Elucidating the Molecular Determinants of Substrate Specificity in Salicylate 1,2-Dioxygenase: A QM/MM Investigation of Salicylate and Gentisate.

Salicylate 1,2-dioxygenase (SDO), a ring-fission nonheme dioxygenase enzyme, catalyzes the regioselective oxidation of gentisic acid (GTQ) and salicylic acid (SAL) in the presence of molecular oxygen. Wild-type SDO from Pseudaminobacter salicylatoxidans exhibits higher catalytic efficiency with GTQ compared to SAL. To elucidate the factors underlying this difference, classical molecular dynamics simulations were performed on wild-type SDO complexed with GTQ and SAL. The simulations revealed that a water molecule anchored by the ARG127 residue adjacent to molecular oxygen is present with the GTQ substrate, unlike with SAL. Further, hybrid quantum mechanics/molecular mechanics calculations indicated that alkylperoxo intermediate formation is more favorable with GTQ, highlighting the crucial role of the 5́'-OH group, which is absent in SAL, in SDO's differential catalytic activity.