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Issue 3, 2014
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Zn-bis-glutathionate is the best co-substrate of the monomeric phytochelatin synthase from the photosynthetic heavy metal-hyperaccumulator Euglena gracilis

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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 Km values of 14–22 mM for GSH and 1.6–2.5 μM for metal-bis-glutathionate (Me-GS2). EgPCS showed the highest Vmax 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.

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

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Supplementary files

Article information


Submitted
18 Oct 2013
Accepted
11 Dec 2013
First published
12 Dec 2013

Metallomics, 2014,6, 604-616
Article type
Paper

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

J. D. García-García, L. Girard, G. Hernández, E. Saavedra, J. P. Pardo, J. S. Rodríguez-Zavala, R. Encalada, A. Reyes-Prieto, D. G. Mendoza-Cózatl and R. Moreno-Sánchez, Metallomics, 2014, 6, 604
DOI: 10.1039/C3MT00313B

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