Ammonium sulphate precipitated crude enzyme from fission yeast (S. pombe) was used to follow incorporation of 35S-labelled glutathione into yGluCys peptides (Hayashi et al. 1991b). The in vitro sequential synthesis of phytochelatin n=2, 3 and 4 was like the model for phytochelatin synthase. Cadmium was not essential for the reactions, perhaps sufficient Cu and Zn were present in the preparation to activate the enzyme. When the available glutathione was reduced from millimolar to low micromolar concentrations, dipeptidyl transfer caused formation of n=2, 3 and 4 oligomers of (yGluCys)n. This is the only in vitro evidence of a biosynthetic origin of the (yGluCys)n family of peptides. In this alternative pathway later addition of glycine and partially purified glutathione synthetase caused conversion of (yGluCys)n oligomers into the respective (yGluCys)nGly oligomers. Phytochelatin synthase enzyme from peas and tomato did not use yGluCys to produce (yGluCys)„ (Klapheck et al. 1995, Chen et al. 1997).
A Cd-sensitive mutant of S. pombe with severely impaired phytochelatin synthesis and only 44 % of the glutathione content of wild type cells was the source of a DNA fragment involved with phytochelatin synthesis (Al-Lahham et al. 1999). Sequence analysis showed that the DNA encoded glutathione synthetase (GSH2, E.C. 220.127.116.11) with the mutant allele having a single base-pair exchange near the 3' end of the reading frame that changed a glycine to aspartate. It was deduced that the GSH2 gene encoded a bifunc-tional enzyme able to synthesize both glutathione and phytochelatins. Com plementation of the mutant with GSH2 from A. thaliana led to a partial restoration of phytochelatin synthesis in the mutant fission yeast (Leuchter et al. 1998). Whether the putative bifunctional activity of glutathione synthetase is a special case for fission yeast or also applies to plants requires further investigation.
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