Characterization of γ-glutamyltransferase- and phytochelatin synthase-mediated catabolism of glutathione and glutathione S-conjugates in Arabidopsis thaliana.

Glutathione (GSH, γ-L-glutamyl-L-cysteinyl-glycine) has been implicated in a multitude of cellular functions, such as protection of cells against oxidative stress, detoxification of xenobiotics via degradation of GSH S-conjugates, and disease resistance. Glutathione also serves as a precursor of phytochelatins, and thereby plays an essential role in heavy metal detoxification. The Arabidopsis genome encodes three functional γ-glutamyltransferase genes (AtGGT1, AtGGT2, AtGGT4) and two phytochelatin synthase genes (AtPCS1, AtPCS2). The function of plant GGT has not yet been clearly defined, although it is thought to be involved in GSH and GSH S-conjugate catabolism. On the other hand, besides its role in heavy metal detoxification, PCS has also been involved in GSH S-conjugate catabolism. Herein we describe the HPLC characterization of GSH and GSH S-conjugate catabolism in Arabidopsis mutants deficient in GSH biosynthesis (pad2-1/gsh1), atggt and atpcs1 T-DNA insertion mutants, atggt pad2-1, atggt atpcs1 double mutants, and the atggt1 atggt4 atpcs1 triple mutant. The results of our HPLC analysis confirm that AtGGT and AtPCS play important roles in two different pathways related with GSH and GSH S-conjugate (GS-bimane) catabolism in Arabidopsis.

Purification and characterization of dipeptidase hydrolyzing L-cysteinylglycine from radish cotyledon.

Dipeptidase activity was detected in the soluble fraction of radish (Raphanus sativus L.) cotyledon, and the purified enzyme had a specific activity of 7.32 nkat/mg protein for hydrolyzing L-cysteinylglycine. The dipeptidase was found to be a hexameric metalloenzyme, composed of homological 55 kDa-subunits. This is the first glutathione catabolism-related dipeptidase isolated from higher plants.

Subcellular localization and possible functions of gamma-glutamyltransferase in the radish (Raphanus sativus L.) plant.

Previously we reported the purification of soluble gamma-glutamyltransferases (GGTs) from radish cotyledon. Subcellular fractionation of radish cells revealed that soluble GGT is a vacuolar enzyme. Acivicin, a GGT inhibitor, mediated the in vivo catabolism inhibition of the glutathione S-conjugate generated from endogenous glutathione and exogenously supplied monochlorobimane. Thus soluble GGT is possibly involved in the catabolism of glutathione S-conjugates.

Purification and properties of soluble and bound gamma-glutamyltransferases from radish cotyledon.

Soluble and cell wall bound gamma-glutamyltransferases (GGTs) were purified from radish (Raphanus sativus L.) cotyledons. Soluble GGTs (GGT I and II) had the same M(r) of 63,000, and were composed of a heavy subunit (M(r), 42,000) and a light one (M(r), 21,000). The properties of GGT I and II were similar. Bound GGTs (GGT A and B) were purified to homogeneity from the pellet after the extraction of soluble GGTs. GGT A and B were monomeric proteins with an M(r) of 61,000. The properties of GGT A and B were similar. Thus, bound GGTs were distinguished from soluble GGTs. The optimal pHs of soluble and bound GGTs were about 7.5. Both soluble and bound GGTs utilized glutathione, gamma-L-glutamyl-p-nitroanilide, oxidized glutathione and the conjugate of glutathione with monobromobimane as substrates, and were inhibited by acivicin, but soluble GGTs were also distinguished from bound GGTs with regard to these properties.

Homoglutathione confers tolerance to acifluorfen in transgenic tobacco plants expressing soybean homoglutathione synthetase.

Homoglutathione (hGSH), which is present in some leguminous plants, is preferred over GSH in in vitro conjugation of acifluorfen and fomesafen by glutathione S-transferase. To investigate the function of hGSH in in vivo detoxification of xenobiotics, we evaluated herbicide tolerance of transgenic tobacco plants expressing soybean homoglutathione synthetase in the cytosol or chloroplasts. Transgenic plants synthesizing hGSH in the cytosol were more tolerant to acifluorfen than wild-type plants, whereas enhanced tolerance to fomesafen was not observed. Transgenic plants synthesizing hGSH in the chloroplasts showed no enhanced tolerance to acifluorfen or fomesafen.

Synthesis of hydroxymethylglutathione from glutathione and L-serine catalyzed by carboxypeptidase Y.

Hydroxymethylglutathione (gamma-L-glutamyl-L-cysteinyl-L-serine; hmGSH) occurs in many species belonging to the family Gramineae, but the biosynthetic pathway for hmGSH has not been identified. We found that carboxypeptidase Y (CPY), but not carboxypeptidase A, catalyzed hmGSH synthesis from glutathione and L-serine in vitro at acidic pH. CPY also catalyzed methylglutathione synthesis from glutathione and L-alanine. These findings suggested that a carboxypeptidase-like enzyme may be involved in hmGSH synthesis in vivo.