Conformational flexibility enables catalysis of phthalate cis-4,5-dihydrodiol dehydrogenase

Phthalate cis-4,5-dihydrodiol dehydrogenase (PhtC), the second enzyme of the phthalate catabolic pathway, catalyzes the dehydrogenation of cis-4,5-dihydrodiol phthalate (DDP). Here, we report the structural and biochemical characterization of PhtC from Comamonas testosteroni KF1 (PhtC_KF1). With biochemical experiments, we have determined the enzyme's catalytic efficiency (k_cat/K_m) with DDP as 2.6 ± 0.5 M^-1s^-1, over 10-fold higher than with cis-3,4-dihydrodiol phthalate (CDP). To understand the structural basis of these reactions, the crystal structures of PhtC_KF1 in apo-form, the binary complex with NAD⁺, and the ternary complex with NAD⁺ and 3-hydroxybenzoate (3HB) were determined. These crystal structures reveal that the binding of 3HB induces a conformational change in the substrate-binding loop. This conformational change causes the opening of the NAD ⁺ binding site while trapping the 3HB. The PhtC_KF1 crystal structures show that the catalytic domain of PhtC_KF1 is larger than that of other structurally characterized homologs and does not align with other cis-diol dehydrogenases. Structural and mutational analysis of the substrate-binding loop residues, Arg164 and Glu167 establish that conformational flexibility of this loop is necessary for positioning the substrate in a catalytically competent pose, as substitution of either of these residues to Ala did not yield the dehydrogenation activity. Further, based on the crystal structures of PhtC_KF1 and related structural homologs, a reaction mechanism is proposed. Finally, with the biochemical analysis of a variant M251LPhtC_KF1, the broader substrate specificity of this enzyme is explained.