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

Abstract

Glutathione transferases (GSTs, EC 2.5.1.18) are ubiquitous proteins in plants that play important roles in stress tolerance and in the detoxification of toxic chemicals and metabolites. In this study, we systematically examined the catalytic diversification of a GST isoenzyme from Phaseolus vulgaris (PvGST) which is induced under biotic stress treatment (Uromyces appendiculatus infection). The full-length cDNA of this GST isoenzyme (termed PvGSTU3-3) with complete open reading frame, was isolated using RACE-RT and showed that the deduced amino acid sequence shares high homology with the tau class plant GSTs. PvGSTU3-3 catalyzes several different reactions and exhibits wide substrate specificity. Of particular importance is the finding that the enzyme shows high antioxidant catalytic function and acts as hydroperoxidase, thioltransferase, and dehydroascorbate reductase. In addition, its K m for GSH is about five to ten times lower compared to other plant GSTs, suggesting that PvGSTU3-3 is able to perform efficient catalysis under conditions where the concentration of reduced glutathione is low (e.g., oxidative stress). Its ability to conjugate GSH with isothiocyanates may provide an additional role for this enzyme to act as a regulator of the released isothiocyanates from glucosinolates as a response of biotic stress. Molecular modeling showed that PvGSTU3-3 shares the same overall fold and structural organization with other plant cytosolic GSTs, with major differences at their hydrophobic binding sites (H-sites) and some differences at the level of C-terminal domain and the linker between the C- and N-terminal domains. PvGSTU3-3, in general, exhibits restricted ability to bind xenobiotics in a nonsubstrate manner, suggesting that the biological role of PvGSTU3-3, is restricted mainly to the catalytic function. Our findings highlight the functional and catalytic diversity of plant GSTs and demonstrate their pivotal role for addressing biotic stresses in Phaseolus vulgaris.

Catalytic features and crystal structure of a tau class glutathione transferase from Glycine max specifically upregulated in response to soybean mosaic virus infections

“…The K m values for GSH, using different electrophilic substrates, lie between a relative narrow range (28.6-87.01 μΜ). The K m values for GSH are about 5-10-times lower compared to other tau class GST isoenzymes, as for example the isoenzyme GmGSTU4-4 [16] or the Phi class enzyme ZmGSTF1-1 [30,53], but is close to that observed for the biotic stress inducible glutathione transferase from Phaseolus vulgaris [11]. The low K m suggests that GmGSTU10-10 is able to work efficiently under oxidative stress conditions where the concentration of reduced GSH is low [54].…”

“…In general, the H-site of GSTs displays a low degree of sequence identity. This reflects differences in substrate specificity, compared to other tau class plant enzymes [3,10,11,16], and suggests that the C-terminal domain of these enzymes may have evolved under differential selective pressures. The H-site of GmGSTU10-10 is typically hydrophobic and built mainly by residues from the C-terminal domain: H4 (His107, Lys111, Trp114, Thr115), H6 (Trp163), H9 (Phe208 and Leu212), and Phe10 from the N-terminal domain.…”

“…Plant glutathione S-transferases (GSTs; EC 2.5.1.18) are enzymes that catalyze the conjugation of reduced glutathione (GSH; γ-Glu-Cys-Gly) to electrophilic centers of a wide variety of, mainly hydrophobic, compounds, both endogenous and xenobiotic [1][2][3][4]. GSTs are implicated in pesticide detoxification [5][6][7], in responses to abiotic and biotic stress (infection, heavy metals, UV radiation, etc) [8][9][10][11], as well as in hormonal regulation and developmental change [12][13][14][15][16]. GSTs can be found in plants from early embryogenesis to senescence [17].…”

“…The G-and H-sites are typically formed from residues of N-and C-terminal domains, respectively. Residues of the H-site are not conserved compared to those of the G-site across cytosolic GSTs [1,[10][11][12]16,21]. GSTs, therefore, display wide substrate specificity toward electrophile molecules including organic halides, organic hydroperoxides, epoxides, arene oxides, α-and βunsaturated carbonyls, organic nitrate esters, and organic thiocyanates [1,10,11,22,23].…”

“…Residues of the H-site are not conserved compared to those of the G-site across cytosolic GSTs [1,[10][11][12]16,21]. GSTs, therefore, display wide substrate specificity toward electrophile molecules including organic halides, organic hydroperoxides, epoxides, arene oxides, α-and βunsaturated carbonyls, organic nitrate esters, and organic thiocyanates [1,10,11,22,23]. Selected GSTs from tau, theta and phi classes exhibit peroxidase activity [12][13][14][15][16] by reducing lipid hydroperoxides directly and by removing lipid peroxidation end products like alkenals, 4-hydroxynonenal, and other α,β-unsaturated aldehydes [3,[9][10][11].…”