Substrate Specificity of Atrazine Chlorohydrolase and Atrazine-Catabolizing Bacteria

Abstract

Bacterial atrazine catabolism is initiated by the enzyme atrazine chlorohydrolase (AtzA) in Pseudomonas sp. strain ADP. Other triazine herbicides are metabolized by bacteria, but the enzymological basis of this is unclear. Here we begin to address this by investigating the catalytic activity of AtzA by using substrate analogs. Purified AtzA from Pseudomonas sp. strain ADP catalyzed the hydrolysis of an atrazine analog that was substituted at the chlorine substituent by fluorine. AtzA did not catalyze the hydrolysis of atrazine analogs containing the pseudohalide azido, methoxy, and cyano groups or thiomethyl and amino groups. Atrazine analogs with a chlorine substituent at carbon 2 and N-alkyl groups, ranging in size from methyl to t-butyl, all underwent dechlorination by AtzA. AtzA catalyzed hydrolytic dechlorination when one nitrogen substituent was alkylated and the other was a free amino group. However, when both amino groups were unalkylated, no reaction occurred. Cell extracts were prepared from five strains capable of atrazine dechlorination and known to contain atzA or closely homologous gene sequences: Pseudomonas sp. strain ADP, Rhizobium strain PATR, Alcaligenes strain SG1, Agrobacterium radiobacter J14a, and Ralstonia picketti D. All showed identical substrate specificity to purified AtzA from Pseudomonas sp. strain ADP. Cell extracts from Clavibacter michiganensis ATZ1, which also contains a gene homologous to atzA, were able to transform atrazine analogs containing pseudohalide and thiomethyl groups, in addition to the substrates used by AtzA from Pseudomonas sp. strain ADP. This suggests that either (i) another enzyme(s) is present which confers the broader substrate range or (ii) the AtzA itself has a broader substrate range.

X-ray Structure and Mutational Analysis of the Atrazine Chlorohydrolase TrzN

“…All other s-triazines used in this study were synthesized in our laboratory as described previously (19).…”

Defining Sequence Space and Reaction Products within the Cyanuric Acid Hydrolase (AtzD)/Barbiturase Protein Family

“…Urea and dioxane were purchased from Fisher Scientific (Pittsburgh, PA). Ammelide and ammeline were synthesized in our laboratory as previously described (31). Identification of AtzD/barbiturase family members.…”

Hydroxyatrazine N -Ethylaminohydrolase (AtzB): an Amidohydrolase Superfamily Enzyme Catalyzing Deamination and Dechlorination

“…With the exception of AtzB, all of the enzymes involved in the conversion of s-triazine herbicide to cyanuric acid have been well characterized (9,11,24,26,27,31). The atzB gene from Pseudomonas sp.…”

“…The work described here outlines a novel approach for cloning the atzB gene that allowed for further enzyme characterization, including a detailed study on substrate specificity. 2-isopropylamino-4-ethylamino-1,3,5-triazine, and 2-mercapto-4-isopropylamino-6-ethylamino-1,3,5-triazine were synthesized as previously described (26,33). Compounds synthesized for this study were analyzed for purity by gas chromatography-mass spectrometry (MS) using an HP 6890/5973 instrument (Hewlett-Packard, San Fernando, CA).…”