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Journal of Bacteriology, October 2007, p. 6989-6997, Vol. 189, No. 19
0021-9193/07/$08.00+0     doi:10.1128/JB.00630-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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

Jennifer L. Seffernick,1,2,§ Asma Aleem,1,2,§ Jeffrey P. Osborne,1,2,{ddagger} Gilbert Johnson,1 Michael J. Sadowsky,2,3,4 and Lawrence P. Wackett1,2,3*

Department of Biochemistry, Molecular Biology and Biophysics,1 Biotechnology Institute,2 Microbial and Plant Genomics Institute,3 Department of Soil, Water, and Climate, University of Minnesota, St. Paul, Minnesota4

Received 23 April 2007/ Accepted 19 July 2007

Hydroxyatrazine [2-(N-ethylamino)-4-hydroxy-6-(N-isopropylamino)-1,3,5-triazine] N-ethylaminohydrolase (AtzB) is the sole enzyme known to catalyze the hydrolytic conversion of hydroxyatrazine to N-isopropylammelide. AtzB, therefore, serves as the point of intersection of multiple s-triazine biodegradative pathways and is completely essential for microbial growth on s-triazine herbicides. Here, atzB was cloned from Pseudomonas sp. strain ADP and its product was purified to homogeneity and characterized. AtzB was found to be dimeric, with subunit and holoenzyme molecular masses of 52 kDa and 105 kDa, respectively. The kcat and Km of AtzB with hydroxyatrazine as a substrate were 3 s–1 and 20 µM, respectively. Purified AtzB had a 1:1 zinc-to-subunit stoichiometry. Sequence analysis revealed that AtzB contained the conserved mononuclear amidohydrolase superfamily active-site residues His74, His76, His245, Glu248, His280, and Asp331. An intensive in vitro investigation into the substrate specificity of AtzB revealed that 20 of the 51 compounds tested were substrates for AtzB; this allowed for the identification of specific substrate structural features required for catalysis. Substrates required a monohydroxylated s-triazine ring with a minimum of one primary or secondary amine substituent and either a chloride or amine leaving group. AtzB catalyzed both deamination and dechlorination reactions with rates within a range of one order of magnitude. This differs from AtzA and TrzN, which do not catalyze deamination reactions, and AtzC, which is not known to catalyze dechlorination reactions.

* Corresponding author. Mailing address: Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, 140 Gortner Lab, 1479 Gortner Ave., St. Paul, MN 55108. Phone: (612) 625-3785. Fax: (612) 625-1700. E-mail: wacke003{at}umn.edu

{triangledown} Published ahead of print on 27 July 2007.

§ These authors contributed equally to this work.

{ddagger} Present address: Department of Chemistry, Manchester College, North Manchester, IN.

Journal of Bacteriology, October 2007, p. 6989-6997, Vol. 189, No. 19
0021-9193/07/$08.00+0     doi:10.1128/JB.00630-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

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