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

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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 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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ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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