Structural and Functional Characterization of a Novel 4,5-Dihydroxyphthalate Decarboxylase for Protocatechuic Acid Biosynthesis

Phthalate esters (PAEs) constitute a widespread class of xenobiotic contaminants characterized by environmental persistence and ecological recalcitrance. Under aerobic conditions, PAE mineralization proceeds via 4,5-dihydroxyphthalate (4,5-DHP) as a key metabolic intermediate, which undergoes stereospecific decarboxylation to protocatechuate (PCA), a versatile pharmacophore with broad therapeutic potential. Here, we elucidate the molecular architecture and catalytic determinants of a previously uncharacterized 4,5-DHP decarboxylase (DhpD) isolated from ultramicrobacteria. Enzymatic assays revealed that PAE-UM2851 exhibits peak activity at pH 7.5 and 45 °C, with a <i>K</i>_m of 911.5 μM. Functional characterization confirmed that PAE-UM2851 catalyzes the decarboxylation of 4,5-DHP to PCA, supported by FTIR spectral evidence showing reduced - CO (1640 cm^-1) and - OH (3450 cm^-1) vibrational modes. Structure-function analysis identified five evolutionarily conserved residues (Ser57, Arg84, Thr115, Lys150, and His223) essential for regulating regioselective decarboxylation. This study not only clarifies the catalytic mechanism of DhpD but also outlines a potential biosynthetic route for PCA.