Evolution of the Catalytic Activity of Arabidopsis thaliana Glutathione Transferase Zeta Class-1 by Saturation Mutagenesis

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  • CHEN Defu
    Laboratory of Molecular Genetics, College of Life Sciences, Nankai University
  • LIU Jia
    Department of Chemistry and Biochemistry, University of Maryland
  • LIU Jian
    Laboratory of Molecular Genetics, College of Life Sciences, Nankai University
  • CHEN Xiwen
    Laboratory of Molecular Genetics, College of Life Sciences, Nankai University

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  • Evolution of the Catalytic Activity of<i>Arabidopsis thaliana</i>Glutathione Transferase Zeta Class-1 by Saturation Mutagenesis

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Abstract

Saturation mutagenesis was performed on three non-catalytic residues, Asn71, Leu108, and Gly177, in and near the active site of Arabidopsis thaliana GSTZ-1 (AtGSTZ-1). Forty unique mutants with more than 10% activity increases, were obtained. Of these, 12 resulted in large activity improvement and were purified for further characterization. Remarkably, 11 of them contained mutations at Leu108, suggesting that Leu108 plays an important role in dichloroacetic acid-dechlorinating (DCA-DC) activity. Kinetic analysis revealed that multiple mutations at these residues increased kcat/Km toward DCA and GSH by as much as 2.2- and 5.8-fold, respectively. Since the catalytic residues were not involved in mutagenesis, the activity enhancements were presumably due to structural change in the active site rather than to a change in catalysis. Our results also suggest that the specific shape of the active site in AtGSTZ-1 is essential to its unique DCA-DC activity.

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