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Dihydroorotase (DHOase) is the third enzyme in the six enzymatic reaction steps of the endogenous pyrimidine nucleotide de novo biosynthesis pathway, which is a metabolic pathway conserved in both bacteria and eukaryotes. However, research on the biological function of DHOase in plant pathogenic fungi is very limited. In this study, we identified and named MoPyr4, a homologous protein of Saccharomyces cerevisiae DHOase Ura4, in the rice blast fungus Magnaporthe oryzae and investigated its ability to regulate fungal growth, pathogenicity, and autophagy. Deletion of MoPYR4 led to defects in growth, conidiation, appressorium formation, the transfer and degradation of glycogen and lipid droplets, appressorium turgor accumulation, and invasive hypha expansion in M. oryzae, which eventually resulted in weakened fungal pathogenicity. Long-term replenishment of exogenous uridine-5'-phosphate (UMP) can effectively restore the phenotype and virulence of the ΔMopyr4 mutant. Further study revealed that MoPyr4 also participated in the regulation of the Pmk1-MAPK signaling pathway, co-localized with peroxisomes for the oxidative stress response, and was involved in the regulation of the Osm1-MAPK signaling pathway in response to hyperosmotic stress. In addition, MoPyr4 interacted with MoAtg5, the core protein involved in autophagy, and positively regulated autophagic degradation. Taken together, our results suggested that MoPyr4 for UMP biosynthesis was crucial for the development and pathogenicity of M. oryzae. We also revealed that MoPyr4 played an essential role in the external stress response and pathogenic mechanism through participation in the Pmk1-MAPK signaling pathway, peroxisome-related oxidative stress response mechanism, the Osm1-MAPK signaling pathway and the autophagy pathway.

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5-Fluorouracil (5-FU) stands as one of the most widely prescribed chemotherapeutics. Despite over 60 years of study, a systematic synopsis of how 5-FU binds to proteins has been lacking. Investigating the specific binding patterns of 5-FU to proteins is essential for identifying additional interacting proteins and comprehending their medical implications. In this review, an analysis of the 5-FU binding environment was conducted based on available complex structures. From the earliest complex structure in 2001 to the present, two groups of residues emerged upon 5-FU binding, classified as P- and R-type residues. These high-frequency interactive residues with 5-FU include positively charged residues Arg and Lys (P type) and ring residues Phe, Tyr, Trp, and His (R type). Due to their high occurrence, 5-FU binding modes were simplistically classified into three types, based on interactive residues (within <4 Å) with 5-FU: Type 1 (P-R type), Type 2 (P type), and Type 3 (R type). In summary, among 14 selected complex structures, 8 conform to Type 1, 2 conform to Type 2, and 4 conform to Type 3. Residues with high interaction frequencies involving the N1, N3, O4, and F5 atoms of 5-FU were also examined. Collectively, these interaction analyses offer a structural perspective on the specific binding patterns of 5-FU within protein pockets and contribute to the construction of a structural interactome delineating the associations of the anticancer drug 5-FU.

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Dihydropyrimidinase (DHPase) plays a crucial role in pyrimidine degradation, showcasing a broad substrate specificity that extends beyond pyrimidine catabolism, hinting at additional roles for this ancient enzyme. In this study, we solved the crystal structure of Pseudomonas aeruginosa DHPase (PaDHPase) complexed with the neurotransmitter γ-aminobutyric acid (GABA) at a resolution of 1.97 Å (PDB ID 8WQ9). Our structural analysis revealed two GABA binding sites in each monomer of PaDHPase. Interactions between PaDHPase and GABA molecules, involving residues within a contact distance of <4 Å, were examined. In silico analyses via PISA and PLIP software revealed hydrogen bonds formed between the side chain of Cys318 and GABA 1, as well as the main chains of Ser333, Ile335, and Asn337 with GABA 2. Comparative structural analysis between GABA-bound and unbound states unveiled significant conformational changes at the active site, particularly within dynamic loop I, supporting the conclusion that PaDHPase binds GABA through the loop-out mechanism. Building upon this molecular evidence, we discuss and propose a working model. The study expands the GABA interactome by identifying DHPase as a novel GABA-interacting protein and provides structural insight into the interaction between a dimetal center in the protein's active site and GABA. Further investigations are warranted to explore potential interactions of GABA with other DHPase-like proteins and to understand whether DHPase may have additional regulatory and physiological roles in the cell, extending beyond pyrimidine catabolism.

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Toxoplasma gondii relies heavily on the de novo pyrimidine biosynthesis pathway for fueling the high uridine-5'-monophosphate (UMP) demand during parasite growth. The third step of de novo pyrimidine biosynthesis is catalyzed by dihydroorotase (DHO), a metalloenzyme that catalyzes the reversible condensation of carbamoyl aspartate to dihydroorotate. Here, functional analyses of TgDHO reveal that tachyzoites lacking DHO are impaired in overall growth due to decreased levels of UMP, and the noticeably growth restriction could be partially rescued after supplementation with uracil or high concentrations of L-dihydroorotate in vitro. When pyrimidine salvage pathway is disrupted, both DHOH35A and DHOD284E mutant strains proliferated much slower than DHO-expressing parasites, suggesting an essential role of both TgDHO His35 and Asp284 residues in parasite growth. Additionally, DHO deletion causes the limitation of bradyzoite growth under the condition of uracil supplementation or uracil deprivation. During the infection in mice, the DHO-deficient parasites are avirulent, despite the generation of smaller tissue cysts. The results reveal that TgDHO contributes to parasite growth both in vitro and in vivo. The significantly differences between TgDHO and mammalian DHO reflect that DHO can be exploited to produce specific inhibitors targeting apicomplexan parasites. Moreover, potential DHO inhibitors exert beneficial effects on enzymatic activity of TgDHO and T. gondii growth in vitro. In conclusion, these data highlight the important role of TgDHO in parasite growth and reveal that it is a promising anti-parasitic target for future control of toxoplasmosis.

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CAD is a large, 2225 amino acid multienzymatic protein required for de novo pyrimidine biosynthesis. Pathological CAD variants cause a developmental and epileptic encephalopathy which is highly responsive to uridine supplements. CAD deficiency is difficult to diagnose because symptoms are nonspecific, there is no biomarker, and the protein has over 1000 known variants. To improve diagnosis, we assessed the pathogenicity of 20 unreported missense CAD variants using a growth complementation assay that identified 11 pathogenic variants in seven affected individuals; they would benefit from uridine treatment. We also tested nine variants previously reported as pathogenic and confirmed the damaging effect of seven. However, we reclassified two variants as likely benign based on our assay, which is consistent with their long-term follow-up with uridine. We found that several computational methods are unreliable predictors of pathogenic CAD variants, so we extended the functional assay results by studying the impact of pathogenic variants at the protein level. We focused on CAD's dihydroorotase (DHO) domain because it accumulates the largest density of damaging missense changes. The atomic-resolution structures of eight DHO pathogenic variants, combined with functional and molecular dynamics analyses, provided a comprehensive structural and functional understanding of the activity, stability, and oligomerization of CAD's DHO domain. Combining our functional and protein structural analysis can help refine clinical diagnostic workflow for CAD variants in the genomics era.

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Dihydroorotase (DHOase) is the third enzyme in the pathway used for the biosynthesis of pyrimidine nucleotides. In mammals, DHOase is active in a trifunctional enzyme, CAD, which also carries out the activities of carbamoyl phosphate synthetase and aspartate transcarbamoylase. Prior to this study, it was unknown whether the FDA-approved clinical drug 5-fluorouracil (5-FU), which is used as an anticancer therapy, could bind to the DHOase domain of human CAD (huDHOase). Here, we identified huDHOase as a new 5-FU binding protein, thereby extending the 5-FU interactome to this human enzyme. In order to investigate where 5-FU binds to huDHOase, we solved the complexed crystal structure at 1.97 Å (PDB ID 8GVZ). The structure of huDHOase complexed with malate was also determined for the sake of comparison (PDB ID 8GW0). These two nonsubstrate ligands were bound at the active site of huDHOase. It was previously established that the substrate N-carbamoyl-L-aspartate is either bound to or moves away from the active site, but it is the loop that is extended towards (loop-in mode) or moved away (loop-out mode) from the active site. DHOase also binds to nonsubstrate ligands via the loop-out mode. In contrast to the Escherichia coli DHOase model, our complexed structures revealed that huDHOase binds to either 5-FU or malate via the loop-in mode. We further characterized the binding of 5-FU to huDHOase using site-directed mutagenesis and the fluorescence quenching method. Considering the loop-in mode, the dynamic loop in huDHOase should be a suitable drug-targeting site for further designing inhibitors and clinical chemotherapies to suppress pyrimidine biosynthesis in cancer cell lines.

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CAD is a 1.5 MDa hexameric protein with four enzymatic domains responsible for initiating de novo biosynthesis of pyrimidines nucleotides: glutaminase, carbamoyl phosphate synthetase, aspartate transcarbamoylase (ATC), and dihydroorotase. Despite its central metabolic role and implication in cancer and other diseases, our understanding of CAD is poor, and structural characterization has been frustrated by its large size and sensitivity to proteolytic cleavage. Recently, we succeeded in isolating intact CAD-like particles from the fungus Chaetomium thermophilum with high yield and purity, but their study by cryo-electron microscopy is hampered by the dissociation of the complex during sample grid preparation. Here we devised a specific crosslinking strategy to enhance the stability of this mega-enzyme. Based on the structure of the isolated C. thermophilum ATC domain, we inserted by site-directed mutagenesis two cysteines at specific locations that favored the formation of disulfide bridges and covalent oligomers. We further proved that this covalent linkage increases the stability of the ATC domain without damaging the structure or enzymatic activity. Thus, we propose that this cysteine crosslinking is a suitable strategy to strengthen the contacts between subunits in the CAD particle and facilitate its structural characterization.

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In this paper, we report the structural analysis of dihydroorotase (DHOase) from the hyperthermophilic and barophilic archaeon Methanococcus jannaschii. DHOase catalyzes the reversible cyclization of N-carbamoyl-l-aspartate to l-dihydroorotate in the third step of de novo pyrimidine biosynthesis. DHOases form a very diverse family of enzymes and have been classified into types and subtypes with structural similarities and differences among them. This is the first archaeal DHOase studied by x-ray diffraction. Its structure and comparison with known representatives of the other subtypes help define the structural features of the archaeal subtype. The M. jannaschii DHOase is found here to have traits from all subtypes. Contrary to expectations, it has a carboxylated lysine bridging the two Zn ions in the active site, and a long catalytic loop. It is a monomeric protein with a large ß sandwich domain adjacent to the TIM barrel. Loop 5 is similar to bacterial type III and the C-terminal extension is long.

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Nepenthes are carnivorous pitcher plants that have several ethnobotanical uses, such as curing stomachache and fever. Here, we prepared different extracts from the stem, leaf, and pitcher of Nepenthes miranda to further investigate their pharmacological potential. The leaf extract of N. miranda obtained by 100% acetone (N. miranda-leaf-acetone) was used in this study to analyze the cytotoxic activities, antioxidation capacity, antibacterial activity, and allantoinase (ALLase) inhibitory effect of this plant. The cytotoxic effects of N. miranda-leaf-acetone on the survival, apoptosis, and migration of the cancer cell lines PC-9 pulmonary adenocarcinoma, B16F10 melanoma, and 4T1 mammary carcinoma cells were demonstrated. Based on collective data, the cytotoxic activities of N. miranda-leaf-acetone followed the order: B16F10 > 4T1 > PC-9 cells. In addition, the cytotoxic activities of N. miranda-leaf-acetone were synergistically enhanced when co-acting with the clinical anticancer drug 5-fluorouracil. N. miranda-leaf-acetone could also inhibit the activity of ALLase, a key enzyme in the catabolism pathway for purine degradation. Through gas chromatography−mass spectrometry, the 16 most abundant ingredients in N. miranda-leaf-acetone were identified. The top six compounds in N. miranda-leaf-acetone, namely, plumbagin, lupenone, palmitic acid, stigmast-5-en-3-ol, neophytadiene, and citraconic anhydride, were docked to ALLase, and their docking scores were compared. The docking results suggested plumbagin and stigmast-5-en-3-ol as potential inhibitors of ALLase. Overall, these results may indicate the pharmacological potential of N. miranda for further medical applications.

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The carnivorous pitcher plant Sarracenia purpurea exhibits many ethnobotanical uses, including the treatments of type 2 diabetes and tuberculosis-like symptoms. In this study, we prepared different extracts from the leaves (pitchers), stems, and roots of S. purpurea and investigated their antioxidant and anticancer properties. To evaluate the extraction efficiency, we individually used different solvents, namely methanol, ethanol, acetone, and distilled water, for S. purpurea extract preparations. The root extract of S. purpurea, obtained by 100% acetone (S. purpurea-root-acetone), had the highest anticancer activities, antioxidation capacity (the DPPH activity with IC50 of 89.3 ± 2.2 µg/mL), antibacterial activities, total phenolic content (33.4 ± 0.7 mg GAE/g), and total flavonoid content (107.9 ± 2.2 mg QUE/g). The most abundant compounds in S. purpurea-root-acetone were identified using gas chromatography-mass spectrometry; 7,8-Dihydro-α-ionone was the major compound present in S. purpurea-root-acetone. In addition, the co-cytotoxicity of S. purpurea-root-acetone (combined with the clinical anticancer drug 5-fluorouracil (5-FU) on the survival, apoptosis, proliferation, and migration of the 4T1 mammary carcinoma) was examined. The combination of 5-FU with S. purpurea-root-acetone could be highly efficient for anti-4T1 cells. We also found that S. purpurea-root-acetone could inhibit the enzymatic activity of human dihydroorotase (huDHOase), an attractive target for potential anticancer chemotherapy. The sic most abundant compounds in S. purpurea-root-acetone were tested using an in silico analysis via MOE-Dock software for their binding affinities. The top-ranked docking conformations were observed for 7,8-dihydro-α-ionone and stigmast-5-en-3-ol, suggesting the inhibition potential against huDHOase. Overall, the collective data in this study may indicate the pharmacological potentials of S. purpurea-root-acetone for possible medical applications.

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Shigella flexneri is the main causative agent of the communicable diarrheal disease, shigellosis. It is estimated that about 80-165 million cases and > 1 million deaths occur every year due to this disease. S. flexneri causes dysentery mostly in young children, elderly and immunocompromised patients, all over the globe. Recently, due to the emergence of S. flexneri antibiotic resistance strains, it is a dire need to predict novel therapeutic drug targets in the bacterium and screen natural products against it, which could eliminate the curse of antibiotic resistance. Therefore, in current study, available antibiotic-resistant genomes (n = 179) of S. flexneri were downloaded from PATRIC database and a pan-genome and resistome analysis was conducted. Around 5059 genes made up the accessory, 2469 genes made up the core, and 1558 genes made up the unique genome fraction, with 44, 34, and 13 antibiotic-resistant genes in each fraction, respectively. Core genome fraction (27% of the pan-genome), which was common to all strains, was used for subtractive genomics and resulted in 384 non-homologous, and 85 druggable targets. Dihydroorotase was chosen for further analysis and docked with natural product libraries (Ayurvedic and Streptomycin compounds), while the control was orotic acid or vitamin B13 (which is a natural binder of this protein). Dynamics simulation of 50 ns was carried out to validate findings for top-scored inhibitors. The current study proposed dihydroorotase as a significant drug target in S. flexneri and 4-tritriacontanone & patupilone compounds as potent drugs against shigellosis. Further experiments are required to ascertain validity of our findings.

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Dihydroorotase (DHOase), a dimetalloenzyme containing a carbamylated lysine within the active site, is a member of the cyclic amidohydrolase family, which also includes allantoinase (ALLase), dihydropyrimidinase (DHPase), hydantoinase, and imidase. Unlike most known cyclic amidohydrolases, which are tetrameric, DHOase exists as a monomer or dimer. Here, we report and analyze two crystal structures of the eukaryotic Saccharomyces cerevisiae DHOase (ScDHOase) complexed with malate. The structures of different DHOases were also compared. An asymmetric unit of these crystals contained four crystallographically independent ScDHOase monomers. ScDHOase shares structural similarity with Escherichia coli DHOase (EcDHOase). Unlike EcDHOase, ScDHOase can form tetramers, both in the crystalline state and in solution. In addition, the subunit-interacting residues of ScDHOase for dimerization and tetramerization are significantly different from those of other DHOases. The tetramerization pattern of ScDHOase is also different from those of DHPase and ALLase. Based on sequence analysis and structural evidence, we identify two unique helices (α6 and α10) and a loop (loop 7) for tetramerization, and discuss why the residues for tetramerization in ScDHOase are not necessarily conserved among DHOases.

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CAD (Carbamoyl-phosphate synthetase 2, Aspartate transcarbamoylase, and Dihydroorotase) is a multifunctional protein that participates in the initial three speed-limiting steps of pyrimidine nucleotide synthesis. Over the past two decades, extensive investigations have been conducted to unmask CAD as a central player for the synthesis of nucleic acids, active intermediates, and cell membranes. Meanwhile, the important role of CAD in various physiopathological processes has also been emphasized. Deregulation of CAD-related pathways or CAD mutations cause cancer, neurological disorders, and inherited metabolic diseases. Here, we review the structure, function, and regulation of CAD in mammalian physiology as well as human diseases, and provide insights into the potential to target CAD in future clinical applications.

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Drug-resistant Staphylococcus aureus is an imminent threat to public health, increasing the importance of drug discovery utilizing unexplored bacterial pathways and enzyme targets. De novo pyrimidine biosynthesis is a specialized, highly conserved pathway implicated in both the survival and virulence of several clinically relevant pathogens. Class I dihydroorotase (DHOase) is a separate and distinct enzyme present in gram positive bacteria (i.e., S. aureus, B. anthracis) that converts carbamoyl-aspartate (Ca-asp) to dihydroorotate (DHO)-an integral step in the de novo pyrimidine biosynthesis pathway. This study sets forth a high-throughput screening (HTS) of 3000 fragment compounds by a colorimetry-based enzymatic assay as a primary screen, identifying small molecule inhibitors of S. aureus DHOase (SaDHOase), followed by hit validation with a direct binding analysis using surface plasmon resonance (SPR). Competition SPR studies of six hit compounds and eight additional analogs with the substrate Ca-asp determined the best compound to be a competitive inhibitor with a KD value of 11 µM, which is 10-fold tighter than Ca-asp. Preliminary structure-activity relationship (SAR) provides the foundation for further structure-based antimicrobial inhibitor design against S. aureus.

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Dihydroorotase (DHOase) is the third enzyme in the de novo biosynthesis pathway for pyrimidine nucleotides, and an attractive target for potential anticancer chemotherapy. By screening plant extracts and performing GC-MS analysis, we identified and characterized that the potent anticancer drug plumbagin (PLU), isolated from the carnivorous plant Nepenthes miranda, was a competitive inhibitor of DHOase. We also solved the complexed crystal structure of yeast DHOase with PLU (PDB entry 7CA1), to determine the binding interactions and investigate the binding modes. Mutational and structural analyses indicated the binding of PLU to DHOase through loop-in mode, and this dynamic loop may serve as a drug target. PLU exhibited cytotoxicity on the survival, migration, and proliferation of 4T1 cells and induced apoptosis. These results provide structural insights that may facilitate the development of new inhibitors targeting DHOase, for further clinical anticancer chemotherapies.

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CAD is a 1.5 MDa particle formed by hexameric association of a 250 kDa protein divided into different enzymatic domains, each catalyzing one of the initial reactions for de novo biosynthesis of pyrimidine nucleotides: glutaminase-dependent Carbamoyl phosphate synthetase, Aspartate transcarbamoylase, and Dihydroorotase. The pathway for de novo pyrimidine synthesis is essential for cell proliferation and is conserved in all living organisms, but the covalent linkage of the first enzymatic activities into a multienzymatic CAD particle is unique to animals. In other organisms, these enzymatic activities are encoded as monofunctional proteins for which there is abundant structural and biochemical information. However, the knowledge about CAD is scarce and fragmented. Understanding CAD requires not only to determine the three-dimensional structures and define the catalytic and regulatory mechanisms of the different enzymatic domains, but also to comprehend how these domains entangle and work in a coordinated and regulated manner. This review summarizes significant progress over the past 10 years toward the characterization of CAD's architecture, function, regulatory mechanisms, and cellular compartmentalization, as well as the recent finding of a new and rare neurometabolic disorder caused by defects in CAD activities.

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Dihydroorotase (DHOase) is the third enzyme in the de novo biosynthesis pathway of pyrimidine nucleotides and considered an attractive target for potential antimalarial, anticancer, and antipathogen chemotherapy. Whether the FDA-approved clinical drug 5-fluorouracil (5-FU) that is used to target the enzyme thymidylate synthase for anticancer therapy can also bind to DHOase remains unknown. Here, we report the crystal structures of DHOase from Saccharomyces cerevisiae (ScDHOase) complexed with malate, 5-FU, and 5-aminouracil (5-AU). ScDHOase shares structural similarity with Escherichia coli DHOase. We also characterized the binding of 5-FU and 5-AU to ScDHOase by using the fluorescence quenching method. These complexed structures revealed that residues Arg18, Asn43, Thr106, and Ala275 of ScDHOase were involved in the 5-FU (PDB entry 6L0B) and 5-AU binding (PDB entry 6L0F). Overall, these results provide structural insights that may facilitate the development of new inhibitors targeting DHOase and constitute the 5-FU and 5-AU interactomes for further clinical chemotherapies.

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Dihydropyrimidinase catalyzes the reversible hydrolytic ring opening of dihydrouracil and dihydrothymine to N-carbamoyl-ß-alanine and N-carbamyl-ß-aminoisobutyrate, respectively. Dihydropyrimidinase from microorganisms is normally known as hydantoinase because of its role as a biocatalyst in the synthesis of d- and l-amino acids for the industrial production of antibiotic precursors and its broad substrate specificity. Dihydropyrimidinase belongs to the cyclic amidohydrolase family, which also includes imidase, allantoinase, and dihydroorotase. Although these metal-dependent enzymes share low levels of amino acid sequence homology, they possess similar active site architectures and may use a similar mechanism for catalysis. By contrast, the five human dihydropyrimidinase-related proteins possess high amino acid sequence identity and are structurally homologous to dihydropyrimidinase, but they are neuronal proteins with no dihydropyrimidinase activity. In this chapter, we summarize and discuss current knowledge and the recent advances on the structure, catalytic mechanism, and inhibition of dihydropyrimidinase.

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The burden of antibiotic resistance necessitates a continued search for new antimicrobials. We evaluated the antimicrobial activities of novel benzothiazoles synthesized by our group. Antibacterial activity was evaluated in vitro in Staphylococcus aureus, Bacillus subtilis, and Escherichia coli, while the antifungal activity was tested in Candida albicans and Aspergillus niger, and expressed as the minimum inhibitory concentration (MIC; µg/mL). MIC values of benzothiazole compounds ranged from 25 to 200 µg/mL. Compounds 3 and 4 gave high antibacterial and moderate antifungal activities, while 10 and 12 showed moderate activity against all tested organisms. In addition, some benzothiazole compounds significantly suppressed the activity of Escherichia coli dihydroorotase and inhibited the dimorphic transition of Candida albicans. Moreover, the active benzothiazole compounds induced DNA and protein leakage in Aspergillus niger spores. Molecular interactions of benzothiazole derivatives with dihydroorotase revealed the formation of hydrogen bonds with the active site residues LEU222 or ASN44. Strong hydrophobic interactions of the bulky thiazole and naphthalene rings at the entrance to the active site might interfere with the access of substrates to their binding sites, which results in dihydroorotase inhibition. Thus, inhibition of dihydroorotase might contribute to the observed antimicrobial actions of these compounds.

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PURPOSE: Pathogenic autosomal recessive variants in CAD, encoding the multienzymatic protein initiating pyrimidine de novo biosynthesis, cause a severe inborn metabolic disorder treatable with a dietary supplement of uridine. This condition is difficult to diagnose given the large size of CAD with over 1000 missense variants and the nonspecific clinical presentation. We aimed to develop a reliable and discerning assay to assess the pathogenicity of CAD variants and to select affected individuals that might benefit from uridine therapy. METHODS: Using CRISPR/Cas9, we generated a human CAD-knockout cell line that requires uridine supplements for survival. Transient transfection of the knockout cells with recombinant CAD restores growth in absence of uridine. This system determines missense variants that inactivate CAD and do not rescue the growth phenotype. RESULTS: We identified 25 individuals with biallelic variants in CAD and a phenotype consistent with a CAD deficit. We used the CAD-knockout complementation assay to test a total of 34 variants, identifying 16 as deleterious for CAD activity. Combination of these pathogenic variants confirmed 11 subjects with a CAD deficit, for whom we describe the clinical phenotype. CONCLUSIONS: We designed a cell-based assay to test the pathogenicity of CAD variants, identifying 11 CAD-deficient individuals who could benefit from uridine therapy.

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