Tetrameric hotdog-fold structure and catalytic mechanism of the SaPaaI thioesterase from Staphylococcus aureus.

Acyl-CoA thioesterases hydrolyse thioester bonds to release free fatty acyl chains and coenzyme A (CoA), thereby regulating lipid metabolism, signaling, and membrane homeostasis. Here, we present the structural and functional characterization of the Paal-like thioesterase SAV0944 (SaPaaI) from Staphylococcus aureus. SaPaaI adopts a class-II hotdog fold comprising six β-strands wrapped around a central α-helix and assembles as a tetrameric dimer-of-dimers, as determined by x-ray crystallography and analytical size-exclusion chromatography. Enzyme assays using a panel of acyl-CoA substrates identify benzoyl-CoA as the preferred substrate. Guided by structural alignment with homologous thioesterases, Gln32 and Glu47 were identified as essential catalytic residues; alanine substitutions at either position abolished activity without perturbing the global fold. The crystal structure revealed asymmetric CoA binding, with only two monomers in the tetramer displaying well-defined ligand density. Comparison of CoA-bound and ligand-free monomers showed that Gln32 undergoes a ~102° conformational rotation that opens the substrate tunnel in the bound state, whereas the corresponding unliganded monomers adopt a closed conformation that sterically occludes the pocket. This mutually exclusive positioning of Gln32 within each dimer provides structural evidence for half-of-the-sites behavior, suggesting that SaPaaI employs a ligand-induced gating mechanism that modulates substrate access. Together, these findings establish SaPaaI as a benzoyl-CoA-selective thioesterase with a noncanonical catalytic configuration and uncover an asymmetric, Gln32-dependent gating mechanism that contributes to substrate specificity in this essential S. aureus enzyme.