Structural and biochemical insights into isophthalate dihydrodiol dehydrogenase from Comamonas testosteroni KF1

Isophthalate serves as a carbon and energy source for several microorganisms through a dedicated catabolic pathway, in which isophthalate dihydrodiol dehydrogenase (IphB) mediates the NAD( P ) ⁺ -dependent oxidation of 1,2-dihydroxy-3,5-cyclohexadiene-1,5-dicarboxylate (1,5-DCD) to protocatechuate. Here, we present the structural and biochemical characterization of isophthalate dihydrodiol dehydrogenase (IphB _KF1 ) from Comamonas testosteroni KF1. Enzyme kinetics revealed IphB _KF1 catalyzes the dehydrogenation of 1,5-DCD substrate with both NAD ⁺ and NADP ⁺ , exhibiting a ~4 fold higher catalytic efficiency ( k _cat / K _M ) with NADP ⁺ . Crystal structures of IphB _KF1 were determined in the apo form, in complex with NADP ⁺ , and as a ternary complex with NADP ⁺ and the product 3,4-dihydroxybenzoic acid (3,4-DHB; protocatechuate) at resolutions of 1.5, 1.8, and 1.5 Å, respectively. Structural analysis reveals an NADP ⁺ -induced conformational change that facilitates cofactor accommodation and identifies key residues involved in NADP ⁺ binding. Sequence and structural analyses highlight two conserved motifs, GXXXGXG and YXXXK, characteristic of the short-chain dehydrogenase/reductase (SDR) superfamily. Ternary complex of IphB _KF1 (NADP ⁺ and 3,4-DHB) and molecular docking with 1,5-DCD suggests that specific active-site residues contribute to substrate recognition and underpin the distinct substrate specificity of IphB _KF1 compared to other SDR family members. Based on structural insights and comparison with homologous enzymes, we propose a catalytic mechanism for isophthalate oxidation. Together, these findings provide a comprehensive framework for understanding substrate specificity and catalysis in isophthalate dihydrodiol dehydrogenases.