9WIN

Crystal structure of the human dihydroorotase domain complexed with 5-fluoroorotic acid

9WIN の概要

• エントリーDOI: 10.2210/pdb9win/pdb
• 分子名称: CAD protein, 5-FLUORO-2,6-DIOXO-1,2,3,6-TETRAHYDROPYRIMIDINE-4-CARBOXYLIC ACID, ZINC ION, ... (4 entities in total)
• 機能のキーワード: dihydroorotase, human cad, hydrolase, zn
• 由来する生物種: Homo sapiens (human)
• タンパク質・核酸の鎖数: 1
• 化学式量合計: 43066.75

構造登録者

Huang, H.Y.,Wang, M.C.,Huang, C.Y. (登録日: 2025-08-28, 公開日: 2026-07-08)

主引用文献

Huang, Y.H.,Huang, T.Y.,Wang, M.C.,Huang, C.Y.
Structural basis of potent inhibition of the human CAD dihydroorotase domain by 5-aminoorotic acid.
Biochem.Biophys.Res.Commun., 787:152804-152804, 2025

PubMed Abstract: 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 (IC = 9.87 μM) compared with weaker inhibition by 5-fluoroorotic acid (5-FOA; IC = 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.

PubMed: 41101239
DOI: 10.1016/j.bbrc.2025.152804

実験手法

X-RAY DIFFRACTION (1.55 Å)