Decoding regioselective reaction mechanism of gentisic acid catalyzed by the gentisate 1,2-dioxygenase enzyme

Abstract

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 hydrocarbon (PAH) 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 a pivotal role in facilitating the oxidative cleavage of an aromatic C–C bond of the GTQ substrate. Three distinct reaction mechanisms using QM/MM calculations have been unearthed to comprehend the regioselective oxidation of GTQ catalyzed by the GDO enzyme. The formation of the main product maleylpyruvate along with pathway A is the most favourable one. The first step for the conversion to an alkyl peroxo intermediate is the rate-determining step with an associated barrier of 21.4 kcal mol⁻¹ 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, and the O–O and C–C bond cleavage steps and additionally uncovered the important anchoring role of Asp174 in the enzymatic cycle. Essentially, our findings paved a new route in the mechanism of degradation processes of PAH pollutants by dioxygenase enzymes, and provide molecular insights into the design of iron-containing biomimetic catalysts.