Hydroxyatrazine N-ethylaminohydrolase (AtzB): An amidohydrolase superfamily enzyme catalyzing deamination and dechlorination

Jennifer L. Seffernick, Asma Aleem, Jeffrey P. Osborne, Gilbert Johnson, Michael J. Sadowsky, Lawrence P. Wackett

Research output: Contribution to journalArticlepeer-review

23 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)6989-6997
Number of pages9
JournalJournal of bacteriology
Volume189
Issue number19
DOIs
StatePublished - Oct 2007

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