7WZD

Crystal Structure of cis-4,5-dihydrodiol phthalate dehydrogenase from Comamonas testosteroni KF1

Summary for 7WZD

• Entry DOI: 10.2210/pdb7wzd/pdb
• Descriptor: 4,5-dihydroxyphthalate dehydrogenase, GLYCEROL (3 entities in total)
• Functional Keywords: dehydrogenase, bacterial protein, nad cofactor, oxidoreductase
• Biological source: Comamonas testosteroni KF-1
• Total number of polymer chains: 2
• Total formula weight: 86369.63

Authors

Sharma, M.,Mahto, J.K.,Kumar, P. (deposition date: 2022-02-17, release date: 2022-09-14, Last modification date: 2023-11-29)

Primary citation

Mahto, J.K.,Sharma, M.,Neetu, N.,Kayastha, A.,Aggarwal, S.,Kumar, P.
Conformational flexibility enables catalysis of phthalate cis-4,5-dihydrodiol dehydrogenase.
Arch.Biochem.Biophys., 727:109314-109314, 2022

PubMed Abstract: 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). With biochemical experiments, we have determined the enzyme's catalytic efficiency (k/K) with DDP as 2.6 ± 0.5 Ms, 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 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 crystal structures show that the catalytic domain of PhtC 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 and related structural homologs, a reaction mechanism is proposed. Finally, with the biochemical analysis of a variant M251LPhtC, the broader substrate specificity of this enzyme is explained.

PubMed: 35667443
DOI: 10.1016/j.abb.2022.109314

Experimental method

X-RAY DIFFRACTION (2.8 Å)