A Reassessment of Substrate Specificity and Activation of Phytochelatin Synthases from Model Plants by Physiologically Relevant Metals

Phytochelatin synthases (PCS) catalyze phytochelatin (PC) synthesis from glutathione (GSH) in the presence of certain metals. The resulting PC-metal complexes are transported into the vacuole, avoiding toxic effects on metabolism. Legumes have the unique capacity to partially or completely replace GSH by homoglutathione (hGSH) and PCs by homophytochelatins (hPCs). However, the synthesis of hPCs has received little attention. A search for PCS genes in the model legume Lotus ( Lotus japonicus ) resulted in the isolation of a cDNA clone encoding a protein (LjPCS1) highly homologous to a previously reported homophytochelatin synthase (hPCS) of Glycine max (GmhPCS1). Recombinant LjPCS1 and Arabidopsis ( Arabidopsis thaliana ) PCS1 (AtPCS1) were affinity purified and their polyhistidine-tags removed. AtPCS1 catalyzed hPC synthesis from hGSH alone at even higher rates than did LjPCS1, indicating that GmhPCS1 is not a genuine hPCS and that a low ratio of hPC to PC synthesis is an inherent feature of PCS1 enzymes. For both enzymes, hGSH is a good acceptor, but a poor donor, of γ -glutamylcysteine units. Purified AtPCS1 and LjPCS1 were activated (in decreasing order) by Cd 2+ , Zn 2+ , Cu 2+ , and Fe 3+ , but not by Co 2+ or Ni 2+ , in the presence of 5 mm GSH and 50 μ m metal ions. Activation of both enzymes by Fe 3+ was proven by the complete inhibition of PC synthesis by the iron-specific chelator desferrioxamine. Plants of Arabidopsis and Lotus accumulated (h)PCs only in response to a large excess of Cu 2+ and Zn 2+ , but to a much lower extent than did with Cd 2+ , indicating that (h)PC synthesis does not significantly contribute in vivo to copper, zinc, and iron detoxification.