- A papain-like enzyme at work: Native and acyl–enzyme intermediate structures in phytochelatin synthesis

A papain-like enzyme at work: Native and acyl-enzyme intermediate structures in phytochelatin synthesis

Denis Vivares, Pascal Arnoux, and David Pignol

Département d'Ecophysiologie Végétale et de Microbiologie, Direction des Sciences du Vivant, Laboratoire de Bioénergétique Cellulaire, Commissariat á l'Energie Atomique/Cadarache, 13108 St Paul lez Durance Cedex, France

Edited by Robert Huber, Max Planck Institute for Biochemistry, Martinsried, Germany, and approved October 28, 2005 (received for review July 12, 2005)



Phytochelatin synthase (PCS) is a key enzyme for heavy-metal detoxification in plants. PCS catalyzes the production of glutathione (GSH)-derived peptides (called phytochelatins or PCs) that bind heavy-metal ions before vacuolar sequestration. The enzyme can also hydrolyze GSH and GS-conjugated xenobiotics. In the cyanobacterium Nostoc, the enzyme (NsPCS) contains only the catalytic domain of the eukaryotic synthase and can act as a GSH hydrolase and weakly as a peptide ligase. The crystal structure of NsPCS in its native form solved at a 2.0-Å resolution shows that NsPCS is a dimer that belongs to the papain superfamily of cysteine proteases, with a conserved catalytic machinery. Moreover, the structure of the protein solved as a complex with GSH at a 1.4-Å resolution reveals a γ-glutamyl cysteine acyl–enzyme intermediate stabilized in a cavity of the protein adjacent to a second putative GSH binding site. GSH hydrolase and PCS activities of the enzyme are discussed in the light of both structures.


* cysteine protease
* heavy-metal detoxification
* phytochelatin synthase 

Phytochelatins (PCs) are known to be the main heavy-metal-detoxifying peptides in the plant kingdom (1). PCs are glutathione (GSH)-derived peptides of the general formula (γ-Glu-Cys)nGly (with n = 2–11), which have been identified in a wide variety of plant species and in some microorganisms. They act as heavy metal (mainly Cd2+) chelators and favor their vacuolar sequestration (24). PCs are synthesized posttranslationally in the presence of heavy-metal ions by the PC synthase (PCS), a γ-glutamyl-cysteine transpeptidase (5).

Genes encoding PCS have been isolated and characterized in Arabidopsis thaliana (AtPCS1) (6), Schizosaccharomyces pombe (SpPCS) (4), and wheat (Triticum aestivum; TaPCS1) (7) but also in the model nematode Caenorhabditis elegans (CePCS1) (8, 9). Sequence alignment of PCS proteins reveals a high degree of similarity in the N-terminal domain whereas the C-terminal region turns out to be extremely variable. Limited proteolysis and mutant analyses confirmed that the N-terminal core domain is sufficient to confer a PCS activity and therefore can be referred to the catalytic domain (7, 10, 11). However, the C-terminal domain was shown to ensure higher PCS activity, improved protein stability, and response to a broader spectrum of heavy-metal ions (10).

Several groups have proposed that PCS is a bisubstrate enzyme, in which the donor molecule is the GSH and the acceptor is another GSH molecule or a PCn molecule (12). Recently, Rea and coworkers (13) have gone further and showed by in vitro radiolabeling experiments on AtPCS1 that this class of enzyme corresponds to a dipeptidyl transferase whose mechanism takes place in two distinct steps: (i) the formation of a γ-glutamyl-cysteine acyl–enzyme intermediate resulting from the GSH deglycination followed by (ii) the transfer of the γ-glutamylcysteine unit from the acyl–enzyme to the acceptor molecule (GSH or PCn).

After the pioneering in vivo experiments of Zenk and coworkers (1, 14), several groups have shown in vitro in various organisms that no significant activity was detectable in the absence of Cd2+ or other heavy-metal ions (46). However, the specific role of divalent metals in PC synthesis is still controversial. Cobbett (12) first proposed that the variable C-terminal domain would bind, via conserved cysteine residues, the heavy-metal ions and then transfer them to the catalytic N-terminal domain. Later, Maier et al. (15) performed Cd-binding assays using peptide libraries of PCS and found that five conserved cysteines in the N-terminal domain of AtPCS1 could bind Cd2+ and were also essential for the PCS activity. Rea and coworkers (13, 16) suggested another model, in which Cd2+ does not directly bind to the protein but rather forms a heavy-metal–GS2 (or PCn) thiolate complex, which would act as the acceptor molecule. According to those authors, three conserved residues in the N-terminal domain, Cys-56, His-162, and Asp-180, when substituted by site-directed mutagenesis, completely abolish the activity of AtPSC1 (17). Their actual assumption is that the first γ-Glu-Cys acyl intermediate of reaction is a thioester, resulting from the nucleophilic attack of Cys-56 on the GSH Cys—Gly peptidic bond. Also based on sequence homology, Rea et al. (17) proposed that the PCS mechanism could be similar to that of papain and, more generally, cysteine proteases. The PCS and cysteine protease mechanisms would only differ in their second step, i.e., the nucleophilic attack on the thioester intermediate: in the PCS case, the acceptor molecule is a second GSH molecule or a PCn molecule, and, in the cysteine protease case, the acceptor is a water molecule, resulting in a net hydrolysis. Such a mechanism is compatible with the ability of PCS to catalyze the cleavage of glycine from GS-conjugated xenobiotics, thus contributing to their degradation (18).

To go beyond these first insights into the PCS mechanism, the three-dimensional structure of PCS or of its nearest homologues is greatly needed. No structural information on PCS is currently available. Recent sequencing has revealed a PCS-related gene in the genome of cyanobacteria (19, 20). The corresponding alr0975 gene derived from Nostoc encodes a protein (called NsPCS) sharing 36% identity at the amino acid level with AtPCS1. The sequence contains only the conserved N-terminal domain of the PCS eukaryotic family. NsPCS has been found to catalyze essentially the first step in PC synthesis, which converts GSH to γ-glutamyl cysteine, whereas only weakly net synthesis of PC2 occurs, i.e., the transfer of the γ-glutamylcysteine unit from the acyl–enzyme to another GSH molecule. No significant difference on the NsPCS activity was found in the presence or in the absence of heavy-metal ions (11, 19, 20). Here, we report crystal structures of (i) NsPCS at 2.0-Å resolution and (ii) the γ-glutamyl cysteine acyl–enzyme intermediate at 1.4-Å resolution. To our knowledge, this enzyme–substrate complex structure under its acyl form is the first one reported for a cysteine protease. The overall structure and a detailed analysis of the active site are described. Our results confirm the previous prediction that PCS belongs to the papain superfamily of cysteine proteases, with the structurally conserved “catalytic triad” and oxyanion hole in the active site. Both structures also cast new light on the putative role of the other eukaryotic conserved cysteines and on the origin of the differences between the cyanobacterial and the eukaryotic PCS catalytic activities. Overall, these structures contribute to a better understanding of the PCS mechanism and provide a structural framework for further functional studies.


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