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Atrazine is a broadly used herbicide in agriculture and it was used here as a model to study the biodegradation of herbicides. The bacterium Pseudomonas sp. ADP metabolizes atrazine to carbon dioxide and ammonia and chloride. The genes encoding atrazine catabolism to cyanuric acid were cloned and expressed in Escherichia coli. The genes were designated atzA, atzB and atzC. Each gene was sequenced. The enzyme activities were characterized. AtzA is atrazine chlorohydrolase which takes atrazine to hydroxyatrizine. AtzB is hydroxyatrazine N-ethylaminohydrolase which produces N-isopropylammelide and N-ethylamine. AtzC is N-isopropylammelide N-isopropylaminohydrolase which produces cyanuric acid and N-isopropylamine. Each product was isolated and characterized to confirm their identity by chromatography and mass spectrometry. Sequence analysis indicated that each of the hydrolytic enzymes AtzA, AtzB and AtzC share identity which the aminohydrolase protein superfamily. Atrazine chlorohydrolase was purified to homogeneity. It was shown to have a kcat of 11 s-1 and a KM of 150 uM. It was shown to require a metal ion, either Fe(II), Mn(II) or Co(II), for activity. The atzA, atzB and atzC genes were shown to reside on a broad-host range plasmid in Pseudomonas sp. ADP. Six other recently isolated atrazine-degrading bacteria obtained from Europe and the United States contained homologs to the atz genes identified in Pseudomonas sp. ADP. The identity of the sequences were very high, being greater than 98% in all pairwise comparisons. This indicates that many atrazine-degrading bacteria worldwide metabolize atrazine via a pathway that proceeds through hydroxyatrazine, a metabolite which is non-phytotoxic and non-toxic to mammals. Enzymes were immobilized and used for degradation of atrazine in aqueous phases. The in-depth understanding of the genomics and biochemistry of the atrazine mineralization pathway enabled us to study factors affecting the prevalence of atrazine degradation in various agricultural soils under conservative and new agricultural practices. Moreover, Pseudomonas sp. ADP and/or its enzymes were added to atrazine-contaminated soils, aquifers and industrial wastewater to increase the rate and extent of atrazine biodegradation above that of untreated environments. Our studies enhance the ability to control the fate of regularly introduced pesticides in agriculture, or to reduce the environmental impact of unintentional releases. |
