Enzyme-Directed Charge Redistribution Controls Substrate Regioselectivity in CO₂ Fixation by Salicylic Acid...

2,4-Dihydroxybenzoic acid is an important intermediate in agricultural and food chemistry. Salicylic acid decarboxylase catalyzes the carboxylation of resorcinol with CO₂ to yield 2,4-dihydroxybenzoic acid. However, this process suffers from poor regioselectivity and low carboxylation activity. Here, we propose that the unique microenvironment created by the enzyme's substrate-binding pocket, referred to here as the enzyme field, induces charge redistribution in resorcinol, thus altering the enzymatic regioselectivity. Charge distributions at carbon atoms were calculated under 14 different enzyme fields, and catalytic assays revealed the correlation between regioselectivity and the electronegativity of the corresponding carbon atom. Furthermore, K23A/Y64T/E291D/Y27A was obtained with 99% C4-carboxylation selectivity from 20%, and a 69-fold increase in k_cat/K_m. The SAD-catalyzed carboxylation mechanism involving HCO₃^--mediated proton transfer was proposed on the basis of QM/MM metadynamics simulations. This work elucidates the repurposing of regioselectivity by enzyme-induced substrate charge distribution and proposes the mechanism of the SAD-catalyzed carboxylation of resorcinol.