Hydroxyatrazine N-ethylaminohydrolase (AtzB): an amidohydrolase superfamily enzyme catalyzing deamination and dechlorination.
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.
Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, 140 Gortner Lab, 1479 Gortner Ave., St. Paul, MN 55108, USA.
Jennifer L
JL
Seffernick
Asma
A
Aleem
Jeffrey P
JP
Osborne
Gilbert
G
Johnson
Michael J
MJ
Sadowsky
Lawrence P
LP
Wackett
D000581:Amidohydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001280:Atrazine / Q000737:chemistry / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002614:Chelating Agents / Q000494:pharmacology; D002850:Chromatography, Gel; D003641:Deamination; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D007700:Kinetics; D015394:Molecular Structure; D011549:Pseudomonas / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity; D015032:Zinc / Q000494:pharmacology
D001426:Bacterial Proteins; D002614:Chelating Agents; D000581:Amidohydrolases; D015032:Zinc; D001280:Atrazine