Laboratory evolution of glutathione biosynthesis reveals natural compensatory pathways

The first and highly conserved step in glutathione (GSH) biosynthesis is formation of gamma-glutamyl cysteine by the enzyme glutamate-cysteine ligase (GshA). However, bioinformatic analysis revealed that many prokaryotic species that encode GSH-dependent proteins lack the gene for this enzyme. To understand how bacteria cope without gshA, we isolated Escherichia coli Delta gshA multigenic suppressors that accumulated physiological levels of GSH. Mutations in both proB and proA, the first two genes in L-proline biosynthesis, provided a new pathway for gamma-glutamyl cysteine formation via the selective interception of ProB-bound gamma-glutamyl phosphate by amino acid thiols, likely through an S-to-N acyl shift mechanism. Bioinformatic analysis suggested that the L-proline biosynthetic pathway may have a second role in gamma-glutamyl cysteine formation in prokaryotes. Also, we showed that this mechanism could be exploited to generate cytoplasmic redox buffers bioorthogonal to GSH.