title: Evolution of the catalytic activity of Arabidopsis thaliana glutathione transferase zeta class-1 by saturation mutagenesis.
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.
authors.affiliation: Laboratory of Molecular Genetics, College of Life Sciences, Nankai University, China.
authors.forename: Defu
authors.initials: D
authors.lastname: Chen
authors.forename: Jia
authors.initials: J
authors.lastname: Liu
authors.forename: Jian
authors.initials: J
authors.lastname: Liu
authors.forename: Xiwen
authors.initials: X
authors.lastname: Chen
mesh_terms: D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000145:classification / Q000235:genetics* / Q000378:metabolism*; D055162:Biocatalysis; D020134:Catalytic Domain; D019143:Evolution, Molecular; D015723:Gene Library; D005982:Glutathione Transferase / Q000737:chemistry / Q000145:classification / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D016296:Mutagenesis; D009154:Mutation; D013329:Structure-Activity Relationship
chemical_list: D029681:Arabidopsis Proteins; C428046:GSTZ1 protein, Arabidopsis; D005982:Glutathione Transferase