Structural basis of potent inhibition of the human CAD dihydroorotase domain by 5-aminoorotic acid

Dihydroorotase (DHOase) catalyzes the reversible conversion of N-carbamoyl-L-aspartate to dihydroorotate in de novo pyrimidine biosynthesis. In humans, DHOase (huDHOase) is part of the CAD enzyme complex and contains a flexible loop that alternates between loop-in and loop-out conformations. Here, we identify 5-aminoorotic acid (5-AOA), previously known as a dihydroorotate dehydrogenase inhibitor, as a potent huDHOase inhibitor. Enzyme assays showed strong inhibition by 5-AOA (IC50 = 9.87 μM) compared with weaker inhibition by 5-fluoroorotic acid (5-FOA; IC50 = 191.59 μM). To elucidate the mechanism, we determined the crystal structures of huDHOase bound to 5-AOA (1.83 Å; PDB ID: 9WIC) and to 5-FOA (1.55 Å; PDB ID: 9WIN) for direct comparison. Structural analysis revealed distinct binding modes: 5-AOA bound in the loop-in conformation, forming extensive stabilizing interactions, whereas 5-FOA bound in the loop-out state with fewer contacts. Consistently, the T1562A mutation decreased the binding affinity of 5-AOA from 18.6 μM to 53.4 μM, an approximately threefold reduction, confirming the role of the loop in inhibitor recognition. These findings establish 5-AOA as a dual inhibitor in pyrimidine biosynthesis and highlight a loop-in binding mechanism for huDHOase inhibition.