Phytochelatin synthase catalyzes key step in turnover of glutathione conjugates.

Conjugation of xenobiotic compounds and endogenous metabolites to glutathione is an ubiquitous process in eukaryotes. In animals, the first and rate-limiting step of glutathione-S-conjugate metabolism is characterized by the removal of the aminoterminal glutamic acid residue of glutathione. In plants, however, glutathione-S-conjugates are generally metabolized by removal of the carboxylterminal glycine residue of the tripeptide glutathione to give rise to the S-glutamylcysteinyl-derivative. Purification of the glutathione-conjugate catabolizing activity from cell suspension cultures of the plant Silene cucubalus indicated that phytochelatin synthase catalyzes the first step of the pathway. Heterologously expressed phytochelatin synthase from Arabidopsis efficiently converted S-bima ne-glutathione to S-bimane-glutamylcysteine, the formation of which was unequivocally identified by mass spectrometry. No further products, such as S-derivatives of phytochelatins, were observed. Several different glutathione-S-conjugates served as substrates for the enzyme and were processed to the corresponding glutamylcysteinyl-adducts. Affinity-purified phytochelatin synthase preparations required divalent heavy metal ions such as Cd(2+), Zn(2+) or Cu(2+) for detectable turnover of glutathione-S-conjugates. Characterization of the enzymatic properties of phytochelatin synthase argues for both cellular functions of the gamma-glutamylcysteinyl-dipeptidyltransferase: (1) formation of heavy-metal binding peptides and (2) degradation of glutathione-S-conjugates. Mechanistically, the former role is the result of gamma-glutamylcysteinyl transpeptidation onto glutathione or derivatives thereof, while the catabolic function reflects transpeptidation of S-glutamylcysteinyl-adducts onto the acceptor molecule water. Thus, phytochelatin synthase seems to fulfil a second crucial role in glutathione metabolism.

Metabolomic analysis of the consequences of cadmium exposure in Silene cucubalus cell cultures via 1H NMR spectroscopy and chemometrics.

Several essential and non-essential metals (typically those from periods 4, 5 and 6 in groups 11-15 in the periodic table) are commonly detoxified in higher plants by complexation with phytochelatin. The genetic and gross metabolic basis of metal tolerance in plants is, however, poorly understood. Here, we have analyzed plant cell extracts using 1H NMR spectroscopy combined with multivariate statistical analysis of the data to investigate the biochemical consequences of Cd(2+) exposure in Silene cucubalus cell cultures. Principal components analysis of 1H NMR spectra showed clear discrimination between control and Cd(2+) dosed groups, demonstrating the metabolic effects of Cd(2+) and thus allowing the identification of increases in malic acid and acetate, and decreases in glutamine and branched chain amino acids as consequences of Cd(2+) exposure. This work shows the value of NMR-based metabolomic approaches to the determination of biochemical effects of pollutants in naturally selected populations.

Increase of free cysteine and citric acid in plant cells exposed to cobalt ions.

Cobalt complexation was investigated in a suspension cell culture of the cobalt hyperaccumulator Crotalaria cobalticola. C. cobalticola cells were more tolerant towards cobalt ions than the suspension cells of the non-accumulators Rauvolfia serpentina and Silene cucubalus. While the concentration of various compounds increased in cells of C. cobalticola challenged with cobalt ions, phytochelatin biosynthesis was not induced. Instead, the exposure to cobalt ions resulted in the increase of citrate and cysteine in cells. Size exclusion chromatography demonstrated the co-elution of cobalt and cysteine in C. cobalticola cell extracts. A significant increase in cysteine was observed also in cells of R. serpentina and S. cucubalus when they were exposed to cobalt ions. These results suggest that free cysteine is involved in cobalt ion complexation in plant cells.

Molecular characterization of the homo-phytochelatin synthase of soybean Glycine max: relation to phytochelatin synthase.

The phytochelatin homologs homo-phytochelatins are heavy metal-binding peptides present in many legumes. To study the biosynthesis of these compounds, we have isolated and functionally expressed a cDNA GmhPCS1 encoding homo-phytochelatin synthase from Glycine max, a plant known to accumulate homo-phytochelatins rather than phytochelatins upon the exposure to heavy metals. The catalytic properties of GmhPCS1 were compared with the phytochelatin synthase AtPCS1 from Arabidopsis thaliana. When assayed only in the presence of glutathione, both enzymes catalyzed phytochelatin formation. GmhPCS1 accepted homoglutathione as the sole substrate for the synthesis of homo-phytochelatins whereas AtPCS1 did not. Homo-phytochelatin synthesis activity of both recombinant enzymes was significantly higher when glutathione was included in the reaction mixture. The incorporation of both glutathione and homoglutathione into homo-phytochelatin, n = 2, was demonstrated using GmhPCS1 and AtPCS1. In addition to bis(glutathionato)-metal complexes, various other metal-thiolates were shown to contribute to the activation of phytochelatin synthase. These complexes were not accepted as substrates by the enzyme, thereby suggesting that a recently proposed model of activation cannot fully explain the catalytic mechanism of phytochelatin synthase (Vatamaniuk, O. K., Mari, S., Lu, Y. P., and Rea, P. A. (2000) J. Biol. Chem. 275, 31451-31459).