Catalytic and structural diversity of the fluazifop-inducible glutathione transferases from Phaseolus vulgaris

Plant glutathione transferases (GSTs) comprise a large family of inducible enzymes that play important roles in stress tolerance and herbicide detoxification. Treatment of Phaseolus vulgaris leaves with the aryloxyphenoxypropionic herbicide fluazifop-p-butyl resulted in induction of GST activities. Three inducible GST isoenzymes were identified and separated by affinity chromatography. Their full-length cDNAs with complete open reading frame were isolated using RACE-RT and information from N-terminal amino acid sequences. Analysis of the cDNA clones showed that the deduced amino acid sequences share high homology with GSTs that belong to phi and tau classes. The three isoenzymes were expressed in E. coli and their substrate specificity was determined towards 20 different substrates. The results showed that the fluazifop-inducible glutathione transferases from P. vulgaris (PvGSTs) catalyze a broad range of reactions and exhibit quite varied substrate specificity. Molecular modeling and structural analysis was used to identify key structural characteristics and to provide insights into the substrate specificity and the catalytic mechanism of these enzymes. These results provide new insights into catalytic and structural diversity of GSTs and the detoxifying mechanism used by P. vulgaris

Isolation of GST isoenzymes from Phaseolus vulgaris L. and characterization of detoxifying mechanism under biotic and abiotic stress

Three Glutathione transferase (GSTs) isoenzymes have been isolated from P. vulgaris leaves after in vivo treatment with 1/250 fluazifop-p-butyl herbicide. The inducible GST isoenzymes were identified and separated by affinity chromatography. They found to belong to phi and tau classes. Moreover, the fluazifop-inducible glutathione transferases from P. vulgaris (PvGSTs, termed PvGSTU2-2, PvGSTF1-1 and PvGSTU3-3) were found to catalyze a broad range of reactions and exhibit quite varied substrate specificity. Another GST isoenzyme was isolated from P. vulgaris (PvGST, termed PvGSTU3-3), after its induction with biotic stress treatment (Uromyces appendiculatus infection). PvGSTU3-3 shares high homology the tau class plant and catalyzes several different reactions and exhibits wide substrate specificity. Of particular importance are the high antioxidant catalytic function and hydroperoxidase, thioltransferase, and dehydroascorbate reductase action of Pv-GSTU3-3. Transgenic tobacco plants over-expressing PvGSTU2-2 isoenzyme have been developed via Agrobacteriun tumefaciens in order to study their in planta potential to confer biotic and abiotic resistance as a means of plant breeding. Our results provide new insights into catalytic and structural diversity of GSTs and the detoxifying mechanism used by P. vulgaris. Moreover, highlight the functional and catalytic diversity of plant GSTs and demonstrate their pivotal role for addressing biotic stresses in P. vulgaris