X-ray structure and mutational analysis of the atrazine chlorohydrolase TrzN

Jennifer L. Seffernick, Erik Reynolds, Alexander A. Fedorov, Elena Fedorov, Steven C. Almo, Michael J. Sadowsky, Lawrence P. Wackett

Research output: Contribution to journalArticle

19 Citations (Scopus)

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 (βα)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 zinc-coordinated water. Mutation of the threonine to a serine resulted in a 12-fold decrease in kcat/Km, largely due to kcat, 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 Glu241 side chain provides a proton to N-1 of the s-triazine substrate to facilitate nucleophilic displacement at the adjacent C-2.

Original languageEnglish (US)
Pages (from-to)30606-30614
Number of pages9
JournalJournal of Biological Chemistry
Volume285
Issue number40
DOIs
StatePublished - Oct 1 2010

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Threonine
Triazines
Amidohydrolases
X-Rays
Atrazine
X rays
Atoms
Protons
Zinc
Catalytic Domain
Oxygen
Kinetics
Carbonic Anhydrases
Water
Chlorine
Herbicides
Hydrolases
Enzymes
Metabolism
Aspartic Acid

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology
  • Medicine(all)

Cite this

Seffernick, J. L., Reynolds, E., Fedorov, A. A., Fedorov, E., Almo, S. C., Sadowsky, M. J., & Wackett, L. P. (2010). X-ray structure and mutational analysis of the atrazine chlorohydrolase TrzN. Journal of Biological Chemistry, 285(40), 30606-30614. https://doi.org/10.1074/jbc.M110.138677

X-ray structure and mutational analysis of the atrazine chlorohydrolase TrzN. / Seffernick, Jennifer L.; Reynolds, Erik; Fedorov, Alexander A.; Fedorov, Elena; Almo, Steven C.; Sadowsky, Michael J.; Wackett, Lawrence P.

In: Journal of Biological Chemistry, Vol. 285, No. 40, 01.10.2010, p. 30606-30614.

Research output: Contribution to journalArticle

Seffernick, JL, Reynolds, E, Fedorov, AA, Fedorov, E, Almo, SC, Sadowsky, MJ & Wackett, LP 2010, 'X-ray structure and mutational analysis of the atrazine chlorohydrolase TrzN', Journal of Biological Chemistry, vol. 285, no. 40, pp. 30606-30614. https://doi.org/10.1074/jbc.M110.138677
Seffernick JL, Reynolds E, Fedorov AA, Fedorov E, Almo SC, Sadowsky MJ et al. X-ray structure and mutational analysis of the atrazine chlorohydrolase TrzN. Journal of Biological Chemistry. 2010 Oct 1;285(40):30606-30614. https://doi.org/10.1074/jbc.M110.138677
Seffernick, Jennifer L. ; Reynolds, Erik ; Fedorov, Alexander A. ; Fedorov, Elena ; Almo, Steven C. ; Sadowsky, Michael J. ; Wackett, Lawrence P. / X-ray structure and mutational analysis of the atrazine chlorohydrolase TrzN. In: Journal of Biological Chemistry. 2010 ; Vol. 285, No. 40. pp. 30606-30614.
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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 {\AA} resolution, is a (βα)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 {\AA} from the zinc atom and 2.6 {\AA} from the oxygen atom of zinc-coordinated water. Mutation of the threonine to a serine resulted in a 12-fold decrease in kcat/Km, largely due to kcat, 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 {\AA} resolution, respectively. Both structure and kinetic determinations suggest that the Glu241 side chain provides a proton to N-1 of the s-triazine substrate to facilitate nucleophilic displacement at the adjacent C-2.",
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