JOURNAL OF BACTERIOLOGY, Aug. 1996, p. 4894–4900 Vol. 178, No. 16 0021-9193/96/$04.0010 Copyright q 1996, American Society for Microbiology Atrazine Chlorohydrolase from Pseudomonas sp. Strain ADP: Gene Sequence, Enzyme Purification, and Protein Characterization† 1,2 2,3,4 1,2,4 MERVYN L. DE SOUZA, MICHAEL J. SADOWSKY, AND LAWRENCE P. WACKETT * Department of Biochemistry and Biological Processes Technology Institute,1 Department of Soil, Water, and Climate,3 Department of Microbiology,4 and Center for Biodegradation Research and Informatics,2 University of Minnesota, St. Paul, Minnesota 55108 Received 26 April 1996/Accepted 30 May 1996 Pseudomonas sp. strain ADP metabolizes atrazine to carbon dioxide and ammonia via the intermediate hydroxyatrazine. The genetic potential to produce hydroxyatrazine was previously attributed to a 1.9-kb AvaI DNA fragment from strain ADP (M. L. de Souza, L. P. Wackett, K. L. Boundy-Mills, R. T. Mandelbaum, and M. J. Sadowsky, Appl. Environ. Microbiol. 61:3373–3378, 1995). In this study, sequence analysis of the 1.9-kb AvaI fragment indicated that a single open reading frame, atzA, encoded an activity transforming atrazine to hydroxyatrazine. The open reading frame for the chlorohydrolase was determined by sequencing to be 1,419 nucleotides and encodes a 473-amino-acid protein with a predicted subunit molecular weight of 52,421. The deduced amino acid sequence matched the first 10 amino acids determined by protein microsequencing. The protein AtzA was purified to homogeneity by ammonium sulfate precipitation and anion-exchange chroma- tography. The subunit and holoenzyme molecular weights were 60,000 and 245,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography, respectively. The puri- 18 18 fied enzyme in H2 O yielded [ O]hydroxyatrazine, indicating that AtzA is a chlorohydrolase and not an oxygenase. The most related protein sequence in GenBank was that of TrzA, 41% identity, from Rhodococcus corallinus NRRL B-15444R. TrzA catalyzes the deamination of melamine and the dechlorination of deethyl- atrazine and desisopropylatrazine but is not active with atrazine. AtzA catalyzes the dechlorination of atrazine, simazine, and desethylatrazine but is not active with melamine, terbutylazine, or desethyldesisopropylatrazine. Our results indicate that AtzA is a novel atrazine-dechlorinating enzyme with fairly restricted substrate specificity and contributes to the microbial hydrolysis of atrazine to hydroxyatrazine in soils and groundwater. The s-triazine ring is found as a constituent of herbicides, zines with one alkylamino group, such as desethylsimazine and dyes, and polymers. The s-triazine herbicides include simazine, desethylatrazine are metabolized by Rhodococcus corallinus terbutylazine, and atrazine [2-chloro-4-(ethylamino)-6-(isopro- NRRL B-15444R (8) via a hydrolytic enzyme that catalyzes pylamino)-1,3,5-triazine)]. The latter is the most environmen- both dechlorination and deamination reactions (28). The gene tally prevalent, being used for the control of broadleaf and encoding this hydrolase, trzA, was recently cloned and se- grassy weeds in major crops like corn, sorghum, and sugarcane. quenced (36). However, both Pseudomonas sp. strain NRRLB Atrazine is relatively persistent in soils (34). Atrazine occasion- 12227 and R. corallinus NRRL B-15444R were incapable of ally exceeds the U.S. Environmental Protection Agency maxi- metabolizing atrazine. Recently, bacteria that metabolize mum contaminant level of 3 ppb in groundwater and surface atrazine have been isolated (29, 32, 35, 36), but little informa- water (1, 5, 6, 14, 21, 22, 23, 30, 31). tion is available about the relevant metabolites, genes, and The metabolism of s-triazine compounds by pure bacterial enzymes. cultures has been studied (3, 4, 8–10, 15, 17, 20, 25–27, 32, 41, We previously reported the isolation of a pure bacterial 42), but most isolates failed to metabolize atrazine (9, 15). In culture, identified as Pseudomonas sp. strain ADP, which de- general, less-substituted s-triazine ring compounds are more graded relatively high concentrations of atrazine (.1,000 ppm) readily metabolized than their heavily substituted counter- under growth and nongrowth conditions (25). Pseudomonas sp. parts. As a result, information about the microbial genetics and strain ADP uses atrazine as the sole source of nitrogen for enzymology of s-triazine compounds has largely been obtained growth and liberates the ring carbon atoms as carbon dioxide. with compounds other than atrazine. For example, ammeline Recently, we reported the cloning, characterization, and ex- (2-hydroxy-4,6-diamino-s-triazine), which is not alkylated on pression of a DNA fragment from strain ADP that confers the ring-substituted amino groups, is metabolized by Pseudo- atrazine dechlorination ability on Escherichia coli DH5a (11). monas sp. strain NRRL B 12227 (12, 13). Its metabolism pro- The data indicate that hydroxyatrazine was the first interme- ceeds to cyanuric acid via two hydrolytic deamination reactions diate in the metabolism of atrazine by Pseudomonas sp. strain that are encoded by the genes trzB and trzC. Diamino-s-tria- ADP. The present study describes the sequence of the gene en- coding the atrazine dechlorination activity, designated atzA, * Corresponding author. Mailing address: Department of Biochem- istry, University of Minnesota, 140 Gortner Laboratories, 1479 Gort- and the purification of the corresponding enzyme (AtzA). ner Ave., St. Paul, MN 55108. Phone: (612) 625-3785. Fax: (612) 625- Atrazine chlorohydrolase was characterized with respect to 5780. Electronic mail address: [email protected]. physical and catalytic properties. This is the first description of † Manuscript number 22,472 in the University of Minnesota Agri- a purified bacterial enzyme capable of catalyzing atrazine de- cultural Experiment Station series. chlorination. 4894 VOL. 178, 1996 CHARACTERIZATION OF ATRAZINE CHLOROHYDROLASE 4895 MATERIALS AND METHODS Bacterial strains, plasmids, and growth conditions. Plasmid pMD4 (11), con- taining a 1.9-kb AvaI fragment in pACYC184 (7), was used in all studies. E. coli DH5a(pMD4) was used for the purification of atrazine chlorohydrolase. The culture was grown in Luria-Bertani LB medium (33), supplemented with 25 mg of chloramphenicol per ml, at 378C with shaking. DNA sequencing. Plasmid DNA was isolated as described previously (33). The nucleotide sequence of the approximately 1.9-kb AvaI DNA fragment, cloned in pMD4, was determined from both strands by using a PRISM Ready Reaction DyeDeoxy Terminator Cycle Sequencing kit (Perkin-Elmer Corp., Norwalk, Conn.) and an ABI model 373A DNA sequencer (Applied Biosystems, Foster City, Calif.). Nucleotide sequence was determined initially by subcloning and subsequently by using primers designed with sequence information obtained from subcloned DNA fragments. The GCG sequence analysis software package (Genetics Computer Group, Inc., Madison, Wis.) was used for all DNA and protein sequence comparisons. DNA and protein sequences were compared with entries in Genbank, EMBL, PIR, and SwissProt sequence databases. Analytical methods. (i) Plate assays. Atrazine or hydroxyatrazine was incor- porated in solid LB (33) or minimal medium (25) at a final concentration of 500 mg/ml to produce an opaque suspension of small particles in the clear agar. The degradation of atrazine or hydroxyatrazine by wild-type and recombinant bacte- ria was indicated by a zone of clearing around atrazine-metabolizing colonies (11). (ii) HPLC analysis. High-performance liquid chromatography (HPLC) anal- ysis was performed by using a Hewlett-Packard HP 1090 Liquid Chromatograph system equipped with a photodiode array detector and interfaced to an HP 79994A Chemstation. Atrazine and its metabolites were resolved by using an analytical C18 reverse-phase NovaPak HPLC column (150 by 3.9 mm; Waters) 4-mm spherical packing; and an acetonitrile (ACN) gradient, in water, at a flow rate of 1.0 ml min21. Linear gradients were used as follows: 0 to 6 min, 10 to 25% ACN; 6 to 21 min, 25 to 65% ACN; 21 to 23 min, 65 to 100% ACN; and 23 to 25 min, 100% ACN. Spectral data of the column eluent were acquired between 200 and 400 nm (12-nm bandwidth per channel), with a sampling frequency of FIG. 1. Nucleotide sequence of atzA. The complete nucleotide sequence of 640 ms. Spectra were referenced against a signal at 550 nm and compared with the approximately 1.9-kb AvaI DNA fragment, cloned in pMD4, was determined those obtained by using authentic samples of atrazine and hydroxyatrazine. on both strands by the primer walking method and PCR. The atzA ORF is 18 16 (iii) MS analysis. H2 O (95%) and H2 O were purchased from Sigma Chem- indicated by the arrow, and a potential Pseudomonas ribosome binding site is ical Company (St. Louis, Mo.). For the labeling experiment, 50 ml of authentic underlined. The start and stop codons are underlined twice. atrazine or hydroxyatrazine at a concentration of 100 mg/ml in 25 mM MOPS buffer (pH 6.9) was added to each vial in two sets of glass vials. The samples were dried-down under vacuum and crimp sealed. The headspaces in the vials were repeatedly evacuated and flushed and finally filled with argon. The contents of The dialyzed protein fraction was loaded onto a Bio-Scale Q20 Anion Ex- 18 16 each vial was resuspended in 50 ml of either H2 OorH2 O by using a gas-tight change column (15 by 118 mm) (Bio-Rad Laboratories, Hercules, Calif.). The Hamilton syringe and allowed to equilibrate at room temperature for 60 min. column was washed with 25 mM MOPS buffer (pH 6.9), and the protein was The experiment was initiated by adding purified atrazine chlorohydrolase (AtzA) eluted using a 400-ml linear gradient of 0.0 to 0.5 M KCl at a flow rate of 2 to each vial. Reaction vials were shaken for 30 s and incubated at room temper- ml/min. Protein eluting from the column was monitored at 280 nm by using a ature for 5 h, and the reaction mixture was dried under vacuum.