Abstract
Glutathione is a ubiquitous thiol tripeptide in land plants, and glutathione-like tripeptides can also be found in some plant species. Rice (Oryza sativa) plants synthesize hydroxymethyl-glutathione, in which the terminal glycine residue of glutathione is replaced by a serine residue; however, the biosynthetic pathway of hydroxymethyl-glutathione has not been identified. We isolated three rice glutathione synthetase homologs, designated OsGS1, OsGS2, and OsGS3, and found that knockdown of OsGS2 via RNA interference markedly decreased hydroxymethyl-glutathione concentration in rice plants. The in vitro enzyme assay, using purified recombinant protein, demonstrated that OsGS2 catalyzed the synthesis of hydroxymethyl-glutathione from γ-glutamylcysteine (γEC) and L-serine in an ATP-dependent manner. OsGS2 could also utilize glycine as a cosubstrate with γEC, but the enzyme-substrate affinity for L-serine was tenfold higher than that for glycine. These results indicate that OsGS2 codes for hydroxymethyl-glutathione synthetase.