JOURNAL OF BACTERIOLOGY, JUlY 1976, p. 536-544 Vol. 127, No. 1 Copyright ©D 1976 American Society for Microbiology Printed in U.S.A. Catechol 1,2-Dioxygenase from Acinetobacter calcoaceticus: Purification and Properties RAMESH N. PATEL,* C. T. HOU, A. FELIX, AND M. 0. LILLARD Corporate Research Laboratories, Exxon Research and Engineering Company, Linden, New Jersey 07036 Received for publication 19 January 1976 Procedures for the purification of catechol 1,2-dioxygenase from extracts of Acinetobacter calcoaceticus strain ADP-96 are described. The purified enzyme was homogeneous as judged by ultracentrifugation and acrylamide gel electro- phoresis. The enzyme contained 2 g-atoms of iron per mol ofprotein. The enzyme had a broad substrate specificity and catalyzed the oxidation of catechol, 4- methylcatechol, 3-methylcatechol, and 3-isopropyl catechol. The activity of the enzyme was inhibited by heavy metals, sulfhydryl inhibitors, and substrate analogues. The molecular weight of the enzyme was 85,000 as estimated by filtration on Bio-Gel agarose and 81,000 as estimated by sedimentation equilib- rium analysis. The subunit size determined by sodium dodecyl sulfate-gel electrophoresis was 40,000. The amino terminal amino acid was methionine. The amino acid composition and spectral properties of 1,2-dioxygenase are also presented. Antisera prepared against the purified enzyme cross-reacted and inhibited enzyme activity in crude extracts from other strain ofA. calcoaceticus, but failed to cross-react and inhibit isofunctional enzyme from organisms of the genera Pseudomonas, Alcaligenes, and Nocardia. Catechol 1,2-dioxygenase (EC 1.13.1.1) (CO), we have been investigating the properties of a nonheme, trivalent, iron-containing enzyme, the protocatechuate 3,4-dioxygenase from Pseu- catalyzes the cleavage of the aromatic ring of domonas aeruginosa and Acinetobacter cal- catechol to cis,cis-muconate with the incorpora- coaceticus (6, 7, 31; C. T. Hou, R. D. Schwartz, tion of 2 atoms of molecular oxygen into the and M. L. Ohaus, Abstr. Annu. Meet. Am. Soc. substrate. It represents the initial enzyme of Microbiol. 1975, RT-1, p. 275; C. T. Hou, M. L. the ,B-ketoadipate pathway, a metabolic se- Ohaus, and A. Felix, Abstr. Annu. Meet. Am. quence used by microorganisms for the degra- Soc. Microbiol. 1975, K156, p. 173). We have dation of aromatic compounds (26). Enzymes of demonstrated that the organic substrate bind- the /3-ketoadipate pathway, including CO, are ing site is distinct from the iron-containing cat- inducible in microorganisms. Comparative alytic site (5). Recently we extended our work studies of the mechanisms of regulation in dif- to CO from A. calcoaceticus. ferent bacterial genera indicate distinctive In this report, we describe procedures for the mechanisms of induction. Thus, regulation of purification of CO from A. calcoaceticus and enzymes of the f3-ketoadipate pathway are dif- describe some of its properties. We have also ferent in Acinetobacter (2) and the fluorescent prepared antisera against the purified enzyme group of Pseudomonas (19). These two groups and present data on the immunological specific- of organisms are taxonomically separated on ity of the enzyme from A. calcoaceticus. We the basis of morphology and deoxyribonucleic hope that studies of these two nonheme, triva- acid (DNA) content. Thus, organisms of the lent, iron-containing, aromatic ring fission en- Acinetobacter genus are nonmotile coccobacilli zymes will provide information on the essential with a DNA content ranging from 40 to 47% active site conformation for intradiol cleavage guanine plus cytosine (1) and are unlike the of aromatic rings. motile rod-shaped Pseudomonas species, which have a DNA content ranging from 58 to 69% MATERIALS AND METHODS guanine cytosine (14, 25). plus Bacterial strain and its growth. Bacterial strain CO has been purified from Pseudomonas spe- ADP-96 was derived from Juni's transformable A. cies (10, 17) and Brevibacterium fuscum (18). calcoaceticus strain BD-413 and was isolated as de- In our continuing effort to understand the scribed previously (21). Strain ADP-96 is a regula- nature of oxygenases, particularly the non- tory gene mutant that produces the enzyme CO heme, trivalent, iron-containing dioxygenases, constitutively in the absence of its inducer. Cultures 536 VOL. 127, 1976 CATECHOL 1,2-DIOXYGENASE 537 of strain ADP-96 were grown at 37 C in a 100-liter 0 to 4 C during sonication. The sonicated cell New Brunswick Fermacell model CF 130 fermenter suspension was centrifuged for 1 h at 15,000 x g. in mineral medium (20) containing 10 mM sodium The supernatant liquid was termed the crude ex- succinate as the sole carbon source. Cells were tract (step 1 in Table 1). Protamine sulfate solution harvested with a refrigerated Sharples centrifuge (70 ml of 2% solution in 0.1 M Tris base) was added and stored at -20 C until used. dropwise with constant stirring to 3.5 liters of crude Cultures of P. aeruginosa strain 45 (26), Alcali- extract. After standing for 30 min, the extract was genes eutrophus ATCC 17697, Nocardia opaca 17039, centrifuged at 15,000 x g for 45 min. The superna- and Acinetobacter calcoaceticus ATCC 14987 were tant solution (step 2 in Table 1) was fractionated grown in 500 ml of mineral medium with 10 mM with ammonium sulfate. Extracts were brought to sodium benzoate as the sole carbon source to induce 30% of saturation with respect to ammonium sulfate CO. by the addition of 176 g ofthe salt per liter ofextract. Chemicals. Benzoate and catechol were pur- Precipitated protein was removed by centrifugation, chased from Matheson Coleman and Bell Co., Nor- and 162 g of ammonium sulfate was added per liter wood, Ohio. 3-Isopropylcatechol, 4-nitrocatechol, 3- of supernatant liquid to bring it to 55% saturation. methylcatechol, 4-methylcatechol, 3-methoxycate- Material precipitating between 30 and 55% satura- chol, and protocatechualdehyde were obtained tion was collected by centrifugation and dissolved in from Aldrich Chemical Co., Milwaukee, Wis. Pyro- buffer A (step 3 in Table 1). This preparation was gallol and protocatechuate were purchased from dialyzed overnight against 4 liters of buffer A, and Eastman Organic Chemical Co., Rochester, N.Y. the dialyzed material was applied to a diethyl- Bio-Gel agarose A-1.5 was obtained from Bio-Rad aminoethyl (DEAE)-cellulose column (5 by 40 cm) Laboratories, Richmond, Calif. Ammonium sulfate that had been equilibrated with buffer A. The sam- (ultrapure) was obtained from Schwarz/Mann Co., ple was washed with 500 ml of buffer A and eluted Orangeburg, N.Y. a,a-Bipyridyl, O-phenanthro- with 3 liters of buffer A that contained NaCl in a line, p-hydroxymercuribenzoate, tiron, dithiothrei- linear gradient running from a concentration of 0 to tol, and 5,5'-dithiobis-2-nitrobenzoic acid were ob- 0.5 M. Fractions of 15 ml were collected at a flow tained from Sigma Chemical Co., St. Louis, Mo. rate of 75 ml/h. Fractions containing CO activity Enzyme assay. Enzyme activity was measured were pooled and were termed DEAE-cellulose eluate routinely either spectrophotometrically, by measur- (step 4 in Table 1). The DEAE-cellulose eluate was ing the increase in absorbance at 260 nm (A2.60,), or brought to 45% saturation by addition of 277 g of polarographically, by measuring oxygen uptake ammonium sulfate per liter. Precipitated protein with an oxygen electrode. The assay system con- was removed, and an additional 65 g of ammonium tained 0.6 ,tmol of catechol in 3.0 ml of 50 mM sulfate was added per liter of supernatant liquid to sodium phosphate buffer, pH 7.5. The reaction was bring it to 55% saturation. Material precipitating started by addition of a suitable amount of enzyme. between 45 and 55% saturation with respect to am- One unit of enzyme activity is defined as the amount monium sulfate was collected by centrifugation and of enzyme that produces 1 ,umol of cis,cis-muconic dissolved in buffer A (step 5 in Table 1). This prepa- acid per min under the standard assay conditions. ration was dialyzed overnight against buffer A, and Protein concentrations were determined spectropho- 7-ml samples were passed through a Bio-Gel agarose tometrically from the A28,, and A260 (12) and by the A-1.5 column (2.5 by 100 cm) that had been equili- method of Lowry et al. (13). brated with buffer A. The flow rate was maintained Purification of CO. Purification was carried out at 30 ml/h, and fractions of 5 ml were collected. in 50 mM tris(hydroxymethyl)aminomethane (Tris)- Fractions containing CO activity were pooled (step 6 hydrochloride buffer, pH 8.0 (buffer A), at about 4 C in Table 1) and precipitated by bringing the solution unless otherwise stated. Crude extract was was pre- to 55% saturation with respect to ammonium sulfate pared from 500 g (wet weight) of frozen cells that by addition of 351 g of the salt per liter of extract. were thawed at 4 C overnight before extraction. The The precipitated protein was dissolved in buffer A cells were suspended in 1 liter of buffer A, placed in and dialyzed overnight against buffer A containing an ice bath, and stirred continuously with a mag- 0.1 M NaCl. In a volume of 15 ml, the enzyme was netic stirrer. The cells were disrupted by 90 min of applied to a QAE-Sephadex column (2.5 by 50 cm) sonication with a Megason ultrasonic disintegrator. that had been equilibrated with buffer A containing Every 5 min, 50 g of cracked ice was added to main- 0.1 M NaCl. Protein was eluted from the column at a tain the temperature of the cell suspension between flow rate of 25 ml/h with buffer A containing NaCl TABLE 1.