. cutaneum. Arrows indicate enzymes operating in - or resorcinol-grown cells. (a) Phenol hydroxylase; (b) catechol 1,2-oxygenase; (c) cis,cis-muconate lactonase; (d-e) delactonizing activities, proposed in analogy to bacterial systems, (+)-muconolactone isomerase, and enol-lactone hydrolase; (t) maleylacetate reductase. gallol at a rate less then 10% of the oxidation rate of catechol (data not included). It is probable that the delactonizing activity of T. cutaneum comprises two different enzymes, an isomerase and a hydrolase. This would resemble what has been found in bacteria (for review see ref. 27). Cain et al. (1) studied the degradation of aromatic acids by a number of yeasts and fungi. In their extensive work they demonstrated METABOLISM OF PHENOLS IN YEAST 19 the existence of the enzymes of the fi-ketoadipate pathway in these organisms. The formation of/f-ketoadipate was also shown by a manometnc method in several cases. Metabolism of resorcinol. Our results show that resorcinol is also converted to,8-ketoadipate. The intermediates are 1,2,4-benzenetriol and maleylacetate. Earlier, we observed that enzymic cleavage of 1,2,4-benzenetriol yields a product with a peak around 330 nm (30). This was probably due to a partly nonenzymic oxidation. Recovery of,-ketoadipate after incubation with resorcinol or 1,2,4-benzenetriol was markedly lower than after incubation with maleylacetate. Alternative metabolism of 1,2,4-benzenetriol (than by the ortho-cleavage) can be excluded. In a control experiment using crude extracts, 2 Mmol of 1,2,4-benzenetriol gave 1.86 umol of maleylacetate. 1,2,4-Benzenetriol is very reactive, especially around neutral ph and in the highly aerobic atmosphere required for ring cleavage. Thus, the low recoveries depend on spontaneous side reactions rather than on alternative routes of resorcinol metabolism. The degradation of resorcinol to maleylacetate by a soil pseudomonad was first reported by Larway and Evans (Biochem. J. 95:52P). Other authors also observed the formation of maleylacetate as an intermediate in aromatic catabolism in various pseudomonads (3, 6, 8). The decarboxylation and isomerization of maleylacetate to trans-acetylacrylic acid agrees with the results of Chapman and Ribbons (3). They have also described and discussed the autooxidation of 1,2,4-benzenetriol. The extracts of the soil pseudomonads described by Larway and Evans (Biochem. J. 95: 52P) did not reduce maleylacetate, not even with NADH- and NADPH-generating systems. Instead, there were indications that fumarate and acetate were formed. The extracts of Pseudomonasputida studied by Chapman and Ribbons (3) did reduce maleylacetate with a requirement for NADH; NADPH was not tested. The cellfree yeast system of T. cutaneum reduces maleylacetate with either NADH or NADPH. Metabolism of other monosubstituted s. The metabolism of o-cresol ceased at an early stage, since the lactonizing enzyme had no activity towards a-methyl-cis,cis-muconate (Table 2). The lactonizing enzyme had considerable activity toward f6-methyl-cis,cis-muconate. Thus, m- and p-cresol are degraded through the stage of lactonization (cf. Table 2). Incubating crude extracts with the cresols gave a negative Rothera reaction (Table 1). For this reason, it is probable that the lactonization product is not metabolized further. This would be similar

20 GAAL AND NEUJAHR to the findings of Catelani et al. (2) that the degradation of 4-methyl-catechol in P. desmolyticum ceases after the formation of (+)-y-carboxymethyl-y-methyl-aa_butenolide. The C02 evolution in incubations of crude extracts with cresols may depend on decarboxylation of some other acid than f,-ketoadipate. It should be stressed that the method of estimating,b-ketoadipate by its catalytic decarboxylation is not specific for,-ketoadipate. We found that, e.g., maleylacetate is decarboxylated even faster than 8-ketoadipate. Maleylacetate does give a positive Rothera reaction, but the sensitivity is at least 10 times lower than towards,b-ketoadipate (data not included). Earlier results with whole cells of T. cutaneum indicate that cresols are metabolized rather vigorously after a lag period (21). This implies that cresols induce a pathway for their own degradation which differs from that induced by. Judging from the reaction(s) with purified enzymes in comparatively high concentrations, the degradation of o-fluoro ceases at the formation of a-fluoro-cis,cis-muconate (Table 2). p-fluoro seems to undergo reactions leading to lactonization giving either fb- or y- fluoro-muconolactone.,8-fluoro-muconolactone should spontaneously hydrolyze to 8-hydroxymuconolactone, analogous to the hydrolysis of,b-chloro-muconolactone (8). However, neither f,-fluoro-muconolactone nor f-hydroxy-muconolactone nor -y-fluoro-muconolactone was metabolized to a,b-ketoacid when we used crude extracts (no C02 evolution). In crude extracts the enzyme concentrations were much lower, however. The possibility of formation of -y-carboxymethylene-aa-butenolide from,b-fluoromuconate must be considered analogous to the results of Evans et al. for f-chloro-cis,cis-muconate (8). Obviously, y-carboxymethylene-a-butenolide (if formed) was not converted to a,bketoacid within the time of the experiment, since no C02 was evolved (Table 1). The high oxygen uptake in the presence of all three fluoros may depend on their further hydroxylation to pyrogallol derivatives and also on spontaneous oxidation of the reaction products. The uncoupling of the electron flow in the hydroxylation reactions is also probable using substrate analogs. The results obtained with chloros indicate that their degradation ceases before ring cleavage (see Table 2). We have not yet studied the induction pattern of the enzymes involved in and resorcinol degradation, but the results of Table 4 reveal that we are dealing with a complex regulatory system where both coordinate and coincident induction may be involved. The low delactoniz- J. BACTFOL. TABLE 4. Enzyme activities in crude extracts of cells grown on and resorcinolp Activity' in cells grown on: Enzyme Phenol Resorcinol Phenol hydroxylase 0.24 0.28 Catechol 1,2-oxygenase 0.56 0.86 cis,cis-muconate-lactionizing en- 0.61 0.48 zyme Delactonizing enzyme(s) 0.33 0.08 Maleylacetate reductase 0.23 0.65 a Cells were grown in Erlenmeyer flasks on a rotary shaker and harvested at midlogarithmic phase. For preparation of crude extracts and enzyme tests, see the text. Expressed as enzyme units per milligram of protein. ing activity in cells grown on resorcinol may be a result of an independent regulatory control of the synthesis of muconolactone isomerase or,bketoadipate enol-lactone hydrolase. In fact, f- ketoadipate enol-lactone hydrolase activity in several bacteria was shown to be expressed by two isofunctional enzymes with independent regulatory control (27). 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