Abstract
Phytochelatin synthase (PCS) is a major determinant of heavy metal tolerance in plants and other organisms. No structural information on this enzyme is as yet available. It is generally believed, however, that the active site region is located in the more conserved N-terminal portion of PCS, whereas various, as yet unidentified (but supposedly less critical) roles have been proposed for the C-terminal region. To gain insight into the structural/functional organization of PCS, we have conducted a limited proteolysis analysis of the enzyme from Arabidopsis (AtPCS1), followed by functional characterization of the resulting polypeptide fragments. Two N-terminal fragments ending at positions 372 (PCS_Nt1) and 283 (PCS_Nt2) were produced sequentially upon V8 protease digestion, without any detectable accumulation of the corresponding C-terminal fragments. As revealed by the results of in vivo and in vitro functional assays, the core PCS_Nt2 fragment is biosynthetically active in the presence of cadmium ions and supports phytochelatin formation at a rate that is only approximately 5-fold lower than that of full-length AtPCS1. The loss of the C-terminal region, however, substantially decreases the thermal stability of the enzyme and impairs phytochelatin formation in the presence of certain heavy metals (e.g. mercury and zinc, but not cadmium or copper). The latter phenotype was shared by PCS_Nt2 and by its precursor fragment PCS_Nt1, which, on the other hand, was almost as stable and biosynthetically active (in the presence of cadmium) as the full-length enzyme. AtPCS1 thus appears to be composed of a protease-resistant (and hence presumably highly structured) N-terminal domain, flanked by an intrinsically unstable C-terminal region. The most upstream part of such a region (positions 284-372) is important for enzyme stabilization, whereas its most terminal part (positions 373-485) appears to be required to determine enzyme responsiveness to a broader range of heavy metals.
Highlights
Phytochelatin synthase (PCS) is a major determinant of heavy metal tolerance in plants and other organisms
It must be noted that the length of different PCS homologs ranges from 360 to 500 amino acids, implying that a rather long, and perhaps functionally important, C-terminal region is present in all PCS enzymes identified to date
This view is reinforced by the observation that the level of sequence conservation in the C-terminal region is similar to that of the N-terminal region, when only plant PCS sequences are considered, and even more so when such analysis is restricted to dicotyledonous plants (Fig. 1, lines b and c, respectively)
Summary
Phytochelatin synthase (PCS) is a major determinant of heavy metal tolerance in plants and other organisms. As revealed by the pioneering studies of Zenk and coworkers [4], PCS is a cytosolic ␥-Glu-Cys dipeptidyl transpeptidase (EC 2.3.2.15) that catalyzes the transpeptidation of the ␥-GluCys moiety of GSH onto a second GSH molecule to form PC2 or onto a preformed PCn molecule to form the corresponding nϩ derivative In both fission yeast and plants, the in vivo synthesis of PCs can be induced by a variety of metal ions, the most effective of which is cadmium [2]. The loss of part of the C-terminal region of AtPCS1, as in the cad mutant [5], definitely leads to a defective cadmium tolerance (and impaired in vitro PC synthesis) phenotype This argues that the putative C-terminal domain, which contains multiple cysteine residues, often arranged in pairs (or near pairs) as in MTs [18], and for which diverse functions have been proposed in recent years, does somehow contribute to PCS functionality
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