Evolution of the catalytic activity of Arabidopsis thaliana glutathione transferase zeta class-1 by saturation mutagenesis

Bioscience, Biotechnology, and Biochemistry
Defu ChenXiwen Chen

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 k(cat)/K(m) 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.

References

Dec 18, 1996·Toxicology·A B DeAngeloG R Olson
Oct 20, 2005·Journal of Bioscience and Bioengineering·Defu ChenTsuneo Yamane
Jan 10, 2006·Methods in Enzymology·Philip G Board, M W Anders
Aug 30, 2008·Journal of Genetics and Genomics = Yi Chuan Xue Bao·Sudan TaoDefu Chen

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