Catalytic Mechanism of the Hotdog-Fold Thioesterase PA1618 Revealed by X-ray Structure Determination of a Substrate-Bound Oxygen Ester Analogue Complex.

Thioesterase activity accounts for the majority of the activities in the hotdog-fold superfamily. The structures and mechanisms of catalysis for many hotdog enzymes have been elucidated by X-ray crystallography and kinetics to probe the specific substrate usage and cellular functions. However, structures of hotdog thioesterases in complexes with substrate analogues reported to date utilize ligands that either represent truncations of the substrate or include additional atoms to prevent hydrolysis. Here we present the synthesis of an isosteric and isoelectronic substrate analogue-benzoyl-OdCoA-and the X-ray crystal structure of a complex of the analogue with Pseudomonas aeruginosa hotdog thioesterase PA1618 (at 1.72 Å resolution). The complex is compared with that of the "imperfect" substrate analogue phenacyl-CoA, refined to a resolution of 1.62 Å. Kinetic and structural results are consistent with Glu64 as the catalytic residue and with the involvement of Gln49 in stabilization of the transition state. Structural comparison of the two ligand-bound structures revealed a crucial ordered water molecule coordinated in the active site of the benzoyl-OdCoA structure but not present in the phenacyl-CoA-bound structure. This suggests a general base mechanism of catalysis in which Glu64 activates the coordinated water nucleophile. Together, our findings reveal the importance of a closely similar substrate analogue to determine the true substrate binding and catalytic mechanism.