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. 2025 Oct 4;26(19):9688.
doi: 10.3390/ijms26199688.

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

Yen-Hua Huang  1 , Tsai-Ying Huang  1   2 , Man-Cheng Wang  1 , Cheng-Yang Huang  1   3
Affiliations

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

Yen-Hua Huang et al. Int J Mol Sci. .

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.

Keywords: 5-fluoroorotate; active site loop; conformational dynamics; dihydroorotase; docking; myricetin.

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Conflict of interest statement

The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Figures

Figure 1
Figure 1
Domain organization of pyrimidine biosynthesis enzymes and the flexible loop region in DHOase across different species. Type I enzymes (e.g., DHOase from Bacillus anthracis, Ba) have separate CPSase, DHOase, and ATCase subunits. Type II enzymes (e.g., DHOase from E. coli, Ec) also have separate CPSase, DHOase, and ATCase subunits. However, type II DHOases are smaller. In yeast type II (e.g., DHOase from S. cerevisiae), DHOase is encoded separately from the multifunctional URA2 protein that harbors CPSase and ATCase functional domains. Type III (e.g., human CAD) integrates CPSase, DHOase, and ATCase domains into a single polypeptide. The DHOase domains (highlighted in yellow) include the flexible active site loops (marked in cyan, red, or blue) with their respective residue ranges indicated. Loop regions are color-coded: red for loop-out state (E. coli DHOase: residues 105–118; human DHOase: residues 1559–1570), cyan for the shorter loop which may restrict movement (B. anthracis DHOase: residues 153–157), and blue for loop-in state (S. cerevisiae DHOase: residues 101–116).
Figure 2
Figure 2
Identification of 5-FOA and myricetin as inhibitors of yeast DHOase from S. cerevisiae. Under standard assay conditions, varying concentrations of (A) 5-FOA and (B) myricetin were tested for their inhibitory effects on S. cerevisiae DHOase activity. Graphical analysis revealed an IC50 of 12.48 ± 0.47 μM for myricetin, whereas 5-FOA exhibited an IC50 value exceeding 600 μM. Error bars represent the standard deviation from three independent measurements.
Figure 3
Figure 3
Distinct 5-FOA binding modes across different DHOase species: (A) S. cerevisiae, (B) human, and (C) E. coli. Different monomers from the solved DHOase structures are shown, including tetrameric S. cerevisiae (PDB ID 7CA0), monomeric human DHOase (PDB ID 4C6L), and dimeric E. coli DHOase (PDB ID 2EG8). The flexible active site loop is shown in red when adopting the loop-out conformation, and in blue when engaging the ligand in the loop-in conformation. The bound inhibitor 5-FOA is shown in lime green, and the metal ions are depicted as black spheres. In E. coli DHOase, the dashed line represents an unresolved region in the structure, possibly reflecting the dynamic transition of the loop. The loop residues are as follows: S. cerevisiae DHOase (residues 102–116), human DHOase (residues 1560–1569), and E. coli DHOase (residues 106–118).
Figure 4
Figure 4
Docking analysis of myricetin to DHOase. Representative DHOase structures with defined loop conformations were used for docking simulations with myricetin. (A) S. cerevisiae DHOase (PDB ID: 6L0A) in the loop-in conformation. (B) E. coli DHOase (PDB ID: 2EG7), monomer in the loop-in conformation. (C) E. coli DHOase (PDB ID: 2EG7), monomer in the loop-out conformation. (D) Salmonella enterica DHOase (PDB ID: 3JZE), monomer in the loop-in conformation. (E) S. enterica DHOase (PDB ID: 3JZE), monomer in the loop-out conformation. (F) Campylobacter jejuni DHOase (PDB ID: 3PNU), monomer in the loop-in conformation. (G) C. jejuni DHOase (PDB ID: 3PNU), monomer in the loop-out conformation. (H) Yersinia pestis DHOase (PDB ID: 6CTY), monomer in the loop-in conformation. (I) Y. pestis DHOase (PDB ID: 6CTY), monomer in the loop-out conformation. (J) Human DHOase (PDB ID: 8GVZ), loop-in conformation. (K) Human DHOase (PDB ID: 4C6C), loop-out conformation. The flexible active site loop is shown in red for loop-out conformations and in blue for loop-in conformations. Myricetin is represented in melon, and metal ions are depicted as black spheres. The two key residues involved in substrate binding and product release, namely Thr109 and Thr110 in E. coli DHOase or their corresponding residues in other DHOases, are also highlighted using stick representation in the structural models. Dashed lines indicate unresolved loop regions, possibly reflecting loop flexibility or transitions between conformational states.
Figure 5
Figure 5
Docking results of myricetin to S. cerevisiae DHOase. Nine different binding poses were predicted by AutoDock Vina. All poses failed to meet the criteria for successful docking, as none involved interactions with the substrate-binding or metal-binding residues.
Figure 6
Figure 6
Docking analysis of 5-FOA to DHOase. Representative DHOase structures with defined loop conformations were used for docking simulations with 5-FOA. (A) S. cerevisiae DHOase (PDB ID: 6L0A) in the loop-in conformation. (B) E. coli DHOase (PDB ID: 2EG7), monomer in the loop-in conformation. (C) E. coli DHOase (PDB ID: 2EG7), monomer in the loop-out conformation. (D) S. enterica DHOase (PDB ID: 3JZE), monomer in the loop-in conformation. (E) S. enterica DHOase (PDB ID: 3JZE), monomer in the loop-out conformation. (F) C. jejuni DHOase (PDB ID: 3PNU), monomer in the loop-in conformation. (G) C. jejuni DHOase (PDB ID: 3PNU), monomer in the loop-out conformation. (H) Y. pestis DHOase (PDB ID: 6CTY), monomer in the loop-in conformation. (I) Y. pestis DHOase (PDB ID: 6CTY), monomer in the loop-out conformation. (J) Human DHOase (PDB ID: 8GVZ), loop-in conformation. (K) Human DHOase (PDB ID: 4C6C), loop-out conformation. The flexible active site loop is shown in red for loop-out conformations and in blue for loop-in conformations. 5-FOA is represented in lime green, and metal ions are depicted as black spheres. The two key residues involved in substrate binding and product release (Thr109 and Thr110 in E. coli DHOase, or their corresponding residues in other DHOases) are also highlighted using stick representation in the structural models. Dashed lines indicate unresolved loop regions, possibly reflecting loop flexibility or transitions between conformational states.
Figure 7
Figure 7
Classification of loop-in conformations in type I DHOases without bound ligands. (A) Structural superimposition of B. anthracis DHOase (BaDHOase) with (PDB ID: 4YIW) and without (PDB ID: 3MPG) the ligand NCA. The loop in the ligand-bound structure is colored blue, while the corresponding loop in the ligand-free structure is in red. Although the interacting residue Gly does not form side-chain contacts with the ligand, the peptide backbone oxygen (also in red) maintains a comparable position in both structures. Due to the nearly identical loop conformations, the unbound structure was also classified as having a loop-in conformation. (B) Structural comparison of BaDHOase-NCA complex with T. thermophilus DHOase (TtDHOase; PDB ID: 2Z00). Based on the similar loop conformation, the ligand-free TtDHOase structure was classified as loop-in. (C) Structural superimposition of BaDHOase-NCA complex with S. aureus DHOase (SaDHOase; PDB ID: 3GRI). Although both enzymes share similar loop lengths, the putative ligand-interacting residue Gly151 in SaDHOase is located 6.9 Å away from the ligand-binding site, indicating that the loop cannot interact with the ligand in this conformation. Thus, this loop was classified as loop-out. (D) Structural superimposition of A. aeolicus DHOase (AaDHOase) with (PDB ID: 4BJH) and without (PDB ID: 1XRF) NCA. Due to the highly similar loop conformations and conserved positioning of the ligand-interacting residue Gly148, the ligand-free structure of AaDHOase was also categorized as loop-in.
Figure 8
Figure 8
Distribution of active site loop conformations (loop-in vs. loop-out) across various ligands bound to DHOase. Pie charts represent the percentage of loop-in (light orange) and loop-out (dark orange) conformations observed for DHOase structures complexed with different ligands, based on structural data from PDB. Ligands include substrates, products, inhibitors, and small molecules: NCA, DHO, a mixture of DHO/NCA, 5-FOA, orotic acid, HDDP, acetic acid (ACY), malic acid, 5-fluorouracil, 5-aminouracil, plumbagin, citrate anion, and cacodylate ion. Percentages within each chart indicate the proportion of DHOase monomers adopting either loop-in or loop-out conformations for a given ligand. Ligands such as malic acid, 5-fluorouracil, 5-aminouracil, plumbagin, citrate, and cacodylate consistently stabilize the loop-in conformation, while 5-FOA predominantly stabilizes the loop-out state. This analysis highlights ligand-specific preferences in loop dynamics across DHOase structures.
Figure 9
Figure 9
AlphaFold 3.0 structural predictions of DHOases from different species, highlighting active site loop conformations. Predicted structures of DHOases were generated using AlphaFold 3.0 for the following species: (A) S. cerevisiae, (B) E. coli, (C) S. enterica, (D) C. jejuni, (E) Y. pestis, (F) human DHOase domain, (G) B. cenocepacia, (H) V. cholerae, (I) P. gingivalis, (J) M. jannaschii, (K) T. thermophilus, (L) A. aeolicus, (M) S. aureus, and (N) B. anthracis. The flexible active site loop in each structure is shown separately beside the core protein for clarity. Prediction confidence, as assigned by AlphaFold, is indicated by color: blue (very high confidence, pLDDT > 90), light blue (confident, 70 < pLDDT ≤ 90), yellow (low confidence, 50 < pLDDT ≤ 70), and orange (very low confidence, pLDDT ≤ 50). In most structures, the active site loops were predicted with high confidence (blue or light blue), except for B. cenocepacia and human DHOases, where terminal disordered regions exhibit lower confidence (yellow or orange). Despite differences in loop sequence and length across species, type II DHOases consistently adopt the loop-in conformation in these models, including E. coli DHOase, which predominantly exhibits the loop-out conformation in crystal structures.
Figure 10
Figure 10
Crystal structures of B. cenocepacia DHOase and V. cholerae DHOase, highlighting active site loop conformations. Ribbon diagrams of (A) B. cenocepacia DHOase (PDB ID: 4LFY) and (B) V. cholerae DHOase (PDB ID: 5VGM) are shown. α-helices are colored in orange, β-strands in green, and loops in gray. The flexible active site loop (loop out) is highlighted in red. Zinc ions within the active site are depicted as black spheres. Both structures exhibit the loop-out conformation, where the active site loop is positioned away from the substrate-binding pocket. This structural observation contrasts with AlphaFold predictions, which suggest a loop-in conformation for these enzymes, underscoring discrepancies between AI-predicted models and experimental crystallographic data. These differences suggest that experimental conditions, ligand occupancy, or intrinsic loop flexibility may influence the observed loop states.
Figure 11
Figure 11
Docking result of myricetin to the loop-deleted (residues 104–108) S. cerevisiae DHOase. To mimic the loop-out conformation, residues 104–108 of the dynamic loop in S. cerevisiae DHOase were manually deleted prior to the docking experiment. The top-ranked binding pose showed successful docking into the active site with an affinity of –8.4 kcal/mol, suggesting that the loop-in conformation is unsuitable as a docking template.
Figure 12
Figure 12
The tetrameric structure of S. cerevisiae DHOase. The loop (colored in dark blue) does not interact with another S. cerevisiae DHOase monomer. This indicates that the loop conformation is not caused by crystal packing or monomer–monomer interactions.
Figure 13
Figure 13
Docking results of myricetin and 5-FOA to EcDHOase and human DHOase. Docking experiments with 5-FOA and myricetin confirmed that the loop-in conformation prevented productive active-site docking.
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