A His-to-Gln mutation in catechol 2,3-dioxygenase catalyzes 2,3-dihydroxybenzoic acid cleavage by an intradiol pathway

Catechol 2,3-dioxygenases (C23DO) catalyze extradiol cleavage of catechol in aromatic hydrocarbon degradation pathways. Here, we report mutagenesis studies on two C23DO isoenzymes from Diaphorobacter sp. strain DS2, aimed at elucidating the mechanism. Docking studies with various substrates on the two isozymes identified critical active-site residues and second-sphere interactions contributing to substrate binding and catalysis. These modeling studies identified eight site-directed mutants, which were designed, produced, and kinetically characterized. Substitution of histidine-to-glutamine (His-to-Gln) (H206Q64/H200Q68) activates the previously inert substrate 2,3-dihydroxybenzoic acid (2,3-DHBA), allowing its intradiol cleavage under strong acidic conditions. The mutants retained ~20–30% of their native activity toward catechol but exhibited a mechanistic switch from Fe2+-assisted extradiol to Fe3+-mediated intradiol cleavage with 2,3-DHBA. These results highlight the catalytic adaptability of acid–base residues and demonstrate how subtle second-sphere mutation can alter dioxygenase regiospecificity, providing new insights for biocatalyst engineering and environmental bioremediation.