Decoding Regioselective Reaction Mechanism of the Gentisic Acid Catalyzed by Gentisate 1,2-Dioxygenase Enzyme

Gentisate 1,2-dioxygenase (GDO), a ring-fission non-heme dioxygenase enzyme, displays a unique regioselective reaction of gentisic acid (GTQ) in the presence of molecular oxygen. GTQ is an important intermediate in the aerobic biodegradation pathways of recalcitrant polyaromatic hydrocarbons (PAHs) pollutants. Classical molecular dynamics simulations of wild-type GDO and its mutated variants (Asp174Glu and Asp174Ala) explored the presence of three active water molecules at the active site which plays pivotal roles in facilitating the oxidative cleavage of an aromatic C-C bond of the GTQ substrate. Three distinct reaction mechanisms using the QM/MM calculations decoded for the regioselective reaction of the GTQ catalyzed by GDO enzyme. The formation of the main product as a maleylpyruvate along with pathway A, which is the most favourable one. The first step for the conversion to an alkyl peroxo intermediate is a rate-determining step with an associated barrier of 21.4 kcal/mol at the uB3LYP-D3/def2-TZVP/OPLS level of theory on the quintet spin surface. Our study illustrates the crucial role of active water molecules in the stabilization of the O2 molecule, the O-O, and C-C bond cleavage steps and additionally uncovered the important anchor role of the Asp174 in the enzymatic cycle. Essentially, our findings paved a new route in the mechanism of degradation processes of PAHs pollutants by dioxygenase enzymes, and provide molecular insights to design iron-containing biomimetic catalysts.