[20] Catechol and chlorocatechol 1,2-Dioxygenases

122

HYDROCARBONS

AND RELATED

[20] C a t e c h o l a n d C h l o r o c a t e c h o l

COMPOUNDS

[9-0]

1,2-Dioxygenases

By KA-LEUNG NGAI, ELLEN L. NEIDLE, and L. NICHOLAS ORNSTON Introduction Catechol and chlorocatechol 1,2-dioxygenases (EC 1.13.11.1; Scheme 1) incorporate molecular oxygen into catechol as they cleave between its hydroxylated carbons to produce cis, cis-muconic acid which is metabolized via the fl-ketoadipate pathway.~ Most broadly distributed in nature is catechol 1,2-dioxygenase, formerly named catechol oxygenase I, which exhibits little or no activity with chlorocatechol. This enzyme normally represents several percent of soluble protein in fully induced cells, and the oxygenase has been purified from a number of bacterial sources. 2-6 The purified enzyme possesses a specific activity of about 20 units/mg protein. Chlorocatechol 1,2-dioxygenase, formerly known as catechol oxygenase II, acts on chlorocatechols to produce the corresponding muconic acid derivatives. This broad specificity, first observed with the enzyme from Brevibacteriumfuscum, 7 was noted with a dioxygenase from Pseudomonas B 13. 3 The purified enzyme exhibits relatively low k~t values with catechol or chlorocatechols. The specific activity of the purified enzyme is about 2 units/mg protein, and it represents about 16% of the soluble protein in fully induced cells, s Some Pseudomonas isolates express catechol oxygenase from the chromosomal catA gene and chlorocatechol oxygenase from the plasmid-borne clcA gene. 3 Plasmids carrying the clcA gene have been isolated, 9 and the chlorocatechol oxygenase gene has been sequencedJ ° The chromosomal 1R. Y. Stanier and L. N. Ornston, Adv. Microb. Physiol. 9, 89 (1973). 2 y. p. Chen, A. R. Glenn, and M. J. Dilworth, Arch. Microbiol. 141,225 (1985). 3 E. Dorn and H. J. Knackmuss, Biochem. J. 174, 73 (1978). 4 y. Kojima, H. Fujisawa, A. Nakazawa, T. Nakazawa, F. Kanetsuna, H. Toniuchi, M. Nozaki, and O. Hayaishi, J. Biol. Chem. 242, 3270 (1967). 5 M. M. Nozaki, M. Iwaki, C. Nakai, Y. Saeki, K. Horiike, H. Kagamiyarna, T. Nakazawa, Y. Ebina, S. Inouye, and A. Nakazawa, in "Oxygenases and Oxygen Metabolism" (M. Nozaki, S. Yamamoto, Y. Ishimura, M. J. Coon, L. Ernster, and R. W. Estabrook, eds.). Academic Press, New York, 1982. 6 R. N. Patel, S. Mazumdar, and L. N. Ornston, J. Biol. Chem. 250, 6567 (1975). 7 H. Nakagawa, H. Inoue, and Y. Takeda, J. Biochem. (Tokyo) 54, 65 (1963). s K.-L. Ngai and L. N. Ornston, J. Bacteriol. 170, 2412 (1988). 9 D. Ghosal, I. S. You, D. K. Chatterjee, and A. M. Chakrabarty, Proc. Natl. Acad. Sci. U.S.A. 82, 1638 (1985). 10B. Frantz and A. M. Chakrabarty, Proc. Natl. Acad. Sci. U.S.A. 84, 4460 (1987).

METHODS IN ENZYMOLOGY, VOL. 188

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[20]

CATECHOLAND CHLOROCATECHOL 1,2-DIOXYGENASES

~

OH OH

Catechol

OH OH

Catechol

oxygenase

catA

C1 ~

123

I

oxygenase

~Co0-CO0-

c l cA

II

C1 ~ C O 0 CO0-

SCHEME1. Reactionsof catecholand chloroeateeholdioxygenases. catechol oxygenase gene from Acinetobacter calcoaceticus has been cloned ~ and sequenced.~2 Because catechol and chlorocatechol oxygenases are expressed at high levels in induced cells, they are relatively amenable to purification. We describe here procedures that allow Acinetobacter catechol oxygenase and Pseudomonas chlorocatechol oxygenase, enzymes for which amino acid sequences can be deduced from DNA sequences, to be purified by high-performance liquid chromatography (HPLC). Our experience suggests that the procedures may be generally applicable to the purification of catechol and chlorocatechol oxygenases from diverse biological origins. Assay Muconatc and chloromuconatcs absorb strongly at 260 nm, a wavelength at which thc absorbance of catcchol or chlorocatechol is slight. Therefore thc activityof the oxygcnascs can bc determined by measuring thc increment in absorbance at 260 n m in the presence of catechol. Muconatc cycloisomcrasc, thc enzyme that acts on muconate, requires M n 2+ and, unlike the catcchol oxygcnascs, is inhibited by EDTA. Thcrcfore quantitative muconatc accumulation from catcchol is assured by adding E D T A to the assay mixture. The assay mixture contains, in a finalvolume of 3.0 ml within a quartz cuvcttc with l-cm light path, 0. I m M catcchol, I m M EDTA, 33 m M Tris-HCl buffer,p H 8.0,and from I to I0 milliunitsof enzyme activity.An enzyme unit is the amount required for transformation of 1.0/zmol of substrate to product per minutc at 25 °. Conversion of 1.0/zmol of sub-

~1E. L. Neidleand L. N. Ornston,J. Bacteriol. 1611,815 (1986). ~2E. L. Neidle,C. Hartnett, S. Bonitz,and L. N. Ornston, J. Bacteriol. 170, 4874 (1988).

124

HYDROCARBONS AND RELATED COMPOUNDS

[20]

strate corresponds to an increment of 5.63 absorbance units under these conditions. The reaction is initiated by addition of enzyme, and activity is monitored spectrophotometrically at 260 nm. The presence of chlorocatechol oxygenase activity is assessed by substitution of chlorocatechol for catechol. It is convenient to prepare mixtures sufficient for several enzyme assays. Stocks of 1.0 M Tris-HC1, pH 8.0, and 0.3 m M sodium EDTA can be prepared in advance and stored at room temperature. Concentrated solutions of 3 m M catechol or chlorocatechol should be prepared on the day of the assay and stored in the cold. Ten assay mixtures can be prepared by mixing 1.0 ml each of the reagent Tris, EDTA, and catechol solutions with 27 ml of distilled water.

Purification of Catechol 1,2-Oxygenase

Growth of Cells.Catcchol 1,2-oxygcnase can be induced by growing A. ca[coaceticus A D P I in mineral medium containing benzoate as the sole carbon and energy source.6,H High concentrations of benzoate are toxic to the organism, and the compound should be added periodically so that its concentration is always below 5 raM. Alternatively,the cam gcne, cloned within pUC19 in Escherichia coiiJMI01(pIBI343), can be expressed to a level of 1.5 units/rag protein by growing the cells with L broth in the presence of I m M IPTG (isopropyl-fl-D-thiogalactopyranosidc).1~ Cells are grown to about 5 m g wet weight/ml of culture; this corresponds to a turbidity of about 300 Klctt units or an apparent absorbancc of about I at 600 n m in a Gilford spectrophotomctcr. The cellsarc harvested, washed with 50 m M sodium cthylenediamine (EDA) buffer,p H 7.3, and frozen if their storage is desired prior to extraction. Preparation of Crude Extract. Cells are suspended in EDA buffer to a final concentration of about 2.5 g wet weight/10 ml and disrupted by sonication. The extract is clarified by centrifugation at 15,000 g for 15 min, treated with 0.25 M NaC1 in order to dissociate enzyme aggregates, and dialyzed against several changes of 10 m M EDA buffer, pH 7.3. The dialyzate is ultracentrifuged at 100,000 g for 1 hr, and the supernatant liquid is filtered through a 0.45/tm Nalgene cellulose acetate membrane filter unit. The resulting solution is the crude extract. High-Performance Liquid Chromatography. A binary gradient HPLC system with a TosoHaas (formerly Toya Soda) TSK DEAE-5PW ( 2 1 . 5 m m × 15cm) semipreparative anion-exchange column is used. HPLC is performed at room temperature; with this exception, enzyme

[20]

CATECHOL AND CHLOROCATECHOL 1,2-DIOXYGENASES

125

preparations are maintained at or below 4 °. A flow rate of 5 ml/min is set, and about 10 ml of crude extract containing 200-250 mg of protein from the crude extract is injected into the HPLC column which has been equilibrated with 10 m M EDA buffer, pH 7.3. The column is washed with the same buffer containing 0-0.25 M NaC1 in a linear gradient over 120 min. Protein in the eluant is monitored at 280 nm; fractions containing catechol oxygenase are readily detected because they have the characteristic red color of the enzyme. Catechol oxygenase is the predominant protein in these fractions, ~t which can be concentrated with a Centricon-30 ultrafilter unit (Amicon, 30,000 MW cutoff). Removal of contaminating proteins can be achieved by hydrophobic interaction chromatography as described below for chlorocatechol oxygenase.

Purification of Chlorocatechol 1,2-Oxygenase

Growth of Cells, Preparation of Crude Extracts, and High-Performance Liquid Chromatography. Cultures ofPseudomonas B13 are preadapted to growth with chlorobenzoate by selection for growth on 5 m M 3-chlorobenzoate plates. It may take 1 week for large colonies to appear, and these should be maintained by streaking on chlorobenzoate plates. Cultures are grown in mineral medium by addition of chlorobenzoate as the sole carbon source so that its concentration does not exceed 5 mM. Procedures for preparation of crude extracts and HPLC DEAE chromatography are the same as those described above for catechol oxygenase. Crude extracts of chlorobenzoate-grown Pseudomonas B 13 contain both catechol oxygenase and chlorocatechol oxygenase, and the presence of the latter enzyme can be established by monitoring activity with 3-chlorocatechol as the substrate. Hydrophobic interaction chromatography (HIC) can be used to purify the oxygenases to homogeneity from fractions emerging from DEAE chromatography. A binary gradient system with a TosoHaas TSK Pbenyl-5PW (7.5 m m × 7.5 cm) phenyl column is used. HPLC is performed at room temperature; the flow rate is set at l ml/min. A volume of 100/~l of concentrate from DEAE chromatography is injected into the HPLC column, which has been equilibrated with l0 m M EDA buffer, pH 7.3, containing 1.0 M ammonium sulfate. The enzyme is eluted with the same buffer containing ammonium sulfate in concentrations extending from 1.0 to 0.0 M in a linear gradient over 60 min. Oxygenase elutes toward the end of the gradient. Fractions containing the enzyme are pooled, desalted, and concentrated to 100 pl with a Centricon-30 ultrafilter unit.

126

HYDROCARBONS AND RELATED COMPOUNDS

[21 ]

Yield, Storage, Stability, and Properties The purification procedures should yield several milligrams of pure enzyme. The oxygenases are stable when stored in 10 mM EDA buffer, pH 7.3, at 4 °. The purified enzymes are burgundy red in color because they contain ferric ion, which participates in catalysis. 13 The amino acid sequences of catecho112 and chlorocatechol1° oxygenases, deduced from the DNA sequences, are evolutionarily homologous with those of P. putida protocatechuate 3,4-dioxygenase, for which the crystal structure has been determined?4 The structural basis for the differences in substrate specificity and catalytic efficiency of the oxygenases is yet to be established. Acinetobacter catechol oxygenase6 and Pseudomonas chlorocatechol oxygenase are dimers with respective subunit sizes of 34,351 and 28,922. Each subunit is associated with ferric ion, and pairs of tyrosyl and histidyl residues that ligate the ion appear to have been conserved by evolution at corresponding positions within the primary sequences of the intradiol oxygenases.~2 ~ L. Que, Jr., Struct. Bonding (Berlin) 40, 38 (1980). 14 D. H. Ohlendorf, J. D. Lipscomb, and P. C. Weber, Nature (London) 336, 403 (1988).

[21 ] M u c o n a t e C y c l o i s o m e r a s e By RJCaARD B. MEAGHER, KA-LEUN6 NGAI, and L. N I C H O L A S O R N S T O N Introduction

••co0-

o~COO-

I.

coo-

c=o

Muconate cycloisomerase,

catB

Muconate cycloisomerase (EC 5.5.1.1) forms muconolactone from cis, cis-muconate during the metabolism of mandelate and benzoate via the fl-ketoadipate pathway? The crystal structure of the enzyme from Pseudomonas putida has been determined2 and has been shown to be similar to the structure of mandelate racemase (Chen, Neidhart, and Petsko, personal communication). Comparison of the amino acid sequences of the racet R. Y. Stanier and L. N. Ornston, Adv. Microb. Physiol. 9, 89 (1973). 2 A. Goldman, D. L. Ollis, IC-L. Ngai, and T. A. Steitz, J. Mol. Biol. 182, 353 (1985).

METHODS IN ENZYMOLOGY, VOL. 188

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