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.
Laboratory of Enzyme Technology, Department of Agricultural Biotechnology, Agricultural University of Athens, 75 Iera Odos Street, 11855 Athens, Greece.
Evangelia
E
Chronopoulou
Panagiotis
P
Madesis
Basiliki
B
Asimakopoulou
Dimitrios
D
Platis
Athanasios
A
Tsaftaris
Nikolaos E
NE
Labrou
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism; D055162:Biocatalysis / Q000187:drug effects*; D003001:Cloning, Molecular; D004137:Dinitrochlorobenzene / Q000737:chemistry / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004790:Enzyme Induction / Q000187:drug effects; D005978:Glutathione / Q000378:metabolism; D005982:Glutathione Transferase / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification; D017879:Isothiocyanates / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D027805:Phaseolus / Q000187:drug effects* / Q000201:enzymology*; D018515:Plant Leaves / Q000187:drug effects / Q000201:enzymology; D011725:Pyridines / Q000737:chemistry / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein; D013379:Substrate Specificity / Q000187:drug effects
D000596:Amino Acids; D004137:Dinitrochlorobenzene; D017879:Isothiocyanates; D011725:Pyridines; D011994:Recombinant Proteins; C050585:fluazifop-butyl; C004471:allyl isothiocyanate; D005982:Glutathione Transferase; D005978:Glutathione