Molecular Insights into How the Dimetal Center in Dihydropyrimidinase Can Bind the Thymine Antagonist 5-Aminouracil: A Different Binding Mode from the Anticancer Drug 5-Fluorouracil

Dihydropyrimidinase (DHPase) is a key enzyme for pyrimidine degradation. DHPase contains a binuclear metal center in which two Zn ions are bridged by a posttranslationally carbamylated lysine. DHPase catalyzes the hydrolysis of dihydrouracil to N-carbamoyl-β-alanine. Whether 5-aminouracil (5-AU), a thymine antagonist and an anticancer drug that can block DNA synthesis and induce replication stress, can interact with DHPase remains to be investigated. In this study, we determined the crystal structure of Pseudomonas aeruginosa DHPase (PaDHPase) complexed with 5-AU at 2.1 Å resolution (PDB entry 7E3U). This complexed structure revealed that 5-AU interacts with Znα (3.2 Å), Znβ (3.0 Å), the main chains of residues Ser289 (2.8 Å) and Asn337 (3.3 Å), and the side chain of residue Tyr155 (2.8 Å). These residues are also known as the substrate-binding sites of DHPase. Dynamic loop I (amino acid residues Pro65-Val70) in PaDHPase is not involved in the binding of 5-AU. The fluorescence quenching analysis and site-directed mutagenesis were used to confirm the binding mode revealed by the complexed crystal structure. The 5-AU binding mode of PaDHPase is, however, different from that of 5-fluorouracil, the best-known fluoropyrimidine used for anticancer therapy. These results provide molecular insights that may facilitate the development of new inhibitors targeting DHPase and constitute the 5-AU interactome.


Introduction
Pyrimidine bases are essential for the replication of genetic information, cellular metabolism, and cell growth in all biological systems [1]. Uracil derivatives, especially 5substituted uracils, play a significant role in pharmacological activities, such as antiviral [2], anticancer drugs [3], antibacterial [4], anti-inflammatory [5,6], and antitumor activities [7][8][9]. For example, 5-fluorouracil (5-FU) is an FDAapproved drug with a remarkable therapeutic effect for the systemic treatment of cancers of the gastrointestinal tract, breast, head, and neck in the clinic [8]. As a thymine antagonist possessing anticancer activities, 5-aminouracil (5-AU) can block DNA synthesis and induce replication stress [10,11]. In addition, recent findings indicate that microbiota can modulate the host response to these chemotherapeutic drugs [12].
us, the whole interactome of these fivesubstituted uracil drugs should be determined for detailed clinical pharmacokinetic and toxicity analyses.
5-FU-associated toxicity was reported [42]. After treatment of 5-FU for anticancer therapy, the asymptomatic patients with DHPase deficiency suffered from severe toxicity, including death [43]. A recent report revealed that DHPase is a novel 5-FU-binding protein [44]. Whether and how other five-substituted uracil drugs such as 5-AU can bind to DHPase remains to be elucidated. In the present study, we found that 5-AU can bind to Pseudomonas aeruginosa DHPase (PaDHPase). We determined the crystal structure of the PaDHPase complex with 5-AU at a 2.1Å resolution (PDB entry 7E3U). Given the structural resemblance between 5-FU and 5-AU, one might conclude that the mode of 5-FU bound by PaDHP must be similar to that of 5-AU. However, we found that their binding modes are different. e fluorescence quenching method and site-directed mutagenesis were also used to confirm the binding mode of 5-AU to PaDHPase revealed by the complex structure. We think that the determination of this complex structure can extend the knowledge of the 5-AU interactome and benefit anticancer development.

Expression and Purification of PaDHPase.
e expression vector pET21b-PaDHPase [16] was transformed into Escherichia coli BL21 (DE3) cells and grown in LB medium at 37°C. e overexpression was induced by incubating with 1 mM isopropyl thiogalactopyranoside for 9 h. Recombinant PaDHPase [36] was purified from the soluble supernatant by using Ni 2+ -affinity chromatography (HiTrap HP; GE Healthcare Bio-Sciences). e recombinant protein was eluted with a linear imidazole gradient and dialyzed against a dialysis buffer (20 mM Tris-HCl and 0.1 M NaCl, pH 7.9; Buffer A).
e resultant enzyme solution was then dialyzed against Buffer A.

Site-Directed Mutagenesis.
e PaDHPase mutants were generated according to the QuikChange Site-Directed Mutagenesis kit protocol (Stratagene; La Jolla, CA, USA). e wild-type plasmid pET21b-PaDHPase was used as a template [16]. e presence of the mutation was verified by DNA sequencing in each construct. e recombinant mutant proteins were purified using the protocol for the wildtype PaDHPase by Ni 2+ -affinity chromatography (Table 1).

Crystallography.
e optimal protein concentration for crystallization of the PaDHPase complex, determined by the Pre-Crystallization Test (Hampton), was 10 mg/mL. Crystals were grown at room temperature by hanging drop vapor diffusion in 16% PEG 8000, 100 mM HEPES, 200 mM calcium acetate, and 200 μM 5-AU, at pH 7.5. Data were collected using an ADSC Quantum-315r CCD area detector at SPXF beamline BL13C1 at the National Synchrotron Radiation Research Center (NSRRC; Hsinchu, Taiwan). e structure of the 5-AU-PaDHPase complex was solved to 2.1Å resolution with the molecular replacement software Phaser-MR [46] using PaDHPase (PDB entry 5E5C) [15] as a model (Table 2). e data were indexed and scaled using HKL-2000 [47]. Models were built and refined with PHE-NIX [48] and Coot [49]. Coordinate and structure factor files have been deposited in the Protein Data Bank (PDB entry 7E3U).

Determination of the Dissociation Constant (K d ).
rough the fluorescence quenching analysis, the K d value of the purified PaDHPase was determined [44]. An aliquot of 5-AU was added into the solution containing PaDHPase (0.8 μM) and 50 mM HEPES at pH 7.0. e decrease in the intrinsic fluorescence of PaDHPase was measured at 336 nm upon excitation at 279 nm and 25°C with a spectrofluorimeter (Hitachi F-2700; Hitachi High-Technologies, Japan). e K d was obtained using the equation:

Crystallization of the PaDHPase-5-AU Complex.
PaDHPase crystals can be obtained by hanging drop vapor diffusion in 28% PEG 6000, 100 mM HEPES, and 200 mM lithium acetate at pH 7.5 [15]. Soaking and cocrystallization of PaDHPase with 5-AU under these conditions were attempted but were unsuccessful. After crystallization screening, the crystals of the PaDHPase-5-AU complex appeared at room temperature in 16% PEG 8000, 100 mM HEPES, 200 mM calcium acetate, and 200 μM 5-AU, at pH 7.5. ese crystals were used for determining the complex structure of PaDHPase.

Structure of PaDHPase in Complex with 5-AU.
e crystals of the 5-AU-PaDHPase complex belong to space group P3 1 2 1 with cell dimensions of a � 112.67, b � 112.67, and c � 161.43Å. e complexed crystal structure of PaDHPase with 5-AU was determined at a 2.1Å resolution ( Table 2). Two monomers of PaDHPase were found in the asymmetric unit (Figure 1(b)). Consistently, PaDHPase functions as a dimer [15]. e electron density of 5-AU was well defined and indicated the presence of 5-AU in the active site of PaDHPase (Figure 1(c)). e orientation of 5-AU could be easily distinguished on the basis of the location of the substituent. However, only one 5-AU molecule was found in the active site of one of the PaDHPase dimers. is is also the case for 5-FU bound to PaDHPase [44]. e binding of 5-AU does not influence the overall structure of PaDHPase. Similar to the apo form, the global architecture of the 5-AU-complexed PaDHPase monomer (subunit A) revealed a TIM-barrel structure embedding the catalytic dimetal center (Znα and Znβ) and a β-sandwich domain, consisting of 17 α-helices, 19 β-sheets, 2 Zn ions, and 1 5-AU molecule. Both of the two zinc ions are involved in binding 5-AU. e dimetal center in the PaDHPase-5-AU complex   consisted of His59, His61, Kcx150, His183, His239, and Asp316 still self-assembles (Figure 1(c)). Lys150 remains carbamylated (Kcx150) regardless of 5-AU binding.

5-AU Binding Mode.
As a uracil derivative possessing many chemotherapeutic and pharmacological activities [11], 5-AU was identified as a ligand bound by PaDHPase in this study. e 5-AU binding mode of PaDHPase was demonstrated by the complexed crystal structure. On the basis of our crystallographic analysis, various interactions between 5-AU and PaDHPase were examined (Figure 2(a)). Residues Tyr155, Ser289, and Asn337 of PaDHPase, crucial for substrate binding [17,19], are also involved in 5-AU binding (Figure 2(b)). 5-AU interacts with Znα (3.2Å), Znβ (3.0Å), the main chains of residues Ser289 (2.8Å) and Asn337 (3.3Å), and the side chain of residue Tyr155 (2.8Å). Dynamic loop I (amino acid residues Pro65-Val70) in PaDHPase is not involved in the binding of 5-AU.

Structural Comparison of the Active Sites between the 5-AU Bound State and the 5-FU Bound State of PaDHPase.
Recently, the crystal structure of PaDHPase in complex with the anticancer drug 5-FU was reported [44]. 5-FU is the bestknown fluoropyrimidine used to target the enzyme thymidylate synthase in anticancer therapy [3,8]. Given the structural similarity between 5-AU and 5-FU (Figure 3(a)), one might conclude that their binding mode by PaDHPase must be similar. e dynamic loops of PaDHPase extend toward the active site when either 5-FU or 5-AU is bound (Figure 3(b)). However, we found that 5-AU and 5-FU binding poses to PaDHPase are different in terms of orientation (Figure 3(b)) and binding residues (Figure 3(c)). e side chain of residue Cys318 in PaDHPase (Figure 3(d)) is involved in binding 5-FU (2.9Å) but not 5-AU ( Figure 2). In addition, the binding contributions from the metal ions in PaDHPase are different. Znβ is not involved in binding 5-FU [44], whereas both Zn ions interact with 5-AU ( Figure 2). us, we concluded that the binding mechanisms of the anticancer drugs 5-FU and 5-AU to PaDHPase are different.

5-AU Binding Analysis.
Prior to this study, whether 5-AU could bind to DHPase remained unknown. To confirm if PaDHPase is capable of binding 5-AU, we used the fluorescence quenching method to determine the binding ability of PaDHPase (Figure 4). Quenching is a complex formation process that decreases the fluorescence intensity of protein.
e fluorescence emission spectra of the wild-type PaDH-Pase was significantly quenched with 5-AU (Figure 4(a)). PaDHPase displayed strong intrinsic fluorescence with a peak wavelength of 336 nm when excited at 279 nm. When 5-AU was titrated into the PaDHPase solution, the intrinsic fluorescence of the protein was progressively quenched. Upon the addition of 300 μM 5-AU, the intrinsic fluorescence of PaDHPase was quenched by 87.3%. Adding 5-AU resulted in a red shift in the PaDHPase emission wavelength (∼6.5 nm; λ max from 336.5 to 343 nm). ese observations indicated that PaDHPase can form a stable complex with 5-AU. As determined through the titration curve, the dissociation constant (K d ) of PaDHPase bound to 5-AU is 97.7 ± 2.0 μM. Based on the K d value of PaDHPase bound to 5-FU (133.2 ± 8.5 μM) [44], PaDHPase may prefer binding to 5-AU over 5-FU.

Structure-Based Mutational Analysis.
e complexed structure revealed Tyr155, Ser289, and Asn337 of PaDHPase as the 5-AU binding sites (Figure 2). 5-AU interacts with the main chains of residues Ser289 and Asn337 and the side chain of residue Tyr155. To investigate the contribution of individual amino acid residues to 5-AU binding, alanine substitution mutants ( Table 2) were constructed and analyzed by fluorescence quenching (Figures 4(b)-4(d)). We found that 300 μM 5-AU quenched the intrinsic fluorescence of mutants Y155A, S289A, and N337A by 68.6%, 61.6%, and 72.4%, respectively. e K d values of Y155A, S289A, and N337A bound to 5-AU were reduced to 247.1 ± 4.0, 256.0 ± 3.2, and 192.1 ± 8.0 μM, respectively. Accordingly, Ser289 is the most effective one of these residues for 5-AU binding.
e K d value of C318A was determined as 103.3 ± 3.1 μM. e abilities of the wild-type PaDHPase and the C318A mutant to bind 5-AU are approximately equal. us, Cys318 is not essential for 5-AU binding.

Role of Znβ Ion in 5-AU Binding.
On the basis of the complex structure of 5-AU-PaDHPase, the Znβ ion is suggested to be critical for 5-AU binding (Figure 2). To confirm the role of Znβ in 5-AU binding and to compare the binding contribution with other mutant proteins (Table 3), we produced mono-Zn PaDHPase (Znα-PaDHPase) for binding analysis. Our crystal structure previously revealed that this mono-Zn enzyme [45] only contains a Znα ion in the active site of PaDHPase. e binding of 5-AU to Znα-PaDHPase was also analyzed by the fluorescence quenching method (Figure 4(f )). Upon the addition of 300 μM 5-AU, the intrinsic fluorescence of Znα-PaDHPase was quenched by 49.3%. e K d value of Znα-PaDHPase for 5-AU binding was calculated to be 281.5 ± 9.0 μM from the titration curve (Figure 4(g)).
Although 5-AU and 5-FU are similar uracil derivatives, no induction of IM cells was detected even using excess 5-FU [10]. erefore, 5-AU and 5-FU might somehow induce   different cellular effects. In this study, we found different binding modes of PaDHPase between 5-AU and 5-FU, and whether the different binding modes can produce distinct signaling pathways has not yet been elucidated. We noticed that other enzymes also respond to 5-AU by binding and/or inhibition.

Conclusion
We identified that PaDHPase can bind 5-AU with a K d value of 97.7 μM (Table 3 and Figure 4). e 5-AU binding mode of PaDHPase, different from that of 5-FU, was determined through structural evidence ( Figure 1) and mutational analysis (Table 3). is structure provides molecular insights into how the dimetal center in PaDHPase can bind 5-AU ( Figure 2). Further research can directly focus on revisiting the role of DHPase in anticancer therapy [42,43,51].

Data Availability
Atomic coordinates and related structure factors were deposited in the PDB with accession code 7E3U. All the data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare that they have no conflicts of interest regarding the publication of this paper.