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 {angstrom} resolution, is a ({beta}{alpha}){sub 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 {angstrom} from the zinc atom and 2.6 {angstrom} from the oxygen atom of zinc-coordinated water. Mutation of the threonine to a serine resulted in a 12-fold decrease in k{sub cat}/K{sub m}, largely due to k{sub 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 {angstrom} resolution, respectively. Both structure and kinetic determinations suggest that the Glu{sup 241} side chain provides a proton to N-1 of the s-triazine substrate to facilitate nucleophilic displacement at the adjacent C-2.
- Research Organization:
- Brookhaven National Lab. (BNL), Upton, NY (United States). National Synchrotron Light Source
- Sponsoring Organization:
- DOE - OFFICE OF SCIENCE
- DOE Contract Number:
- DE-AC02-98CH10886
- OSTI ID:
- 1019663
- Report Number(s):
- BNL-95509-2011-JA; JBCHA3; TRN: US1103673
- Journal Information:
- Journal of Biological Chemistry, Vol. 285, Issue 40; ISSN 0021-9258
- Country of Publication:
- United States
- Language:
- English
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