X-ray Structure and Mutational Analysis of the Atrazine Chlorohydrolase TrzN

Abstract

Atrazine chlorohydrolase, TrzN (triazine hydrolase or atrazine chlorohydrolase 2), initiates bacterial metabolism of the herbicide atrazine by hydrolytic displacement of a chlorine substituent from the s-triazine ring. The present study describes crystal structures and reactivity of wild-type and active site mutant TrzN enzymes. The homodimer native enzyme structure, solved to 1.40 Å resolution, is a (␤␣) 8 barrel, characteristic of members of the amidohydrolase superfamily. TrzN uniquely positions threonine 325 in place of a conserved aspartate that ligates the metal in most mononuclear amidohydrolases superfamily members. The threonine side chain oxygen atom is 3.3 Å from the zinc atom and 2.6 Å from the oxygen atom of zinccoordinated water. Mutation of the threonine to a serine resulted in a 12-fold decrease in k cat /K m , largely due to k cat , whereas the T325D and T325E mutants had immeasurable activity. The structure and kinetics of TrzN are reminiscent of carbonic anhydrase, which uses a threonine to assist in positioning water for reaction with carbon dioxide. An isosteric substitution in the active site glutamate, E241Q, showed a large diminution in activity with ametryn, no detectable activity with atratone, and a 10-fold decrease with atrazine, when compared with wild-type TrzN. Activity with the E241Q mutant was nearly constant from pH 6.0 to 10.0, consistent with the loss of a proton-donating group. Structures for TrzN-E241Q were solved with bound ametryn and atratone to 1.93 and 1.64 Å resolution, respectively. Both structure and kinetic determinations suggest that the Glu 241 side chain provides a proton to N-1 of the s-triazine substrate to facilitate nucleophilic displacement at the adjacent C-2.There is substantial evidence that microbes rapidly evolve new enzymes to metabolize anthropogenic chemicals (1, 2). The s-triazine herbicides, such as atrazine, were first introduced into the environment 50 years ago and Ͼ2 billion pounds have been applied globally. s-Triazine compounds were initially found to be poorly biodegradable, but more rapid biodegradation is observed today (3). Many atrazine-degrading bacteria have now been isolated (4 -6). They invariably contain highly conserved genes encoding enzymes that hydrolytically displace substituents from the s-triazine ring carbon atoms to generate cyanuric acid (Fig. 1). The genes, trzN, atzA, atzB, and atzC, are found on plasmids and now are distributed globally (7, 8). These observations are consistent with the idea that a new metabolic pathway for atrazine catabolism may have evolved and spread in recent evolutionary times (9).The s-triazine herbicides, such as atrazine (2-chloro-4-isopropylamino-6-ethylamino-1,3,5-triazine) and ametryn (2-thiomethyl-4-isopropylamino-6-ethylamino-1,3,5-triazine), are metabolized readily by dedicated enzymes. The enzymes have been purified to homogeneity and shown to be inactive with structurally analogous pyrimidines and other closely related compounds (10 -13). Moreover, the enzymes are isolated from bacteria...

Aerobic Biodegradation of 2,4-Dinitroanisole by Nocardioides sp. Strain JS1661

“…5). Atrazine hydrolase (TrzN) from Arthrobacter has been reported to catalyze the cleavage of the methoxy group of the heterocyclic pesticide atratone at a low rate (41,42). However, TrzN had no amino acid similarity to DNAN hydrolase.…”

300-Fold Increase in Production of the Zn 2+ -Dependent Dechlorinase TrzN in Soluble Form via Apoenzyme Stabilization

“…Hydrolysis of atrazine was quantified spectrophotometrically (SpectraMax 190) by the decrease in absorbance at 264 nm that accompanies atrazine dechlorination, as described previously (38). Assays and standard curves used an analytical standard of Ͼ99.5% pure atrazine (Sigma), and all measurements were made in triplicate.…”

“…Both proteins were concentrated to 20 mg/ml in crystallization buffer (50 mM HEPES, pH 7.5, 100 mM NaCl). The crystallization used a protocol slightly modified from that previously used to obtain crystals of native TrzN (38). For holo-TrzN-G3, the crystallization buffer also included 100 M ZnCl 2 .…”

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

“…In other bacterial genera, such as Arthrobacter and Nocardioides, the function of AtzA is conducted by TrzN (32)(33)(34)(35)(36). Although TrzN is physiologically analogous to AtzA, it appears to have evolved independently, as the two enzymes share low sequence identity and have significantly different reaction mechanisms and substrate ranges.…”

“…Recently, there have been concerted efforts to understand the structural biology of the hydrolases of the atrazine catabolic pathway, with the structure of TrzN being obtained in 2010 (33) and the structure of AtzD (15), which possesses a previously unreported Toblerone protein fold, being reported in 2013. A structure for AtzC has also been deposited in the Protein Data Bank (PDB accession number 2QT3).…”