Extradiol Cleavage of 3-Substituted Catechols by an Intradiol Dioxygenase, Pyrocatechase, from a Pseudomonad*

l), a ferric ion-containing dioxygenase from Pseudomonas arvilla C-l, catalyzes the intradiol cleavage of catechol with insertion of 2 atoms of molecular oxygen to form cis,cis-muconic acid. The enzyme also catalyzed the oxidation of various catechol derivatives, including 4-methyl-catechol, 4-chlorocatechol, 4-formylcatechol (protocatechu-aldehyde), 4,5-dichlorocatechol, 3,5-dichlorocatechol, 3-methylcatechol, 3-methoxycatechol, and 3-hydroxycatechol (pyrogallol). All of these substrates gave products having an absorption maximum at around 260 nm, which is characteristic of cis, cis-muconic acid derivatives. However, when used the product formed two

All of these substrates gave products having an absorption maximum at around 260 nm, which is characteristic of cis, cis-muconic acid derivatives. However, when 3-methylcatechol was used as substrate, the product formed showed two absorption maxima at 390 and 260 nm. These two absorption maxima were found to be attributable to two different products, 2-hydroxy-6-oxo-2,4-heptadienoic acid and 5-carboxy-Z-methyl-2,4-pentadienoic acid (Z-methylmuconic acid). The former was produced by the extradiol cleavage between the carbon atom carrying the hydroxyl group and the carbon atom carrying the methyl group; the latter by an intradiol cleavage between two hydroxyl groups. Since these products were unstable, they were converted to and identified as 6-methylpyridine-2-carboxylic acid and 2methylmuconic acid dimethylester, respectively. Similarly, d-methoxycatechol gave two products, namely, 2-hydroxy-5methoxycarbonyl-2,4-pentadienoic acid and 5-carboxy-2methoxy-2,4-pentadienoic acid (Z-methoxymuconic acid). With 3-methylcatechol as substrate, the ratio of i&radio1 and extradiol cleavage activities of Pseudomonas pyrocatechase during purification was almost constant and was about 17. The final preparation of the enzyme was homogeneous when examined by disc gel electrophoresis and catalyzed both reactions simultaneously with the same ratio as during purification.
All attempts to resolve the enzyme into two components with separate activities, including inactivation of the enzyme with urea or heat, treatment with * This work has been supported in part by grants from the Matsunaga Science Foundation, the Mishima Kaiun Foundation, the Japan Foundation for Applied Enzymology, the Medical Research Foundation for Geriatric Diseases, and from the Scientific Research Fund of the Ministry of Education of Japan.
1 Recipient of Sigma Chemical Co. Postgraduate Fellowship. 5 On leave from, and present address, Institute of Biochemistry and Physiology of Microorganisms, U.S.S.R. Academy of Sciences, Pustchino on the Oka, Moscow Region, U.S.S.R. sulfhydryl-blocking reagents or chelating agents, and inhibition of the enzyme with various inhibitors, proved unsuccessful.
These results strongly suggest that Pseudomonas pyrocatechase is a single enzyme, which catalyzes simultaneously both intradiol and extradiol cleavages of some a-substituted catechols.
Dioxygenases are a group of enzymes that catalyze the incorporation of 2 atoms of molecular oxygen into their substrates. The cleavage of the aromatic ring in nature is a function that depends largely or entirely upon this type of enzyme (1). When o-dihydroxylphenyl compounds (catechols) are cleaved by an individual dioxygenase, three modes of ring fission have been demonstrated so far (2) : (a) cleavage of the bond between carbon atoms bearing the hydroxyl groups (intradiol cleavage), (b) cleavage of the bond between the carbon atoms of positions 2 and 3 (extradiol cleavage, proximal), and (c) that of positions 1 and 6 (extradiol cleavage, distal) ( Fig. 1). Pyrocatechase (catechol:oxygen 1,2-oxidoreductase (decycliaing) EC 1.13.11.1)) which catalyzes the conversion of catechol to cis, cis-5-carboxy-2,4-pentadienoic acid (cis ,cismuconic acid) (Equation l), is an enzyme that catalyzes a typical intradiol cleavage (3).
Metapyrocatechase (catechol : oxygen 2,3-oxidoreductase (decyclizing) EC 1.13.11.2), which catalyzes the conversion of catechol to 2-hydroxy-6-oxo-2,4-hexadienoic acid (a-hydroxymuconic c-semialdehyde) (Equation 2), is an enzyme that carries out a typical extradiol cleavage (4). Although both enzymes contain nonheme iron as a sole cofactor, the intradiol type of enzyme contains the ferric form of iron, whereas the extradiol type contains the ferrous form of iron (5,6). It has been believed previously that the site of cleavage of an aromatic ring is strictly specific for each enzyme, namely, the ferric ion-containing dioxygenases would exclusively cleave the catechol ring in the intradiol manner, whereas the ferrous ioncontaining dioxygenases would cleave it in the extradiol manner (7,8).

CHO
In this paper, however, we present some evidence indicating that a ferric ion-containing dioxygenase, pyrocatechase, from a pseudomonad catalyzes not only an intradiol cleavage, but also an extradiol proximal cleavage when 3-substituted catechols are used as substrates (Scheme lj.

Spectra of Reaction Products
Among the above-mentioned substrates, catechol, 4-substituted catechol derivatives, and disubstituted catechol derivatives gave products having an absorption peak at around 260 nm. However, when P-pyrocatechase was incubated with 3-methylcatechol, a yellow colored reaction product (or products) was obtained which showed two absorption peaks, one at 260 nm and the other at 390 nm. The latter peak increased in absorbance upon addition of an alkaline solution (Fig. 2~) catechase gave a colorless product having a peak at 260 nm, and no measurable absorption at 390 nm was observed, even after addition of an alkaline solution (data not shown). Metapyrocatechase gave a yellow product having two absorption peaks at 390 and 320 nm at pH 7.5 when reacted with 3-methylcatechol. The peak at 390 nm increased upon addition of an alkaline solution with simultaneous disappearance of the peak at 320 nm (Fig. 2b). The molar extinction coefficients (E) of the products by B-pyrocatechase and metapyrocatechase were 18,000 at 260 nm (pH 7.5) and 44,600 at 390 nm (pH 12.0), respectively.
Further, when P-pyrocatechase was incubated with 3-methoxycatechol, a product having an absorption peak at around 280 nm was obtained (Fig. 3a). On the addition of an alkaline solution, a new absorption peak appeared at 370 nm accompanied by a decrease in absorption at around 300 nm. When metapyrocatechase was incubated with 3-methoxycatechol, a product was obtained with an absorption peak at 305 nm, which shifted to 370 nm upon the addition of an alkaline solution (Fig. 3b). Molar extinction coefficient of the product at 370 nm was about 21,400, at pH 12. The reaction product of 3-methoxycatechol with Bpyrocatechase showed an absorption peak at 285 nm (t = 17,800  (17).

Thin Layer Chromatography and Paper Electrophoresis of Reaction Products
In order to determine whether the above-mentioned spectra are attributable to a single product or two products, each formed by intradiol and extradiol cleavages, respectively, thin layer chromatography and high voltage electrophoresis of the products were carried out.
When 3-methylcatechol was used as substrate, each product obtained by the action of B-pyrocatechase and metapyrocatechase gave only a single spot on cellulose thin layer chromatogram.
RF values of each spot were 0.9 and 0.75, respectively. However, P-pyrocatechase gave two spots which coincided in RF values with those obtained with B-pyrocatechase and metapyrocatechase, respectively. One of the products with an RF value of 0.75 as well as the product by metapyrocatechase gave a redbrown spot with 2,4-dinitrophenylhydrazinc, suggesting that both products have a carbonyl moiety. Similarly, the reaction products of P-pyrocatechase with 3-methoxycatechol yielded two spots. Essentially the same results were obtained on high voltage paper electrophoresis.

Zdentijication of Reaction Products of S-ilbethylcatechol
For the identification of the reaction products, they were converted to their stable derivatives according to the following scheme (Scheme 2).
Zsolation and ZdentiJication of Reaction Product Formed by Intradiol Cleavage-Incubation was carried out at 25" in a final volume of 60 ml containing 50 mM potassium phosphate buffer, pH 7.5, 70 mg of P-pyrocatechase, and 100 mg (0.8 mmol) of 3-methylcatechol (I). The reaction was performed by stepwise additions (0.5 ml each) of 80 mM 3-methylcatechol in order to prevent the inhibition by excess substrate. During the reaction, the pH was maintained at 7.5 by occasional additions of 5 N NaOH. After completion of the reaction, pH was lowered to 1.5 by the addition of 6 s HCl. The reaction product was extracted five times with 100 ml each of ethyl acetate. The ethyl acetate extracts were combined, dried over anhydrous Na2S04, and evaporated to dryness. The residue was dissolved in a small amount of methanol and applied zonally on a silica gel thin layer plate (0.5 mm thickness). The plate was developed with a solvent system consisting of chloroform-methanol-acetic acid (9 : 3 : 1). One main band with an RF value of 0.48 was detected with an ultraviolet lamp and a broad brown band was visible near the origin with an RF value of 0.1. The silica gel with the main ba.nd was cut out from the plate, and extracted with methanol. The extract was evaporated to dryness (41.5 mg). The product thus obtained (II) showed an absorption peak at 260 nm in 0.05 M Tris-acetate buffer, pH 7.5.
The product (II) was then methylated with diazomethane in methanol. After evaporation of the solvent, the residue was subjected to preparative thin layer chromatography on silica gel F254 (Merck) using chloroform as a developing solvent. The band of an RF value of 0.53, which was visualized under ultraviolet light, was scraped and extracted with chloroform. After evaporation of the solvent, the product (IV) was crystallized from chloroform (m.p. 57-59"). Its infrared spectrum had an ester carbonyl absorption band at 1,708 cm-i in chloroform. The electron impact mass spectrum showed a molecular ion peak at m/e 184 which corresponded to a molecular formula CsH1204, and a base ion peak at m/e 125 resulted from the loss of COOCH3 from parent ion. Furthermore, the fragment ion peaks, suggesting the presence of methyl and methoxycarbonyl groups in the molecule, were observed at m/e 170 (YW+ -CHz), 153 (Mf -OCH,), 139 (M+ -CH,-OCH,), 110 (If+ -COOCH,-CHI), and 97 (M+ -COOCH,-CO). TMS, tetramethylsilane.
The nuclear magnetic resonance spectrum (Fig. 4) showed a broad methyl singlet at 2.05 ppm, two methoxyls at 3.77 ppm (singlet) and 3.83 ppm (singlet), and three signals assignable to olefinic protons at 5.97 ppm (doublet, J = 16 Hz), 6.47 ppm (broad doublet, J = 12 Hz), and 8.10 ppm (double doublet, J = 12 and 16 Hz). The pattern of spin-spin couplings among three olefinic protons and the presence of long range coupling between methyl (2.05 ppm) and one of the olefinic protons (6.47 ppm) established the arrangement of these protons above Cz through O5 as shown by the formula in Fig. 4. Moreover, the rather large coupling constant (16 Hz) between protons at 5.97 ppm (C,i and at 8.10 ppm (C,) suggested that these olefinic protons behaved in a trans geometry to each other (18). However, the geometry of the double bond between CZ and Ca has not been determined.
Thus, the structure (IV) was tentatively assigned to this product.
Isolation and Identification of Reaction Product Formed by Extradiol Cleavage-Since the expected product (V) of extradiol cleavage was extremely unstable, the product was converted to its corresponding picolinic acid derivative (VZ) for final identification. Incubation was carried out in essentially the same manner as above using 250 mg (2.0 mmol) of 3-methylcatechol (I) and 140 mg of P-pyrocatechase in a total volume of 200 ml. After completion of the reaction, the enzyme was separated from the product by filtering the mixture through a Diaflo membrane (PM-lo), using a Diaflo apparatus. To the filtrate, 600 ml of a 28% aqueous ammonia solution were added and the mixture was allowed to stand for 40 hours at 25" to convert the product (V) to the corresponding picolinic acid derivative (I'Z). The mixture was evaporated to dryness and the product was extracted with methanol. The extract was applied in a zone to a silica gel thin layer plate (0.5 mm thickness) and developed with a mixture of chloroform/methanol/acetic acid (9/3/l). One thick zone with an RF value of 0.48 and another with an RF value of 0.24 were detected by an ultraviolet lamp. The former zone was found to consist of the product formed by the intradiol cleavage and the latter of the picolinic acid derivative. The silica gel containing the latter zone was removed from the plate, extracted with chloroform/methanol (9/l). The extract was evaporated to dryness to give crystals (6.4 mg). Recrystallization from methanol gave needles, m.p. 127".
Ultraviolet absorption spectra of the crystals were the same as that previously reported by Nozaki el al. (2). It was further identified as 6-methylpyridine-2-carboxylic acid (VZ) by com-FIQ. 5. Infrared spectra of the picolinic acid derivative (VI) (lower tracing) from the extradiol cleavage product of a-methylcatechol (I) and authentic 6-methylpyridine-2-carboxylic acid (upper tracing) in KBr pellet.
parison with the authentic sample, prepared from 2,6lutidine, by infrared spectroscopy as shown in Fig. 5.

Isolation and Identijicalion of Reaction Products of-S-Methoxycatechol
Conversion of the reaction products to their stable derivatives was carried out according to the following scheme (Scheme 3). The reaction mixture contained in a final volume of 60 ml, 50 mM potassium phosphate buffer, pH 7.5, 140 mg of P-pyrocatechase, and 100 mg (0.7 mmol) of 3-methoxycatechol (VII). Incubation of the mixture and extraction of the reaction products by ethyl acetate were carried out in essentially the same manner as those for the reaction product of 3-methylcatechol formed by the intradiol cleavage. The dried extract was dissolved in 20 ml of ethyl acetate and applied on a Sephadex LH-20 column (3 X 70 cm) equilibrated with ethyl acetate. The products were eluted in separate fractions when the column was developed by ethyl acetate. The first fraction showed an absorption peak at 305 nm, which shifted to 370 nm upon alkalization, whereas the second fraction had an absorption peak at 285 nm which did not shift on alkalization.
Each fraction was pooled separately and evaporated to dryness to give crystals. The first fraction (8.0 mg) gave Compound IX (m.p. 144-146") and the second fraction (46 mg) Compound . Each compound showed a single spot on a silica gel thin layer chromatogram with a solvent system of chloroform/methanol/triethylamine (50/15/l) having Rp values of 0.38 (IX) and 0.27 (F1Z1), respectively. Judging from the absorption spectra of the products (Fig. 3) and their molar extinction coefficients mentioned above, the ratio of the products formed by the intradiol and extradiol cleavage of 3methoxycatechol was about 5. Mass spectra of these products were shown in Fig. 6, a and  afforded another methyl ester (XI), m.p. 52-53". Each compound showed a single spot on a silica gel thin layer chromatogram using chloroform as a developing solvent with RF values of 0.37 (X) and 0.27 (XI), respectively. The mass spectra of both compounds showed the same molecular ion peak at m/e 200 (M+, CgHlzOs) with a different fragmentation pattern as shown in Fig.  6, c and d. However, the ester X was gradually converted into the ester XI in chloroform at 60". Thus, it was suggested that the ester X should be a geometric isomer of the ester XI.
Nuclear magnetic resonance spectrum of ester X (Fig. 7) showed olefinic proton on OS at 5.72 ppm as a doublet with a coupling constant of 9.5 Hz, in addition to three methoxyl singlets at 3.73, 3.78, and 3.87 ppm. From the value of the coupling constant, the geometry of the double bond between Cd and Cs appeared to be in the cis configuration (18). On the other hand, Cb proton of the XI appeared at 6.08 ppm as a doublet with coupling constant of 16 Hz. Thus, the geometry of the double bond between C4 and Cs in the Compound XI was assigned to be the trans configuration (18). However, the stereochemistry of the double bond between Cz and Cs has not been determined. Consequently, the structures of these methylated compounds derived from VIII and IX were tentatively assigned as X and XI, respectively.

P@jication of Enzyme
The above-mentioned results indicated that two products were produced from 3-methylcatechol by the action of P-pyrocatechase. One with an absorption peak at 260 nm was formed by the intradiol cleavage and the other with a peak at 390 nm formed by the extradiol cleavage. The question remained as to whether the extradiol cleavage was due to contamination by an extradiol enzyme or whether a single enzyme catalyzed both intradiol and extradiol enzyme or whether a single enzyme catalyzed both intradiol and extradiol cleavages simultaneously.
In order to answer this question, ratios of the intradiol and extradiol cleavage activities were determined at each step during the purification. As shown in Table I, the ratios were almost constant throughout purification. The final preparation obtained with Sephadex G-200 was homogeneous on ultracentrifugation and disc gel electrophoresis.
Since the possibility existed that the extradiol cleavage activity was due to contamination or to mutation, the bacteria were isolated by a plating out technique and the enzyme was purified from severa, different single colonies. In all cases, the enzymes showed both activities simultaneously with a constant ratio.

Znactivation and Inhibition Studies
Stability-The time course of the inactivation of these two activities was studied with various concentrations of urea (Fig. 8). As the urea concentration increased, the rate of inactivation increased. The time course of inactivation at various concentrations of urea were essentially identical for both activities. Likewise, inactivation on heating at various temperatures or on treating with a proteinase, Nagase, paralleled each other for both activities. pH profiles for stability of both activities (45", 10 min) were also superimposed and the optimal pH was about 8.5.
E$ect of Inhibitors-As shown in Table II, when the enzyme was pretreated with various inhibitors including oxidizing agents, sulfhydryl inhibitors, and metal-chelating agents, both activities decreased to about the same extent. Metapyrocatechase was completely inact.ivated by , but this concentration of Hz02 did not show any effect on the extradiol cleavage activity of P-pyrocatechase.
The incubation of the enzyme with 5 x 10F3 M Tiron (4,5-dihydroxy-m-benzenedisulfonic acid disodium salt), a specific chelator for ferric ion, caused complete inactivation of both activities (Table II). As shown in Fig. 9, however, the absorbance at 475 nm due to the formation of a ferric ion-Tiron complex increased with concomitant loss of both activities.
All other attempts to separate the components responsible for the two activities have been unsuccessful, indicating that a single enzyme is involved in catalyzing both activities simultaneously. The next question studied was whether or not the active sites for the two activities were identical. To answer this question, the  Reaction mixture contained in a final volume of 3 ml, 50 mM Trisacetate buffer, pH 8.5, 12 mg of P-pyrocatechase, and 5 mM Tiron.
The reaction was carried out at 24". At the indicated time, the absorbance at 475 nm (X-X) which indicates the formation of a chelate complex between the ferric ion and Tiron, and extra.diol (O--O) and intradiol (O--O) cleavage activities were measured as described in Fig. 8. nature of the inhibition by the substrate analogue, o-nitrophenol, was examined. This compound was not metabolized by the enzyme, but acted as a competitive inhibitor for both activities. The K, values of 3-methylcatechol for intradiol and extradiol activities were almost the same, being 9.5 and 8.8 PM, respectively. The Ki values of o-nitrophenol for the two activities were also of the same order of magnitude, being 20 and 34 p&r, respectively. These results seem to suggest that only a single active site is responsible for both activities.
On the other hand, the K, values for the other substrate, oxygen, were found to be 47 and 200 pM for intradiol and extradiol activities, respectively. DISCUSSIOX P-Pyrocatechase, which has long been believed to cleave the catechol ring exclusively in an intradiol manner, appears to act on X-methylcatechol to produce two products: a yellow one with an absorption maximum at 390 nm and a second with an absorption maximum at 260 nm. A yellow product is normally produced by the extradiol cleavage of catechol (8), and in fact, the yellow product produced by the action of P-pyrocatechase on 3-methylcatechol was found to be identical with that produced by metapyrocatechase, an extradiol dioxygenase. The product absorbing at 260 nm was found to be identical with that produced by Bpyrocatechase. All attempts to separate the two activities of P-pyrocatechase by such means as purification of the enzyme, inactivation under various conditions and inhibition by substrate analogues, have so far been unsuccessful. Dioxygenase reactions are believed to be highly specific with respect to the cleavage site in the catcchol ring, with the ferric ion-containing dioxygenases catalyzing an intradiol cleavage and the ferrous ion-containing dioxygenases catalyzing an extradiol cleavage. However, the data prcscntcd in this paper indicate that P-pyrocatechase, a single enzyme, catalyzes two different cleavage reactions of S-methylcatechol simultaneously.
The physiological substrate, catechol, and the 4-substituted catechol derivatives were cleaved exclusively by the enzyme in the intradiol manner. However, some 3-substituted catechol derivatives, including 3-methylcatechol and 3-methoxycatechol, were cleaved not only in the intradiol but also in the extradiol, proximal manner, but not in the extradiol, distal manner. It is of interest that. 3-or 4-substituted catechol derivatives were cleaved exclusively by metapyrocatechase in the extradiol, proximal manner, but not in the distal manner (2). The ratios of intradiol and extradiol cleavages for 3-mcthylcatechol and 3-methoxycatechol were 17 and 5, respectively. Whether differences in the specificity of the cleavage site and in the ratio of two cleavages for different substrates are due to the electron distribution in the molecule or steric properties of these substrates needs further investigation.
The fact that the K, value for 3-methylcatechol as well as the Ki value for a competitive inhibitor, o-nitrophenol, was the same order of magnitude for intradiol and extradiol activities indicates that a single substrate binding site is probably responsible for both activities. However, the K, value for oxygen was different for the two activities. This is consistent with the reaction sequence proposed previously for dioxygenases (8), in which the organic substrate reacts first with the enzyme and then with oxygen. These results suggest that the initial step for both reactions may be identical, but that the reaction with oxygen is different for each. Whether or not a nonenzymatic reaction is involved in the latter step remains unsolved.
There are several other examples of enzymes that catalyze more than one reaction with a single substrate. Peroxidase is known to catalyze a variety of reactions, including peroxidation and hydroxylation of various substrates. A mixture of reaction products is formed from a single substrate (19,20). The exact nature of the reaction has not been elucidated fully, but the formation of a free radical from the substrate appears to occur during the reaction (21). Lysine monooxygenase, a flavoprotein, which catalyzes the monooxygenation of lysine to form d-aminonorvaleramide, catalyzes both monooxygenation and oxidation reactions simultaneously with certain substrates.'J Poor fit of the substrate analogues with the active site of the enzyme appears to affect the activation mechanism of oxygen by this oxygenase, so that some of the substrate molecules are oxidized rather than oxygenated.' Likewise, another flavoprotein monooxygenase, lactate oxidase (decarboxylating) catalyzes the formation of two different products, chloroacetate and pyruvate, from a novel substrate, /I-chlorolactate (22). It is also well known that several vitamin B6 enzymes can catalyze more than a single reaction (23, 24). z-Aspartate-fidecarboxylase was one of the first enzymes which was shown to catalyze additional reactions. In the presence of pyruvate, the enzyme catalyzes the P-decarboxylation and transamination of aspartate (25). With P-chloralanine as substrate, the enzyme catalyzes the formation of pyruvate, ammonia, and Cl-, as well as an inactive alkylated form of the enzyme (26).
Thus, a number of enzymes are not as specific in terms of substrate and reaction pathway as originally thought. The data presented in this paper are the first to show that a nonheme ironcontaining enzyme can catalyze more than a single reaction. These results may provide a clue to better understanding of the reaction mechanism of dioxygenases.