Characterization of phytochelatin synthase produced by the primitive red alga Cyanidioschyzon merolae.

Phytochelatins (PCs), non-protein peptides with the general structure [(γ-Glu-Cys)n-Gly (n≥ 2)], are involved in the detoxification of toxic heavy metals mainly in higher plants. The synthesis of the peptides is mediated by phytochelatin synthase (PCS), which is activated by a range of heavy metals. CmPCS, a PCS-like gene found in the genomic DNA of the primitive red alga Cyanidioschyzon merolae, was isolated and a recombinant protein (rCmPCS) fused with a hexahistidine tag at the N-terminus of CmPCS was produced. The finding that this protein mediated PC synthesis from glutathione in a metal-dependent way clearly establishes that rCmPCS is functional. The maximum activity was attained at a reaction temperature of 50 °C, considerably higher than the temperature required for the maximal activity of PCS isolated from the higher plant Silene cucubalus, probably due to the alga being a thermophile. CmPCS showed optimal pH in a slightly higher region than higher plant PCSs, probably due to the less effective charge relay network in the catalytic triad. In addition, the pattern of enzyme activation by metal ions was specific to rCmPCS, with Ag+, Cu2+, and Hg2+ showing only limited activation. In contrast to other eukaryotic PCSs, CmPCS has an extra domain in the N-terminal region from residues 1 to 109, and contains fewer cysteine residues in the C-terminal domain. These differences may be responsible for the metal specificity of the activation of CmPCS. Although the enzyme preparation lost PCS activity progressively when stored at 4 °C, the inclusion of Cd2+ in the preparation effectively prevented the reduction of activity. Furthermore, Cd2+ effectively restored the activity of the inactivated enzyme. These results indicate that Cd2+ ions bind the enzyme to maintain the structural integrity of the peptides.

Dequenching of Cu(I)-bathocuproine disulfonate complexes for high-performance liquid chromatographic determination of phytochelatins, heavy-metal-binding peptides produced by the primitive red alga Cyanidioschyzon merolae.

A novel method has been devised for the determination of phytochelatins (PCs), heavy-metal-tolerant peptides produced by higher plants and algae. The method is based on the facts that fluorescence of bathocuproine disulfonate (BCS) is quenched by Cu(I) ions as a result of Cu(I)-BCS complex formation and that PCs compete with BCS for Cu(I). Detection of PCs via recovered fluorescence of BCS using the Cu(I)-BCS complex as a postcolumn reagent, following separation of peptides on an octyldecylsilane column, demonstrated a highly sensitive method for determination of PCs. PCs in the primitive red alga, Cyanidioschyzon merolae, grown in the presence or absence of added Cd(II) were successfully determined by this protocol. Unlike other methods for the determination of PCs, which rely on the SH groups in the peptides, the proposed method is unique in that detection is based on the chemical nature of PCs, which favors the formation of complexes with Cu(I). In this context, the new method yields chromatograms based on the strength of binding Cu(I) ions.