Beyond peptidase activity of cyanobacterial phytochelatin synthases: the case of Nostoc sp., Gleiterinema sp., Gloeobacter violaceous

The enzyme phytochelatin synthase (PCS) is a γ-glutamylcysteine dipeptidyl (trans)peptidase (EC 2.3.2.15), belonging to the clan CA of the papain-like cysteine proteases. The PCS catalyzes the prompt formation of some peculiar thiol-peptide compounds, the so-called “phytochelatins”, starting from the reduced form of glutathione (GSH) via a transpeptidation reaction. Phytochelatins (PCs) are thiol-peptide compounds whose general structure is (γ-glutamate–cysteine)n–glycine, with n usually ranging from 2 to 5 (Grill et al., 1985). Due to the thiol group of the cysteine residues, PCs can bind cadmium (Cd) and other thiophilic heavy metals and prevent them from circulating in the cytosol, thus dramatically reducing their toxicity. It is now well known that land plants, as well as several marine and freshwater algae (e.g. some members of Chlorophyta, Chrysophyceae, Phaeophyceae, Rhodophyta), some fungi, lichens and even some animal species do actually produce PCs in response to heavy metal stress, in particular Cd. PCS is indeed of particular interest from an evolutionary prospect, due to its constitutive expression and its widespread presence in nature. Recently, some PCS-like enzymes, sharing significant sequence homologies with land plant PCSs, were identified in cyanobacteria and in some gamma- and beta- proteobacteria. Previous evidences suggested that predicted product of PCS gene of Nostoc sp. (alr0975) contains the conserved N-terminal domain, but not the variable C-terminal domain found in eukaryotic PCSs (Tsuji et al., 2004; Vivares et al., 2005). Proteins encoded by the cyanobacterial genes seem to be primitive forms of PCS and to represent an early stage in the evolution of the enzyme in photoautotrophic organisms, since up to now only peptidase and almost absent transpeptidase activities have been reported for cyanobacteria (Tsuji et al., 2005). However, studies on functional characterization of prokaryotic PCS are scant, despite their importance as fundamental landmarks in evolution. Thus, the aim of our study is to investigate possible functional and molecular differences between phototrophic eukaryotic and prokaryotic PCS, by studying the enzymes in three cyanobacterial strains (Nostoc sp. PCC 7120, Geitlerinema sp. PCC 7407 and Gloeobacter violaceous PCC 7421). Preliminary HPLC-mass-spectrophotometry results evidenced a noteworthy PC synthesis in all the strains upon Cd-exposure, thus demonstrating that cyanobacterial PCS-like enzymes do possess transpeptidase activity, likewise eukaryotic PCSs. This evidence highlights a remarkable evolutionary conservation of PCS functionality between cyanobacteria, algae and land plants.