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Open AccessArticle
Conformational Dynamics of the Active Site Loop in Dihydroorotase Highlighting the Limitations of Loop-In Structures for Inhibitor Docking
by
Yen-Hua Huang
Yen-Hua Huang
,
Tsai-Ying Huang
Tsai-Ying Huang
Man-Cheng Wang
Man-Cheng Wang
Cheng-Yang Huang
Cheng-Yang Huang
1
Department of Biomedical Sciences, Chung Shan Medical University, Taichung City 40201, Taiwan
2
The Affiliated Senior High School of National Chung Hsing University, Taichung City 412011, Taiwan
3
Department of Medical Research, Chung Shan Medical University Hospital, Taichung City 40201, Taiwan
*
Author to whom correspondence should be addressed.
Int. J. Mol. Sci. 2025, 26(19), 9688; https://doi.org/10.3390/ijms26199688 (registering DOI)
Submission received: 29 August 2025
/
Revised: 29 September 2025
/
Accepted: 3 October 2025
/
Published: 4 October 2025
Abstract
Dihydroorotase (DHOase) catalyzes the reversible cyclization of N-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 Saccharomyces cerevisiae 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, S. cerevisiae 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.
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MDPI and ACS Style
Huang, Y.-H.; Huang, T.-Y.; Wang, M.-C.; Huang, C.-Y.
Conformational Dynamics of the Active Site Loop in Dihydroorotase Highlighting the Limitations of Loop-In Structures for Inhibitor Docking. Int. J. Mol. Sci. 2025, 26, 9688.
https://doi.org/10.3390/ijms26199688
AMA Style
Huang Y-H, Huang T-Y, Wang M-C, Huang C-Y.
Conformational Dynamics of the Active Site Loop in Dihydroorotase Highlighting the Limitations of Loop-In Structures for Inhibitor Docking. International Journal of Molecular Sciences. 2025; 26(19):9688.
https://doi.org/10.3390/ijms26199688
Chicago/Turabian Style
Huang, Yen-Hua, Tsai-Ying Huang, Man-Cheng Wang, and Cheng-Yang Huang.
2025. "Conformational Dynamics of the Active Site Loop in Dihydroorotase Highlighting the Limitations of Loop-In Structures for Inhibitor Docking" International Journal of Molecular Sciences 26, no. 19: 9688.
https://doi.org/10.3390/ijms26199688
APA Style
Huang, Y.-H., Huang, T.-Y., Wang, M.-C., & Huang, C.-Y.
(2025). Conformational Dynamics of the Active Site Loop in Dihydroorotase Highlighting the Limitations of Loop-In Structures for Inhibitor Docking. International Journal of Molecular Sciences, 26(19), 9688.
https://doi.org/10.3390/ijms26199688
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