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

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 k(cat) and K(m) of AtzB with hydroxyatrazine as a substrate were 3 s(-1) and 20 microM, 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.

FIG. 1.

AtzB represents the convergence of multiple s-triazine degradative pathways. Reactions catalyzed by AtzA and TrzN funnel numerous s-triazines through the common metabolite hydroxyatrazine. AtzB is present in both of these pathways to further degrade hydroxyatrazine to N-isopropylammelide.

FIG. 2.

SDS-PAGE analysis of protein samples at various stages of AtzB purification. Lanes: 1, crude cell extract; 2, 0 to 30% ammonium sulfate fraction; 3, peak from ceramic hydroxyapatite column; 4, standard molecular mass markers. Numbers to the right of the gel are in kilodaltons.

FIG. 3.

Substrate analogs of hydroxyatrazine. (A) Dechlorination by AtzB: substrate specificity with 2-chloro-4-hydroxy-s-triazines. (B) Deamination by AtzB: substrate specificity with 2-hydroxy-s-triazine analogs. R₁ and R₂ refer to the 4 and 6 positions of the s-triazine ring, in which the analysis of substrates suggests that AtzB requires two amino groups for deamination activity. (C) Tested compounds lacking a hydroxy substituent that were not AtzB substrates. (D) Tested compounds with two hydroxy substituents that were not AtzB substrates.

FIG. 4.

Selectivity of AtzB for N-alkyl groups removed from asymmetric 2-hydroxy-4,6-di-N-alkyl-1,3,5-triazines. The preferentially removed groups are surrounded by dashed lines.