Conformational Dynamics of the Active Site Loop in Dihydroorotase Highlighting the Limitations of Loop-In Structures for Inhibitor Docking

Dihydroorotase (DHOase) catalyzes the reversible cyclization of <i>N</i>-carbamoyl-L-aspartate to dihydroorotate, a key step in de novo pyrimidine biosynthesis. A flexible active site loop in DHOase undergoes conformational switching between loop-in and loop-out states, influencing substrate binding, catalysis, and inhibitor recognition. In this study, we identified 5-fluoroorotate (5-FOA) and myricetin as inhibitors of <i>Saccharomyces cerevisiae</i> DHOase and systematically analyzed 97 crystal structures and AlphaFold 3.0 models of DHOases from 16 species representing types I, II, and III. Our results demonstrate that loop conformation is not universally ligand-dependent and varies markedly across DHOase types, with type II enzymes showing the greatest flexibility. Notably, <i>S. cerevisiae</i> DHOase consistently adopted the loop-in state, even with non-substrate ligands, restricting accessibility for docking-based inhibitor screening. Docking experiments with 5-FOA and myricetin confirmed that the loop-in conformation prevented productive active-site docking. These findings highlight the importance of selecting appropriate loop conformations for structure-based drug design and underscore the need to account for loop dynamics in inhibitor screening.