The structure of the hexameric atrazine chlorohydrolase AtzA

Atrazine chlorohydrolase (AtzA) was discovered and purified in the early 1990s from soil that had been exposed to the widely used herbicide atrazine. It was subsequently found that this enzyme catalyzes the first and necessary step in the breakdown of atrazine by the soil organism Pseudomonas sp. strain ADP. Although it has taken 20 years, a crystal structure of the full hexameric form of AtzA has now been obtained. AtzA is less well adapted to its physiological role (i.e. atrazine dechlorination) than the alternative metal-dependent atrazine chlorohydrolase (TrzN), with a substrate-binding pocket that is under considerable strain and for which the substrate is a poor fit

High-resolution X-ray structures of two functionally distinct members of the cyclic amide hydrolase family of Toblerone fold enzymes

The Toblerone fold was discovered recently when the first structure of the cyclic amide hydrolase, AtzD (a cyanuric acid hydrolase), was elucidated. We surveyed the cyclic amide hydrolase family, finding a strong correlation between phylogenetic distribution and specificity for either cyanuric acid or barbituric acid. One of six classes (IV) could not be tested due to a lack of expression of the proteins from it, and another class (V) had neither cyanuric acid nor barbituric acid hydrolase activity. High-resolution X-ray structures were obtained for a class VI barbituric acid hydrolase (1.7 Å) from a Rhodococcus species and a class V cyclic amide hydrolase (2.4 Å) from a Frankia species for which we were unable to identify a substrate. Both structures were homologous with the tetrameric Toblerone fold enzyme AtzD, demonstrating a high degree of structural conservation within the cyclic amide hydrolase family. The barbituric acid hydr olase structure did not contain zinc, in contrast with early reports of zinc-dependent activity for this enzyme. Instead, each barbituric acid hydrolase monomer contained either Na+ or Mg2+, analogous to the structural metal found in cyanuric acid hydrolase. The Frankia cyclic amide hydrolase contained no metal but instead formed unusual, reversible, intermolecular vicinal disulfide bonds that contributed to the thermal stability of the protein. The active sites were largely conserved between the three enzymes, differing at six positions, which likely determine substrate specificit

Reactions catalyzed by AtzC and its closest relatives, TrzC, CodA and D-AAase.

<p>Reactions catalyzed by AtzC and its closest relatives, TrzC, CodA and D-AAase.</p

X-Ray Structure and Mutagenesis Studies of the <i>N</i>-Isopropylammelide Isopropylaminohydrolase, AtzC

<div><p>The <i>N</i>-isopropylammelide isopropylaminohydrolase from <i>Pseudomonas</i> sp. strain ADP, AtzC, provides the third hydrolytic step in the mineralization of <i>s-</i>triazine herbicides, such as atrazine. We obtained the X-ray crystal structure of AtzC at 1.84 Å with a weak inhibitor bound in the active site and then used a combination of <i>in silico</i> docking and site-directed mutagenesis to understand the interactions between AtzC and its substrate, isopropylammelide. The substitution of an active site histidine residue (His249) for an alanine abolished the enzyme’s catalytic activity. We propose a plausible catalytic mechanism, consistent with the biochemical and crystallographic data obtained that is similar to that found in carbonic anhydrase and other members of subtype III of the amidohydrolase family</p></div

A comparison between the previously reported AtzC structure and the high resolution AtzC structure with bound inhibitor.

<p>A: The AtzC is shown as multicolored: helices in red, strands in yellow and loops in green whereas the 2QT3 structure is shown in cyan. The amino (N) and carboxy (C) termini of the proteins are indicated, as is the location of malonate (mal). B: Residues in the active site of AtzC are shown in stick representation and named along with malonate (mal). 2QT3 carbons are shown in cyan whereas the structure reported here have green carbons. The Zn ion is shown as a grey sphere coordinated to His60, His62, His217 and Asp303. The Trp309 and Gln160 residues are seen to be in a different orientation between the ligand bound and apo structures.</p

X-ray structure of the amidase domain of AtzF, the allophanate hydrolase from the cyanuric acid-mineralizing multienzyme complex

The activity of the allophanate hydrolase from Pseudomonas sp. strain ADP, AtzF, provides the final hydrolytic step for the mineralization of s-triazines, such as atrazine and cyanuric acid. Indeed, the action of AtzF provides metabolic access to two of the three nitrogens in each triazine ring. The X-ray structure of the N-terminal amidase domain of AtzF reveals that it is highly homologous to allophanate hydrolases involved in a different catabolic process in other organisms (i.e., the mineralization of urea). The smaller C-terminal domain does not appear to have a physiologically relevant catalytic function, as reported for the allophanate hydrolase of Kluyveromyces lactis, when purified enzyme was tested in vitro. However, the C-terminal domain does have a function in coordinating the quaternary structure of AtzF. Interestingly, we also show that AtzF forms a large, ca. 660-kDa, multienzyme complex with AtzD and AtzE that is capable of mineralizing cyanuric acid. The function of this complex may be to channel substrates from one active site to the next, effectively protecting unstable metabolites, such as allophanate, from solvent- mediated decarboxylation to a dead-end metabolic product

Crystallographic Data.

<p>Crystallographic Data.</p

Reaction mechanism of AtzC and comparison with Carbonic anhydrase.

<p>The reaction mechanisms for: A) AtzC (proposed here) and B) carbonic anhydrase [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137700#pone.0137700.ref045" target="_blank">45</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137700#pone.0137700.ref047" target="_blank">47</a>] are shown.</p