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

Abstract

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.

Insights into ligand binding to a glutathione S-transferase from mango: Structure, thermodynamics and kinetics

“…A rapid equilibrium random sequential bi-bi mechanism [35, 56], has been reported in the GSTs from other plants, such as sorghum, as well as in three tomato GSTUs [57]. The MiGSTU showed a Michaelis-Menten kinetics, and a bi-bi mechanism type that could be sequential (ordered) is suggested, in accordance to that reported for other GSTs [3, 35, 57]. Since the kinetic mechanism is related to the enzyme structure, in the ordered mechanism the binding of the first substrate to the active site induces a conformational change, needed for the binding of the second substrate.…”

“…Even though some exceptions have been found within this family, a Michaelis-Menten behavior has been revealed by most of the GSTs. Differences in two enzymes catalytic mechanisms of the same class (Tau) were reported in Phaseolus vulgaris [3], where a Michaelis–Menten kinetics was followed by the PvGSTU2-2 using CDNB as the substrate, while a sigmoid substrate dependence was revealed by the PvGSTU1-1. A rapid equilibrium random sequential bi-bi mechanism [35, 56], has been reported in the GSTs from other plants, such as sorghum, as well as in three tomato GSTUs [57].…”

“…These electrophiles include both endogenous and xenobiotics compounds, which are targeted at specific sites either intra or extracellular [3–7]. The GSTs are ubiquitous proteins, mainly cytosolic, classified into different classes [8–10].…”

“…The GSTs are ubiquitous proteins, mainly cytosolic, classified into different classes [8–10]. The theta, zeta, phi and tau GST classes have been reported mainly in plants, being phi and tau classes the most abundant between them [3, 10–12]. The GST family has multiple functions in plants besides herbicide resistance and xenobiotic detoxification; they can be induced by pathogens or dehydration, and may even bind auxins and anthocyanin [13] [7, 14, 15].…”

Cloning and Characterization of a Biotic-Stress-Inducible Glutathione Transferase from Phaseolus vulgaris

“…Glutathione transferases (GSTs, EC 2.5.1.18) are multifunctional enzymes that are involved in phase II cellular detoxification mechanism by catalyzing the nucleophilic attack of reduced glutathione (GSH) on the electrophilic center of xenobiotics and toxic metabolites [1,2]. These GSH adducts are more water soluble and can be excreted more easily [3,4].…”