Structural and Mechanistic Insights into Aspartate Decarboxylase PanD and a Pyrazinoic Acid-Derived Inhibitor.

() aspartate decarboxylase PanD is required for biosynthesis of the essential cofactor coenzyme A and targeted by the first line drug pyrazinamide (PZA). PZA is a prodrug that is converted by a bacterial amidase into its bioactive form pyrazinoic acid (POA). Employing structure-function analyses we previously identified POA-based inhibitors of PanD showing much improved inhibitory activity against the enzyme. Here, we performed the first structure-function studies on PanD encoded by the nontuberculous mycobacterial lung pathogen (), shedding light on the differences and similarities of and PanD. Solution X-ray scattering data provided the solution structure of the entire tetrameric PanD, which in comparison to the structure of the derived C-terminal truncated PanD mutant revealed the orientation of the four flexible C-termini relative to the catalytic core. Enzymatic studies of PanD explored the essentiality of the C-terminus for catalysis. A library of recombinant PanD mutants based on structural information and PZA/POA resistant PanD mutations in illuminated critical residues involved in the substrate tunnel and enzymatic activity. Using our library of POA analogues, we identified (3-(1-naphthamido)pyrazine-2-carboxylic acid) (analogue 2) as the first potent inhibitor of PanD. The inhibitor shows mainly electrostatic- and hydrogen bonding interaction with the target enzyme as explored by isothermal titration calorimetry and confirmed by docking studies. The observed unfavorable entropy indicates that significant conformational changes are involved in the binding process of analogue 2 to PanD. In contrast to PZA and POA, which are whole-cell inactive, analogue 2 exerts appreciable antibacterial activity against the three subspecies of .