A Medicago truncatula Homoglutathione Synthetase Is Derived from Glutathione Synthetase by Gene Duplication

Abstract

Glutathione (GSH) and homo-GSH (hGSH) are the major low-molecular weight thiols synthesized in Medicago truncatula. Two M. truncatula cDNAs (gshs1 and gshs2) corresponding to a putative GSH synthetase (GSHS) and a putative hGSH synthetase (hGSHS) were characterized. Heterologous expression of gshs1 and gshs2 cDNAs in an Escherichia coli strain deficient in GSHS activity showed that GSHS1 and GSHS2 are a GSHS and an hGSHS, respectively. Leucine-534 and proline-535 present in hGSHS were substituted by alanines that are conserved in plant GSHS. These substitutions resulted in a strongly stimulated GSH accumulation in the transformed E. coli strain showing that these residues play a crucial role in the differential recognition of ␤-alanine and glycine by hGSHS. Phylogenetic analysis of GSHS2 and GSHS1 with other eukaryotic GSHS sequences indicated that gshs2 and gshs1 are the result of a gene duplication that occurred after the divergence between Fabales, Solanales, and Brassicales. Analysis of the structure of gshs1 and gshs2 genes shows they are both present in a cluster and in the same orientation in the M. truncatula genome, suggesting that the duplication of gshs1 and gshs2 occurred via a tandem duplication.

Perturbations of Amino Acid Metabolism Associated with Glyphosate-Dependent Inhibition of Shikimic Acid Metabolism Affect Cellular Redox Homeostasis and Alter the Abundance of Proteins Involved in Photosynthesis and Photorespiration

“…In the first reaction, common to the synthesis of both molecules, g-glutamylCys is formed from Glu and Cys by g-glutamylcysteinyl synthetase. In the second reaction, either Gly or b-Ala is added to the C-terminal site of g-glutamyl-Cys by GSH synthetase or hGSH synthetase, respectively (Frendo et al, 1999(Frendo et al, , 2001. Although GSH and hGSH are synthesized by distinct enzymes encoded by different genes (Frendo et al, 2001), the homologs are considered to fulfill similar functions in regulating cellular redox state (Noctor et al, 2011).…”

Structural Basis for Evolution of Product Diversity in Soybean Glutathione Biosynthesis

“…Although the interplay between genomes, protein function, and a plant's environment shapes the evolution of new metabolism, it is unclear why legumes required evolution of hGS and hGSH production in nodules. Aside from the shared localization of hGS in nodules (Moran et al, 2000;Frendo et al, 2001;Iturbe-Ormaetxe et al, 2002;Skipsey et al, 2005), there appears to be no correlation between the presence of hGS in a legume species and the position of that species in the legume phylogeny (Wojciechowski et al, 2004). Nonetheless, given the conservation of hGS in the legumes examined so far, it seems likely that environmental factors, such as nodulation and/or habitat, contributed to the diversification of GSH metabolism.…”

“…In each enzyme, residues in the Ala-rich loop contact the terminal residue of the tripeptide product ( Figure 5). A Leu and Pro in the hGS from soybean and other legumes replaces the invariant double Ala sequence of the eukaryotic GS (Moran et al, 2000;Frendo et al, 2001;Iturbe-Ormaetxe et al, 2002;Skipsey et al, 2005). Structurally, the Ala-rich loop of hGS shifts relative to the same loop in GS to allow space for binding of the larger hGSH product and b-Ala substrate ( Figure 5A).…”

“…The flexible and mutable nature of loops allows for the sampling of the new sequences and localized structures that generate shifts in substrate specificity or new catalytic activity. Frendo et al (2001) originally proposed that legumes evolved hGS from gene duplication of GS after the divergence of the order Fabales, which includes the legumes, from other flowering plants. Our results suggest a molecular mechanism underpinning the evolution of hGS from GS.…”

“…In nearly all organisms, glutathione synthetase (GS) catalyzes the addition of Gly to gEC (Meister, 1995;Herrera et al, 2007). In legumes, homoglutathione synthetase (hGS) uses b-Ala instead of Gly to form hGSH (Matamoros et al, 1999;Frendo et al, 2001;Iturbe-Ormaetxe et al, 2002). Although GS and hGS share similar reaction mechanisms based on biochemical and structural studies, the molecular basis for the difference in substrate specificity is unclear due to no available structural data for any plant GS or hGS.…”