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.IMPORTANCEIsophthalate is a recalcitrant aromatic compound linked to the environmental release of phthalate-based plastics and plasticizers. However, the microbial degradation of isophthalate is relatively less known in comparison to other phthalate isomers. Efficient bacterial utilization of isophthalate requires a coordinated pathway in which the cis-dihydrodiol dehydrogenase IphB converts intermediates into protocatechuate, a central carbon metabolism intermediate. Despite its importance, the structural basis of this transition has remained unclear. Here, we provide a comprehensive structural and biochemical investigation of isophthalate dihydrodiol dehydrogenase (IphB_KF1), revealing the structural basis for cofactor binding, active-site architecture, and conformational dynamics leading to the selective processing of isophthalate-derived substrates. These findings provide a framework for the design of enzymes with improved activity and selectivity for pollutant removal.