Computational Study on the Reaction Mechanism of LigW-Catalyzed Carboxylation of Monohydroxybenzoic Acids

Abstract

Enzymatic carboxylation of phenols via the Kolbe-Schmitt reaction represents a promising sustainable strategy for $CO_2$ fixation and synthesis of high-value chemicals. This study investigates the reaction mechanism of 5-carboxyvanillate decarboxylase (LigW)-catalyzed carboxylation of non-natural monohydroxybenzoic acids, employing the quantum chemical cluster approach. First, we investigate the carboxylation mechanism of 4-hydroxybenzoate (4-HBA) to produce 4-hydroxyisophthalate (4-HIPA), a dicarboxylic acid with potent antioxidant and neuroprotective applications. The calculations reveal that the $CO_2$-binding mediates the preferred binding mode of the substrates and the incorporation of $CO_2$ to the active site favors the mode beneficial for the following reaction. The chemical reaction is initiated by the formation of a carbon-carbon bond between $CO_2$ and 4-HBA, followed by the rate-limiting proton transfer from the active site residue Asp314 to the resulting intermediate of the first step with a calculated barrier of 19.2 kcal/mol. Additionally, the potential of LigW in catalyzing the carboxylation of 3-hydroxybenzoate (3-HBA) is evaluated, and the calculations show that the reaction is energetically unfeasible due to the prohibitively high barrier of chemical steps. These mechanistic insights, together with the previous studies on the natural substrate, provide important information for the rotational design of LigW variants for industrial biocatalysis and $CO_2$ utilization.