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The structure of the hexameric atrazine chlorohydrolase AtzA.

Peat TS, Newman J, Balotra S, Lucent D, Warden AC, Scott C - Acta Crystallogr. D Biol. Crystallogr. (2015)

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: CSIRO Biomedical Manufacturing, Parkville, Australia.

ABSTRACT
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.

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The entrance to the central cavity of the AtzA hexamer and the amino-acid residues of the hexamer-stabilizing interface. The entrance to the AtzA hexamer central cavity is largely formed by Arg163 contributed by each monomer. The hole formed in the hexamer is approximately 14 Å across at the entrance. Amino-acid substitutions at residues Ala170, Met256, Pro258 and Tyr261 destabilize the hexamer. The interactions between three monomers on one face of the hexamer (left) and at a single monomer–monomer interface (right) are shown.
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fig3: The entrance to the central cavity of the AtzA hexamer and the amino-acid residues of the hexamer-stabilizing interface. The entrance to the AtzA hexamer central cavity is largely formed by Arg163 contributed by each monomer. The hole formed in the hexamer is approximately 14 Å across at the entrance. Amino-acid substitutions at residues Ala170, Met256, Pro258 and Tyr261 destabilize the hexamer. The interactions between three monomers on one face of the hexamer (left) and at a single monomer–monomer interface (right) are shown.

Mentions: The AtzA hexamer (about 315 kDa in molecular weight) is a trimer of dimers (Fig. 2 ▶a), with the dimer interface being significantly larger than the individual protomer interfaces used to make up the hexamer (Figs. 2 ▶a and 2 ▶b): 3295 Å2 compared with 585 Å2. The hexamer was confirmed by DLS and size-exclusion chromatography (SEC; Supplementary Fig. S2), consistent with previous reports for the solution state of the protein (de Souza et al., 1996 ▶; Scott et al., 2009 ▶). The dimer surface covers 17–18% of the monomer (a single monomer has a surface area of ∼18 600 Å2; Fig. 2 ▶c). The dimer interface is similar to that found in the other amidohydrolases and the dimer is similar enough in overall structure that the search model used for molecular replacement (PDB entry 3hpa) gave a significantly more robust solution when the dimer was used instead of the monomer. Two full hexamers (over 600 kDa) were found in the asymmetric unit of the crystal structure in space group P22121, whereas a single hexamer was found in space group P212121. The two structures are essentially identical (r.m.s.d. of 0.3 Å), with the higher resolution data giving greater clarity in amino-acid positions and having significantly more water molecules added to the model. The most obvious interaction seen when looking down the threefold axis of the hexamer is the 11-amino-acid insertion (relative to PDB entry 3hpa) that forms the loop and helix encompassing residues 160–183 that has not been seen in the other amidohydrolase structures to date. The ‘cavity’ in the hexamer is approximately 14 Å across at the entrance, with one residue in particular, Arg163, forming much of the surface of this entrance (Fig. 3 ▶).


The structure of the hexameric atrazine chlorohydrolase AtzA.

Peat TS, Newman J, Balotra S, Lucent D, Warden AC, Scott C - Acta Crystallogr. D Biol. Crystallogr. (2015)

The entrance to the central cavity of the AtzA hexamer and the amino-acid residues of the hexamer-stabilizing interface. The entrance to the AtzA hexamer central cavity is largely formed by Arg163 contributed by each monomer. The hole formed in the hexamer is approximately 14 Å across at the entrance. Amino-acid substitutions at residues Ala170, Met256, Pro258 and Tyr261 destabilize the hexamer. The interactions between three monomers on one face of the hexamer (left) and at a single monomer–monomer interface (right) are shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4356373&req=5

fig3: The entrance to the central cavity of the AtzA hexamer and the amino-acid residues of the hexamer-stabilizing interface. The entrance to the AtzA hexamer central cavity is largely formed by Arg163 contributed by each monomer. The hole formed in the hexamer is approximately 14 Å across at the entrance. Amino-acid substitutions at residues Ala170, Met256, Pro258 and Tyr261 destabilize the hexamer. The interactions between three monomers on one face of the hexamer (left) and at a single monomer–monomer interface (right) are shown.
Mentions: The AtzA hexamer (about 315 kDa in molecular weight) is a trimer of dimers (Fig. 2 ▶a), with the dimer interface being significantly larger than the individual protomer interfaces used to make up the hexamer (Figs. 2 ▶a and 2 ▶b): 3295 Å2 compared with 585 Å2. The hexamer was confirmed by DLS and size-exclusion chromatography (SEC; Supplementary Fig. S2), consistent with previous reports for the solution state of the protein (de Souza et al., 1996 ▶; Scott et al., 2009 ▶). The dimer surface covers 17–18% of the monomer (a single monomer has a surface area of ∼18 600 Å2; Fig. 2 ▶c). The dimer interface is similar to that found in the other amidohydrolases and the dimer is similar enough in overall structure that the search model used for molecular replacement (PDB entry 3hpa) gave a significantly more robust solution when the dimer was used instead of the monomer. Two full hexamers (over 600 kDa) were found in the asymmetric unit of the crystal structure in space group P22121, whereas a single hexamer was found in space group P212121. The two structures are essentially identical (r.m.s.d. of 0.3 Å), with the higher resolution data giving greater clarity in amino-acid positions and having significantly more water molecules added to the model. The most obvious interaction seen when looking down the threefold axis of the hexamer is the 11-amino-acid insertion (relative to PDB entry 3hpa) that forms the loop and helix encompassing residues 160–183 that has not been seen in the other amidohydrolase structures to date. The ‘cavity’ in the hexamer is approximately 14 Å across at the entrance, with one residue in particular, Arg163, forming much of the surface of this entrance (Fig. 3 ▶).

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: CSIRO Biomedical Manufacturing, Parkville, Australia.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus