ATRAZINE CHLOROHYDROLASE FROM PSEUDOMONAS SP STRAIN ADP - GENE SEQUENCE, ENZYME-PURIFICATION, AND PROTEIN CHARACTERIZATION

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 DN A fragment from strain ADP (M. L. de Souza, L. P. Wackrtt, R. L. Bound y-Mills, R. T. Mandelbaum, and M. J. Sadowsky, Appl. Environ. Microbio l. 61:3373-3378, 1995). In this study, sequence analysis of the 1,9-kb AvaI fragment indicated that a single open reading frame, atzA, encod ed an activity transforming atrazine to hydroxyatrazine. The open read ing frame for the chlorohydrolase was determined by sequencing to be 1 ,419 nucleotides and encodes a 173-amino-acid protein with a predicted subunit molecular weight of 52,421. The deduced amino acid sequence m atched the first 10 amino acids determined by protein microsequencing. The protein AtzA was purified to homogeneity by ammonium sulfate prec ipitation and anion-exchange chromatography. The subunit and holoenzym e molecular weights were 60,000 and 245,000 as determined by sodium do decyl sulfate-polyacrylamide gel electrophoresis and gel filtration ch romatography, respectively. The purified enzyme in (H2O)-O-18 yielded [O-18] hydroxyatrazine, indicating that AtzA is a chlorohydrolase and not an oxygenase. The most related protein sequence in GenBank was tha t of TrzA, 41% identity, from Rhodococcus corallinus NRRL B-15444R . T rzA catalyzes the deamination of melamine and the dechlorination of de ethylatrazine and desisopropylatrazine but is not active with atrazine . AtzA catalyzes the dechlorination of atrazine, simazine, and desethy latrazine but is not active with melamine, terbutylazine, or desethyld esisopropylatrazine. Our results indicate that AtzA is a novel atrazin e-dechlorinating enzyme with fairly restricted substrate specificity a nd contributes to the microbial hydrolysis of atrazine to hydroxyatraz ine in soils and groundwater.