Selective Binding of Glutathione Conjugates of Fatty Acid Derivatives by Plant Glutathione Transferases

Abstract

Proteomic studies with Arabidopsis thaliana have revealed that the plant-specific Tau (U) class glutathione transferases (GSTs) are selectively retained by S-hexylglutathione affinity supports. Overexpression of members of the Arabidopsis GST superfamily in Escherichia coli showed that 25 of the complement of 28 GSTUs caused the aberrant accumulation of acylated glutathione thioesters in vivo, a perturbation that was not observed with other GST classes. Each GSTU caused a specific group of fatty acyl derivatives to accumulate, which varied in chain length (C 6 to C 18 ), additional oxygen content (0 or 1), and desaturation (0 or 1). Thioesters bound tightly to recombinant GSTs (K d ϳ 1 M), explaining their accumulation. Transient expression of GSTUs in Nicotiana benthamiana followed by recovery by Strep-tag affinity chromatography allowed the respective plant ligands to be extracted and characterized. Again, each GST showed a distinct profile of recovered metabolites, notably glutathionylated oxophytodienoic acid and related oxygenated fatty acids. Similarly, the expression of the major Tau protein GSTU19 in the endogenous host Arabidopsis led to the selective binding of the glutathionylated oxophytodienoic acid-glutathione conjugate, with the enzyme able to catalyze the conjugation reaction. Additional ligands identified in planta included other fatty acid derivatives including divinyl ethers and glutathionylated chlorogenic acid. The strong and specific retention of various oxygenated fatty acids by each GSTU and the conservation in binding observed in the different hosts suggest that these proteins have selective roles in binding and conjugating these unstable metabolites in vivo.In plants, the glutathione transferases (GSTs 2 ; EC 2.5.1.18) are a superfamily of proteins with the dominant Phi (F) and Tau (U) classes having largely undefined functions in endogenous metabolism (1). This is in contrast to their relatively well studied role in catalyzing the glutathione conjugation of herbicides (2). Because of their importance in determining herbicide selectivity, these proteins have been purified from a range of major crops including maize (3), soybean (4), and wheat (5, 6). One approach which has been commonly applied to isolate these proteins is affinity chromatography using a range of glutathione (GSH) derivatives as ligands (7). These ligands often display a surprising degree of selectivity in capturing specific GSTs. Thus, in wheat extracts, whereas GSH-agarose effectively isolated GSTFs, S-hexylglutathione (GS-hexyl) proved to be a selective ligand for GSTUs (5). Similarly, glutathionylated dyes have proved to be highly selective for specific GSTs in maize (8).In each application, the use of GSH derivatives as affinity ligands has been developed through serendipity, with limited attention directed to the potential significance of such binding selectivity in relating to the nature of in vivo substrates and reaction products. At a molecular level, crystallographic studies with both plant GSTFs (9) and GSTUs...

Arabidopsis Defense against Botrytis cinerea: Chronology and Regulation Deciphered by High-Resolution Temporal Transcriptomic Analysis

“…Seven glutathione S-transferase (GST) genes are present in two clusters (26 and 30) overrepresented for this term and are upregulated around 14 HAI. All seven genes (GSTU3, GSTU7, GSTU8, GSTU10, GSTU19, GSTU24, and GSTU25) are members of the Tau family of GSTs, which are plant specific and can bind glutathione conjugated fatty acid derivatives (Dixon and Edwards, 2009). Although the precise function of these GSTs is not known, GSTU19 and GSTU25 both have high conjugating activity toward the xenobiotic 1-chloro-2,4-dinitrobenzene, used as an indicator of detoxifying activity.…”

“…Although the precise function of these GSTs is not known, GSTU19 and GSTU25 both have high conjugating activity toward the xenobiotic 1-chloro-2,4-dinitrobenzene, used as an indicator of detoxifying activity. However, GSTU19 can conjugate glutathione onto oxylipins, suggesting that it may play a role in modulating jasmonate signaling (Dixon and Edwards, 2009). In addition to toxin detoxification, cells are able to prevent toxin accumulation through sequestration in the vacuole or active transport out of cells.…”

Structural basis of jasmonate-amido synthetase FIN219 in complex with glutathione S-transferase FIP1 during the JA signal regulation

“…The excess light-induced ROS defense mechanism is also linked to JA signaling through transcriptional regulation (43). Previous research revealed that overexpression of members of the Arabidopsis GST superfamily in Escherichia coli resulted in 25 of the 28 GST tau (U) proteins (GSTUs) causing an aberrant accumulation of acylated GSH thioesters in vivo, a perturbation that was not observed with other GST classes (47). In particular, the study demonstrated that GSTU members catalyzed the S-glutathionylation of 12-oxo-phytodienoic acid (OPDA), an early biosynthetic precursor of JA (28,47).…”

“…Previous research revealed that overexpression of members of the Arabidopsis GST superfamily in Escherichia coli resulted in 25 of the 28 GST tau (U) proteins (GSTUs) causing an aberrant accumulation of acylated GSH thioesters in vivo, a perturbation that was not observed with other GST classes (47). In particular, the study demonstrated that GSTU members catalyzed the S-glutathionylation of 12-oxo-phytodienoic acid (OPDA), an early biosynthetic precursor of JA (28,47). Further research is required to determine whether FIP1 plays a role in other regulatory mechanisms in jasmonic acid signaling, such as preventing epimerization of the active (+)-7-iso-jasmonoyl-L-isoleucine attaching to the inactive (−)-jasmonoyl-L-isoleucine.…”

Catalases may play different roles in influencing resistance to virus-induced hypersensitive necrosis

“…GST isoenzymes are encoded by a gene family divided into different classes based on sequence similarities and biochemical function (Edwards et al, 2000;Wagner et al, 2002). Most GSTs belong to the phi and theta classes and participate in detoxification reactions by catalyzing the conjugate formation between electrophilic compounds and the antioxidant glutathione (GSH) (Wagner et al, 2002;Dixon and Edwards, 2009a;Dixon et al, 2011). However, certain tau class GSTs possess a glutathione-peroxidase activity and are able to catalyze the breakdown of H 2 O 2 and lipid hydroperoxides (Bartling et al, 1993;Dixon et al, 2009b).…”