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. IMPORTANCE Isophthalate 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.