Glutathione biosynthesis in bacteria by bifunctional GshF is driven by a modular structure featuring a novel hybrid ATP-grasp fold.

Glutathione is an intracellular redox-active tripeptide thiol with a central role in cellular physiology across all kingdoms of life. Glutathione biosynthesis has been traditionally viewed as a conserved process relying on the sequential activity of two separate ligases, but recently, an enzyme (GshF) that unifies both necessary reactions in one platform has been identified and characterized in a number of pathogenic and free-living bacteria. Here, we report crystal structures of two prototypic GshF enzymes from Streptococcus agalactiae and Pasteurella multocida in an effort to shed light onto the structural determinants underlying their bifunctionality and to provide a structural framework for the plethora of biochemical and mutagenesis studies available for these enzymes. Our structures reveal how a canonical bacterial GshA module that catalyzes the condensation of L-glutamate and L-cysteine to γ-glutamylcysteine is linked to a novel ATP-grasp-like module responsible for the ensuing formation of glutathione from γ-glutamylcysteine and glycine. Notably, we identify an unprecedented subdomain in the ATP-grasp module of GshF at the interface of the GshF dimer, which is poised to mediate intersubunit communication and allosteric regulation of enzymatic activity. Comparison of the two GshF structures and mapping of structure-function relationships reveal that the bifunctional GshF structural platform operates as a dynamic dimeric assembly.