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BIOCHEMICAL PROCESSES AND MACROMOLECULAR STRUCTURES

A Reassessment of Substrate Specificity and Activation of Phytochelatin Synthases from Model Plants by Physiologically Relevant Metals¹

Departamento de Nutrición Vegetal, Estación Experimental de Aula Dei, Consejo Superior de Investigaciones Científicas, 50080 Zaragoza, Spain

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 -glutamylcysteine units. Purified AtPCS1 and LjPCS1 were activated (in decreasing order) by Cd²⁺, Zn²⁺, Cu²⁺, and Fe³⁺, but not by Co²⁺ or Ni²⁺, in the presence of 5 mM GSH and 50 µM metal ions. Activation of both enzymes by Fe³⁺ 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 Cu²⁺ and Zn²⁺, but to a much lower extent than did with Cd²⁺, indicating that (h)PC synthesis does not significantly contribute in vivo to copper, zinc, and iron detoxification.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Manuel Becana (becana{at}eead.csic.es).

Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.105.073635.

^* Corresponding author; e-mail becana{at}eead.csic.es; fax 34–976–716145.

Received November 4, 2005; returned for revision February 6, 2006; accepted February 6, 2006.

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