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 M, 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.

Bacterial Ammeline Metabolism via Guanine Deaminase

“…Figure 2 shows a cluster network diagram where the nodes represent protein sequences, and the edges represent BLASTP linkages at an e-score cutoff of e Ϫ5 . The enzymes previously shown to act on melamine, TriA (28) and TrzA (20), and hydroxyatrazine, AtzB (27), clustered most closely with the guanine deaminases. TrzN, an enzyme that hydrolytically displaces a chlorine substituent from the s-triazine herbicide atrazine, also clusters with the guanine deaminases.…”

“…TriA (melamine deaminase) is related to AtzA (28,31) and TrzN (35), enzymes that catalyze the dechlorination of atrazine. AtzB, which catalyzes the second step in the atrazine metabolic pathway, was reported to also deaminate ammeline as a side reaction, but the rate of the reaction was not measured in that study (27).…”

“…Creatinine deaminase was purchased from MP Biomedicals (Solon, OH), adenosine deaminase was purchased from Roche Applied Science (Mannheim, Germany), and D-aminoacylase and urease were purchased from Sigma-Aldrich (St. Louis, MO). The TriA (28), AtzB (27), and TrzN (35) proteins were purified as described elsewhere.…”

An unexpected vestigial protein complex reveals the evolutionary origins of an s-triazine catabolic enzyme

“…1A) is comprised of three hydrolytic steps: dechlorination, followed by two sequential N-alkyl chain hydrolyses. The upper pathway is catalyzed by AtzA (4,7), AtzB (8) and AtzC (9), respectively; all three enzymes are metalloenzymes of the amidohydrolase superfamily (9,10).…”

X-Ray Structure of the Amidase Domain of AtzF, the Allophanate Hydrolase from the Cyanuric Acid-Mineralizing Multienzyme Complex

“…strain ADP in the mid-1990s and is comprised of six hydrolases: atrazine chlorohydrolase (AtzA; EC 3.8.1.8) (20,21), N-ethylaminohydrolase (AtzB; EC 3.5.99.3) (22,23), N-isopropylammelide isopropylaminohydrolase (AtzC; EC 3.5.99.4) (24,25), cyanuric acid amidohydrolase (AtzD; EC 3.5.2.15) (15,26,27), biuret amidohydrolase (AtzE; EC 3.5.1.84) (28), and allophanate hydrolase (AtzF; EC 3.5.1.54) (29)(30)(31). These hydrolases sequentially dechlorinate (AtzA) and remove the two N-alkyl side groups (AtzB and AtzC) to produce cyanuric acid, which is then further hydrolyzed to biuret, allophanate, and ammonia via AtzD, AtzE, and AtzF, respectively (Fig.…”