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All living organisms have a complex network of metabolic pathways for biosynthesis of building blocks for proteins, nucleic acids, and lipids, and breakdown of organic compounds to provide energy and/or sources of carbon, nitrogen, and phosphorous. The assembly of these metabolic pathways was critical for the evolution of the earliest microbes and their divergence into ever more complex and efficient organisms adapted to the varied ecological niches on Earth. We have studied the evolution of the pathway for biosynthesis of glutathione, an important tri-peptide that contributes reducing equivalents in reactions involved in metabolism, regulation, and defense against oxidative stress. To evaluate this hypothesis, we used bioinformatics approaches to analyze sequences of the two enzymes in the glutathione biosynthesis pathway, g-glutamylcysteine ligase (GshA) and glutathione synthetase (GshB). GshA catalyzes the synthesis of g-glutamylcysteine from glutamate and cysteine, while GshB catalyzes the addition of glycine to g-glutamylcysteine to give glutathione.

GshA sequences fall into three distinct groups. Group 1 includes sequences primarily from gamma proteobacteria, Group 2 includes sequences from non-plant eukaryotes, and Group 3 includes sequences primarily from alpha proteobacteria and plants. Although pairwise sequence identities between the groups are insignificant, conserved sequence motifs can be found, suggesting that the proteins are distantly related. Numerous lateral gene transfers are suggested by the data, the most dramatic of which is a transfer from an alpha proteobacterium to a plant more than 300 million years ago. GshB sequences fall into two distinct groups, comprising bacterial and eukaryotic sequences. Proteins in both groups have a common structural fold, but the sequences are so divergent that it is uncertain whether these proteins are homologous or arose by convergent evolution.

Our analysis suggests that the genes in the glutathione biosynthesis pathway were acquired independently. The GshA gene most likely arose in cyanobacteria and was subsequently transferred to other bacteria, eukaryotes, and at least one Archaeon. Because of the high degree of divergence in GshA sequences, the data neither support nor refute the hypothesis that the eukaryotic gene was transferred from the progenitor of mitochondria. After the acquisition of a gene for GshA, eukaryotes and most bacteria apparently recruited a protein with the ATP grasp superfamily structural fold to serve as GshB. It is clear that the eukaryotic GshB did not evolve directly from the bacterial enzyme.