The s-triazine ring is found as a constituent of herbicides, dyes, and polymers. The s-triazine herbicides include simazine, terbutylazine, and atrazine [2-chloro-4-(ethylamino)-6-(isopropylamino)-1,3,5-triazine)]. The latter is the most environmentally prevalent, being used for the control of broadleaf and grassy weeds in major crops like corn, sorghum, and sugarcane. Atrazine is relatively persistent in soils (34). Atrazine occasionally exceeds the U.S. Environmental Protection Agency maximum contaminant level of 3 ppb in groundwater and surface water (1,5,6,14,21,22,23,30,31).The metabolism of s-triazine compounds by pure bacterial cultures has been studied (3, 4, 8-10, 15, 17, 20, 25-27, 32, 41, 42), but most isolates failed to metabolize atrazine (9, 15). In general, less-substituted s-triazine ring compounds are more readily metabolized than their heavily substituted counterparts. As a result, information about the microbial genetics and enzymology of s-triazine compounds has largely been obtained with compounds other than atrazine. For example, ammeline (2-hydroxy-4,6-diamino-s-triazine), which is not alkylated on the ring-substituted amino groups, is metabolized by Pseudomonas sp. strain NRRL B 12227 (12, 13). Its metabolism proceeds to cyanuric acid via two hydrolytic deamination reactions that are encoded by the genes trzB and trzC. Diamino-s-triazines with one alkylamino group, such as desethylsimazine and desethylatrazine are metabolized by Rhodococcus corallinus NRRL B-15444R (8) via a hydrolytic enzyme that catalyzes both dechlorination and deamination reactions (28). The gene encoding this hydrolase, trzA, was recently cloned and sequenced (36). However, both Pseudomonas sp. strain NRRLB 12227 and R. corallinus NRRL B-15444R were incapable of metabolizing atrazine. Recently, bacteria that metabolize atrazine have been isolated (29,32,35,36), but little information is available about the relevant metabolites, genes, and enzymes.We previously reported the isolation of a pure bacterial culture, identified as Pseudomonas sp. strain ADP, which degraded relatively high concentrations of atrazine (Ͼ1,000 ppm) under growth and nongrowth conditions (25). Pseudomonas sp. strain ADP uses atrazine as the sole source of nitrogen for growth and liberates the ring carbon atoms as carbon dioxide. Recently, we reported the cloning, characterization, and expression of a DNA fragment from strain ADP that confers atrazine dechlorination ability on Escherichia coli DH5␣ (11). The data indicate that hydroxyatrazine was the first intermediate in the metabolism of atrazine by Pseudomonas sp. strain ADP.The present study describes the sequence of the gene encoding the atrazine dechlorination activity, designated atzA, and the purification of the corresponding enzyme (AtzA). Atrazine chlorohydrolase was characterized with respect to physical and catalytic properties. This is the first description of a purified bacterial enzyme capable of catalyzing atrazine dechlorination.

The gene encoding melamine deaminase (TriA) from Pseudomonas sp. strain NRRL B-12227 was identified, cloned into Escherichia coli, sequenced, and expressed for in vitro study of enzyme activity. Melamine deaminase displaced two of the three amino groups from melamine, producing ammeline and ammelide as sequential products. The first deamination reaction occurred more than 10 times faster than the second. Ammelide did not inhibit the first or second deamination reaction, suggesting that the lower rate of ammeline hydrolysis was due to differential substrate turnover rather than product inhibition. Remarkably, melamine deaminase is 98% identical to the enzyme atrazine chlorohydrolase (AtzA) from Pseudomonas sp. strain ADP. Each enzyme consists of 475 amino acids and differs by only 9 amino acids. AtzA was shown to exclusively catalyze dehalogenation of halo-substituted triazine ring compounds and had no activity with melamine and ammeline. Similarly, melamine deaminase had no detectable activity with the halo-triazine substrates. Melamine deaminase was active in deamination of a substrate that was structurally identical to atrazine, except for the substitution of an amino group for the chlorine atom. Moreover, melamine deaminase and AtzA are found in bacteria that grow on melamine and atrazine compounds, respectively. These data strongly suggest that the 9 amino acid differences between melamine deaminase and AtzA represent a short evolutionary pathway connecting enzymes catalyzing physiologically relevant deamination and dehalogenation reactions, respectively.

Pseudomonas strain ADP metabolizes the herbicide atrazine via three enzymatic steps, encoded by the genesatzABC, to yield cyanuric acid, a nitrogen source for many bacteria. Here, we show that five geographically distinct atrazine-degrading bacteria contain genes homologous toatzA, -B, and -C. The sequence identities of the atz genes from different atrazine-degrading bacteria were greater than 99% in all pairwise comparisons. This differs from bacterial genes involved in the catabolism of other chlorinated compounds, for which the average sequence identity in pairwise comparisons of the known members of a class ranged from 25 to 56%. Our results indicate that globally distributed atrazine-catabolic genes are highly conserved in diverse genera of bacteria.

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