Zn-bis-glutathionate is the best co-substrate of the monomeric phytochelatin synthase from the photosynthetic heavy metal-hyperaccumulator Euglena gracilis.
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 Zn(2+) or Cd(2+), 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 Cd(2+), whereas its heterologous expression in ycf-1 yeast cells provided resistance to Cd(2+) 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.
Departamento de Bioquímica, Instituto Nacional de Cardiología, Juan Badiano No. 1, Sección XVI, Tlalpan, México D.F. 14080, México. rafael.moreno@cardiologia.org.mx morenosanchez@hotmail.com.
Jorge D
JD
García-García
Lourdes
L
Girard
Georgina
G
Hernández
Emma
E
Saavedra
Juan P
JP
Pardo
José S
JS
Rodríguez-Zavala
Rusely
R
Encalada
Adrián
A
Reyes-Prieto
David G
DG
Mendoza-Cózatl
Rafael
R
Moreno-Sánchez
D000595:Amino Acid Sequence; D019881:Aminoacyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005056:Euglena gracilis / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D005978:Glutathione / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D010802:Phylogeny; D016415:Sequence Alignment; D015032:Zinc / Q000737:chemistry / Q000378:metabolism*
D019881:Aminoacyltransferases; C093784:glutathione gamma-glutamylcysteinyltransferase; D005978:Glutathione; D015032:Zinc