Zn-bis-glutathionate is the best co-substrate of the monomeric phytochelatin synthase from the photosynthetic heavy metal-hyperaccumulator Euglena gracilis

Abstract

The phytochelatin synthase from photosynthetic Euglena gracilis (EgPCS) was analyzed at the transcriptional, kinetic, functional, and phylogenetic levels. Recombinant EgPCS was a monomeric enzyme able to synthesize, in the presence of Zn²⁺ or Cd²⁺, phytochelatin2–phytochelatin4 (PC₂–PC₄) using GSH or S-methyl-GS (S-methyl-glutathione), but not γ-glutamylcysteine or PC₂ as a substrate. Kinetic analysis of EgPCS firmly established a two-substrate reaction mechanism for PC₂ synthesis with K_m values of 14–22 mM for GSH and 1.6–2.5 μM for metal-bis-glutathionate (Me-GS₂). EgPCS showed the highest V_max and catalytic efficiency with Zn-(GS)₂, and was inactivated by peroxides. The EgPCS N-terminal domain showed high similarity to that of other PCSases, in which the typical catalytic core (Cys-70, His-179 and Asp-197) was identified. In contrast, the C-terminal domain showed no similarity to other PCSases. An EgPCS mutant comprising only the N-terminal 235 amino acid residues was inactive, suggesting that the C-terminal domain is essential for activity/stability. EgPCS transcription in Euglena cells was not modified by Cd²⁺, whereas its heterologous expression in ycf-1 yeast cells provided resistance to Cd²⁺ stress. Phylogenetic analysis of the N-terminal domain showed that EgPCS is distant from plants and other photosynthetic organisms, suggesting that it evolved independently. Although EgPCS showed typical features of PCSases (constitutive expression; conserved N-terminal domain; kinetic mechanism), it also exhibited distinct characteristics such as preference for Zn-(GS)₂ over Cd-(GS)₂ as a co-substrate, a monomeric structure, and ability to solely synthesize short-chain PCs, which may be involved in conferring enhanced heavy-metal resistance.