X-ray structure and mutational analysis of the atrazine Chlorohydrolase TrzN.
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 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.
Seffernick
Jennifer L
JL
Department of Biochemistry, University of Minnesota, St Paul, Minnesota 55108, USA.
Reynolds
Erik
E
Fedorov
Alexander A
AA
Fedorov
Elena
E
Almo
Steven C
SC
Sadowsky
Michael J
MJ
Wackett
Lawrence P
LP
D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D018360:Crystallography, X-Ray; D006863:Hydrogen-Ion Concentration; D006867:Hydrolases / Q000737:chemistry* / Q000235:genetics; D007700:Kinetics; D020125:Mutation, Missense; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D040681:Structural Homology, Protein; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014227:Triazines / Q000737:chemistry*; D015032:Zinc / Q000737:chemistry*
D001426:Bacterial Proteins; D014227:Triazines; C100057:ametryne; C017035:atratone; D006867:Hydrolases; C448782:trzN protein, Nocardioides sp. C190; D015032:Zinc