Enzymes responsible for deamination reactions are widespread throughout intermediary metabolism and serve to incorporate and recycle nitrogen among key metabolites essential for DNA and protein synthesis. Some are members of an amidohydrolase protein superfamily, which catalyze at least 30% of the steps in four intermediary metabolic pathways (22). Recently, a new class of bacterial amidohydrolases has been identified; they catalyze the hydrolytic displacement of amino groups and chlorine substituents from s-triazine ring compounds (22, 32).

The s-triazine compounds have numerous applications throughout industry and agriculture (10, 19, 25, 30). Those containing N-alkyl substituents, like atrazine (2-chloro-4-N-ethylamino-6-N-isopropylamino-1,3,5-triazine), have been applied successfully as herbicides (3). Atrazine and analogous chlorinated s-triazines were initially considered to be incompletely metabolized by microorganisms (10, 19). However, 40 years after the initial introduction of atrazine into the environment, bacteria with the ability to completely mineralize this herbicide have been isolated (12, 26, 31, 40). Subsequently, bacteria were shown to initiate atrazine metabolism via dechlorination to yield hydroxyatrazine (2, 8, 12, 26, 31, 40). In 1996, the dechlorinating enzyme atrazine chlorohydrolase (AtzA) was purified and shown, via [18O]water experiments, to catalyze a hydrolytic displacement reaction (Fig. ​(Fig.1)1) (13). 



FIG. 1

Comparison of the reactions catalyzed by melamine deaminase (TriA) from Pseudomonas sp. strain NRRL B-12227 (A) and AtzA from Pseudomonas sp. strain ADP (B).



The substrate specificity of AtzA from Pseudomonas sp. strain ADP was recently investigated (35). AtzA catalyzes the hydrolytic removal of a chlorine or fluorine substituent but does not remove cyano, azido, methoxy, thiomethyl, or amino substituents from compounds structurally analogous to atrazine. AtzA is also not active with any of the pyrimidine substrates tested (35).

Melamine (2,4,6-triamino-1,3,5-triazine), a related s-triazine that predates the use of atrazine (29), is also metabolized by soil bacteria. Worldwide production of melamine in 1994 was estimated to be 900 million lb (21). Melamine is most commonly used in the production of melamine-formaldehyde resins, which are used in laminates, adhesives, fire retardants, molding compounds, coatings, and concrete plasticizers (29). Prior to the identification of atrazine-mineralizing bacteria, Cook and Hutter isolated melamine-metabolizing pseudomonads (11). One of these, Pseudomonas sp. strain NRRL B-12227, catalyzes consecutive hydrolysis of the three amino substituents of melamine, producing the intermediates ammeline, ammelide, and cyanuric acid (Fig. ​(Fig.1).1). Another bacterium, Pseudomonas sp. strain NRRL B-12228, was unreactive with melamine but catalyzed deamination of ammeline to ammelide and of ammelide to cyanuric acid (11). Genes for ammeline and ammelide deamination, trzB and trzC, respectively, have been cloned from Pseudomonas sp. strain NRRL B-12228 (17). Detailed restriction site pattern analysis revealed conservation of trzC but not trzB in Pseudomonas sp. strain NRRL B-12227 (17). The genes encoding the enzyme for melamine or ammeline deamination in Pseudomonas sp. strain NRRL B-12227, however, were not reported. Furthermore, Pseudomonas sp. strain NRRL B-12227 was shown not to metabolize atrazine (11).

Given the similarity in structure between melamine and atrazine and their similar hydrolytic metabolism, it was hypothesized here that AtzA gene probes and antibodies might be used to identify the melamine deaminase gene and protein, respectively, in Pseudomonas sp. strain NRRL B-12227. Using this strategy, the melamine deaminase gene, designated triA, was identified, cloned, and sequenced. The melamine deaminase gene from Pseudomonas sp. strain NRRL B-12227 was 99% identical to the atzA gene from Pseudomonas sp. strain ADP. The cloned melamine deaminase was expressed in Escherichia coli DH5α and shown to catalyze the deamination of melamine and ammeline. Melamine deaminase had no activity with any of the chlorotriazine substrates tested. Taken together with the known substrate specificity of AtzA, these studies identified two nearly identical proteins that catalyze clearly distinct biochemical reactions.

Melamine Deaminase and Atrazine Chlorohydrolase: 98 Percent Identical but Functionally Different

A bacterium that was capable of metabolizing atrazine at very high concentrations (>1,000 ppm) was isolated from a herbicide spill site. The organism was differentiated by observing clearing zones on indicator agar plates containing 1,000 ppm atrazine. Detailed taxonomic studies identified the organism as a Pseudomonas sp., designated ADP, that was dissimilar to currently known species. Pseudomonas sp. strain ADP metabolized atrazine as its sole nitrogen source. Nongrowing suspended cells also metabolized atrazine rapidly; for example, 9 x 10(sup9) cells per ml degraded 100 ppm of atrazine in 90 min. Atrazine was metabolized to hydroxyatrazine, polar metabolites, and carbon dioxide. When uniformly ring-labeled [(sup14)C]atrazine was used, 80% of the radioactivity was liberated as (sup14)CO(inf2). These data indicated the triazine ring was completely mineralized. The isolation and characterization of Pseudomonas sp. strain ADP may contribute to efforts on atrazine bioremediation, particularly in environments containing very high pesticide levels.

Isolation and Characterization of a Pseudomonas sp. That Mineralizes the s-Triazine Herbicide Atrazine

Isolation and Characterization of aPseudomonassp. That Mineralizes thes-Triazine Herbicide Atrazine

The first phase of intensive data collection for the National Water-Quality Assessment (NAWQA) was completed during 1993−1995 in 20 major hydrologic basins of the United States. Groundwater land-use studies, designed to sample recently recharged groundwater (generally within 10 years) beneath specific land-use and hydrogeologic settings, are a major component of the groundwater quality as sessment for NAWQA. Pesticide results from the 41 land-use studies conducted during 1993−1995 indicate that pesticides were commonly detected in shallow groundwater, having been found at 54.4% of the 1034 sites sampled in agricultural and urban settings across the United States. Pesticide concentrations were generally low, with over 95% of the detections at concentrations less than 1 μg/L. Of the 46 pesticide compounds examined, 39 were detected. The compounds detected most frequently were atrazine (38.2%), deethylatrazine (34.2%), simazine (18.0%), metolachlor (14.6%), and prometon (13.9%). Statistically significant rel...

/pdf/occurrence-of-pesticides-in-shallow-groundwater-of-the-32qi4eye5y.pdf

Occurrence of Pesticides in Shallow Groundwater of the United States: Initial Results from the National Water-Quality Assessment Program

Understanding pesticide metabolism in plants and microorganisms is necessary for pesticide development, for safe and efficient use, as well as for developing pesticide bioremediation strategies for contaminated soil and water. Pesticide biotransformation may occur via multistep processes known as metabolism or cometabolism. Cometabolism is the biotransformation of an organic compound that is not used as an energy source or as a constitutive element of the organism. Individual reactions of degradation–detoxification pathways include oxidation, reduction, hydrolysis, and conjugation. Metabolic pathway diversity depends on the chemical structure of the xenobiotic compound, the organism, environmental conditions, metabolic factors, and the regulating expression of these biochemical pathways. Knowledge of these enzymatic processes, especially concepts related to pesticide mechanism of action, resistance, selectivity, tolerance, and environmental fate, has advanced our understanding of pesticide scienc...

Pesticide metabolism in plants and microorganisms

Atrazine, simazine, and propazine are closely related herbicides that can be used for weed control. The degradation of s‐triazines in soil, water, and sediments has received considerable attention. In this review, the literature related to degradation of atrazine and related s‐triazines is reviewed. Particular emphasis is directed toward those substances formed along the degradative pathways and to the biodegradation aspects of mineralization. The s‐triazines are unusual in that the carbon and nitrogen ring contains no available electrons for aerobic biodegradation. The only sources of energy for aerobic biodegradation of atrazine are found on the ethyl and isopropy I groups. There is some evidence to support the concept that dealkylation of s‐triazines occurs to support the bioenergetic needs of microorganisms. Biodegradation studies with simazine or atrazine as the carbon and energy source have generally shown biodegradation of the alkyl side chains with evolution of radioactive carbon dioxide from chai...

Darrell D. Sumner

Papers

Degradation of atrazine and related s‐triazines