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Applied and Environmental Microbiology, October 2000, p. 4247-4252, Vol. 66, No. 10
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Substrate Specificity of Atrazine Chlorohydrolase and Atrazine-Catabolizing Bacteria

Jennifer L. Seffernick,1,2 Gilbert Johnson,1 Michael J. Sadowsky,2,3,4 and Lawrence P. Wackett1,2,3,*

Department of Biochemistry, Molecular Biology, and Biophysics,1 Biological Process Technology Institute,3 Center for Microbial and Plant Genomics,2 and Department of Soil, Water, and Climate,4 University of Minnesota, St. Paul, Minnesota 55108

Received 20 April 2000/Accepted 17 July 2000

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.

* Corresponding author. Mailing address: Department of Biochemistry, Molecular Biology, and Biophysics, Biological Process Technology Institute, 1479 Gortner Ave., University of Minnesota, St. Paul, MN 55108. Phone: (612) 625-3785. Fax: (612) 625-1700. E-mail: wackett{at}biosci.cbs.umn.edu.

Applied and Environmental Microbiology, October 2000, p. 4247-4252, Vol. 66, No. 10
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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