Dihydroorotate Dehydrogenase mRNA and Protein Expression Analysis in Normal and Drug‐Resistant Cells

To follow the expression of the fourth enzyme of pyrimidine de novo synthesis dihydroorotate dehydrogenase (DHODH) in cells and tissues, we studied the DHODH mRNA expression by means of RT‐PCR in rat tissues. Rabbit polyclonal anti‐DHODH immunoglobulins were applied for immunochemical quantification of the enzyme protein by Western blotting. In mouse B‐lymphocytes, which were adapted to tolerate up to a 50‐fold concentration of the DHODH inhibitor leflunomide, a 20 fold protein overexpression was measured. Southern blotting indicated DHODH gene amplification.     

Requirements for the mitochondrial import and localization of dihydroorotate dehydrogenase.

In animals, dihydroorotate dehydrogenase (DHODH) is a mitochondrial protein that carries out the fourth step in de novo pyrimidine biosynthesis. Because this is the only enzyme of this pathway that is localized to mitochondria and because the enzyme is cytosolic in some bacteria and fungi, we carried out studies to understand the mode of targeting of animal DHODH and its submitochondrial localization. Analysis of fractionated rat liver mitochondria revealed that DHODH is an integral membrane protein exposed to the intermembrane space. In vitro-synthesized Drosophila, rat and human DHODH proteins were efficiently imported into the intermembrane space of isolated yeast mitochondria. Import did not alter the size of the in vitro synthesized protein, nor was there a detectable size difference when compared to the DHODH protein found in vivo. Thus, there is no apparent proteolytic processing of the protein during import either in vitro or in vivo. Import of rat DHODH into isolated yeast mitochondria required inner membrane potential and was at least partially dependent upon matrix ATP, indicating that its localization uses the well described import machinery of the mitochondrial inner membrane. The DHODH proteins of animals differ from the cytosolic proteins found in some bacteria and fungi by the presence of an N-terminal segment that resembles mitochondrial-targeting presequences. Deletion of the cationic portion of this N-terminal sequence from the rat DHODH protein blocked its import into isolated yeast mitochondria, whereas deletion of the adjacent hydrophobic segment resulted in import of the protein into the matrix. Thus, the N-terminus of the DHODH protein contains a bipartite signal that governs import and correct insertion into the mitochondrial inner membrane.     

Drosophila melanogaster dihydroorotate dehydrogenase: the N-terminus is important for biological function in vivo but not for catalytic properties in vitro

Dihydroorotate dehydrogenase (DHODH, EC 1.3.99.11), the fourth enzyme of pyrimidine de novo synthesis, is an integral flavoprotein of the inner mitchondrial membrane and is functionally connected to the respiratory chain. Here, experiments have been directed toward determining the roles of the N-terminal sequence motifs both in enzymatic properties of insect DHODH produced in vitro and the in vivo function of the protein. Full-length and three N-terminal truncated derivatives of the Drosophila melanogaster enzyme were expressed in Escherichia coli and purified. For identification on Western blots of recombinant DHODH as well as the native enzyme from flies polyclonal anti-DHODH immunoglobulins were generated and affinity-purified. The enzymatic characteristics of the four versions of DHODH were very similar, indicating that the N-terminus of the enzyme does not influence its catalytic function or its susceptibility to prominent DHODH inhibitors: A77-1726, brequinar, dichloroallyl-lawsone and redoxal. Whereas the efficacy of A77-1726 and dichloroallyl-lawsone were similar with Drosophila and human DHODH, that of brequinar and redoxal differed significantly. The differences in responses of insect DHODH and the enzyme from other species may allow the design of new agents that will selectively control insect growth, due to pyrimidine nucleotide limitation. In vivo expression of the full-length and N-truncated DHODHs from engineered transgenes revealed that the truncated proteins could not support normal de novo pyrimidine biosynthesis during development of the fly (i.e., failure to complement dhod-null mutations), apparently due to instability of the truncated proteins. It is concluded that the proper intracellular localization, directed by the N-terminal targeting and transmembrane motifs, is required for stability and subsequent proper biological function in vivo.     

Characterization of Trypanosoma brucei dihydroorotate dehydrogenase as a possible drug target; structural, kinetic and RNAi studies

Nucleotide biosynthesis pathways have been reported to be essential in some protozoan pathogens. Hence, we evaluated the essentiality of one enzyme in the pyrimidine biosynthetic pathway, dihydroorotate dehydrogenase (DHODH) from the eukaryotic parasite Trypanosoma brucei through gene knockdown studies. RNAi knockdown of DHODH expression in bloodstream form T. brucei did not inhibit growth in normal medium, but profoundly retarded growth in pyrimidine-depleted media or in the presence of the known pyrimidine uptake antagonist 5-fluorouracil (5-FU). These results have significant implications for the development of therapeutics to combat T. brucei infection. Specifically, a combination therapy including a T. brucei -specific DHODH inhibitor plus 5-FU may prove to be an effective therapeutic strategy. We also show that this trypanosomal enzyme is inhibited by known inhibitors of bacterial Class 1A DHODH, in distinction to the sensitivity of DHODH from human and other higher eukaryotes. This selectivity is supported by the crystal structure of the T. brucei enzyme, which is reported here at a resolution of 1.95 Å. Additional research, guided by the crystal structure described herein, is needed to identify potent inhibitors of T. brucei DHODH.     

Functional expression of human dihydroorotate dehydrogenase (DHODH) in pyr4 mutants of ustilago maydis allows target validation of DHODH inhibitors in vivo

Dihydroorotate dehydrogenase (DHODH; EC 1.3.99.11) is a central enzyme of pyrimidine biosynthesis and catalyzes the oxidation of dihydroorotate to orotate. DHODH is an important target for antiparasitic and cytostatic drugs since rapid cell proliferation often depends on the de novo synthesis of pyrimidine nucleotides. We have cloned the pyr4 gene encoding mitochondrial DHODH from the basidiomycetous plant pathogen Ustilago maydis. We were able to show that pyr4 contains a functional mitochondrial targeting signal. The deletion of pyr4 resulted in uracil auxotrophy, enhanced sensitivity to UV irradiation, and a loss of pathogenicity on corn plants. The biochemical characterization of purified U. maydis DHODH overproduced in Escherichia coli revealed that the U. maydis enzyme uses quinone electron acceptor Q6 and is resistant to several commonly used DHODH inhibitors. Here we show that the expression of the human DHODH gene fused to the U. maydis mitochondrial targeting signal is able to complement the auxotrophic phenotype of pyr4 mutants. While U. maydis wild-type cells were resistant to the DHODH inhibitor brequinar, strains expressing the human DHODH gene became sensitive to this cytostatic drug. Such engineered U. maydis strains can be used in sensitive in vivo assays for the development of novel drugs specifically targeted at either human or fungal DHODH.     

Modifications induced by plasma of gestational hypertensive women on the Na+/K+-ATPase obtained from human placenta

Pyrimidines and purine (deoxy)nucleotides are the building blocks of DNA and RNA. Nucleoside diphosphate sugars, e.g. UDP-glucose, are the reactive intermediates in the synthesis of nearly all glycosidic bonds between sugars.In mammals the requirement for pyrimidines is met by UMP de novo synthesis and, to a greater or lesser extent, by salvage of free nucleosides. The exceptional compartmentation of the de novo synthesis with respect to mitochondrially-bound dihydroorotate dehydrogenase ('DHOdehase' or 'DHODH', EC 1.3.99.11) is one focus of the present work. DHODH activity was determined by the dihydroorotate-dependent oxygen consumption or by the UV absorption of the product orotate with mitochondria isolated from rodent and porcine tissues. For comparison, the cytochrome c and choline-dependent oxygen consumption of mitochondria from different tissues was measured. The highest specific activity of the rat DHODH was found in liver (2.3 × 10-3 µmol/min × mg protein) > kidney > heart. The application of known enzyme inhibitors Brequinar Sodium and Leflunomide for DHODH and sodium cyanide for cytochrome c oxidase verified the specificity of the activity tests used. The relation of DHODH activity versus that of cytochrome c oxidase revealed the lowest ratios in heart mitochondria and the highest in liver mitochondria. Since disorders in the mitochondrial energy metabolism could entail severe impairment of pyrimidine biosynthesis via respiratory-chain coupled DHODH, it is suggested to include improvement of pyrimidine nucleotide status in therapy protocols. (Mol Cell Biochem 174: 125–129, 1997)     

Inhibition of pyrimidine synthesis reverses viral virulence factor-mediated block of mRNA nuclear export

The NS1 protein of influenza virus is a major virulence factor essential for virus replication, as it redirects the host cell to promote viral protein expression. NS1 inhibits cellular messenger ribonucleic acid (mRNA) processing and export, down-regulating host gene expression and enhancing viral gene expression. We report in this paper the identification of a nontoxic quinoline carboxylic acid that reverts the inhibition of mRNA nuclear export by NS1, in the absence or presence of the virus. This quinoline carboxylic acid directly inhibited dihydroorotate dehydrogenase (DHODH), a host enzyme required for de novo pyrimidine biosynthesis, and partially reduced pyrimidine levels. This effect induced NXF1 expression, which promoted mRNA nuclear export in the presence of NS1. The release of NS1-mediated mRNA export block by DHODH inhibition also occurred in the presence of vesicular stomatitis virus M (matrix) protein, another viral inhibitor of mRNA export. This reversal of mRNA export block allowed expression of antiviral factors. Thus, pyrimidines play a necessary role in the inhibition of mRNA nuclear export by virulence factors.     

Protein instability and functional defects caused by mutations of dihydro-orotate dehydrogenase in Miller syndrome patients

Miller syndrome is a recessive inherited disorder characterized by postaxial acrofacial dysostosis. It is caused by dysfunction of the DHODH (dihydroorotate dehydrogenase) gene, which encodes a key enzyme in the pyrimidine de novo biosynthesis pathway and is localized at mitochondria intermembrane space. We investigated the consequence of three missense mutations, G202A, R346W and R135C of DHODH, which were previously identified in patients with Miller syndrome. First, we established HeLa cell lines stably expressing DHODH with Miller syndrome-causative mutations: G202A, R346W and R135C. These three mutant proteins retained the proper mitochondrial localization based on immunohistochemistry and mitochondrial subfractionation studies. The G202A, R346W DHODH proteins showed reduced protein stability. On the other hand, the third one R135C, in which the mutation lies at the ubiquinone-binding site, was stable but possessed no enzymatic activity. In conclusion, the G202A and R346W mutation causes deficient protein stability, and the R135C mutation does not affect stability but impairs the substrate-induced enzymatic activity, suggesting that impairment of DHODH activity is linked to the Miller syndrome phenotype.     

The isolation and characterization of a cDNA encoding phospholipid-specific inositol polyphosphate 5-phosphatase

We report the cDNA cloning and characterization of a novel human inositol polyphosphate 5-phosphatase (5-phosphatase) that has substrate specificity unlike previously described members of this large gene family. All previously described members hydrolyze water soluble inositol phosphates. This enzyme hydrolyzes only lipid substrates, phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 4,5-bisphosphate. The cDNA isolated comprises 3110 base pairs and predicts a protein product of 644 amino acids and M(r) = 70,023. We designate this 5-phosphatase as type IV. It is a highly basic protein (pI = 8.8) and has the greatest affinity toward phosphatidylinositol 3,4,5-trisphosphate of known 5-phosphatases. The K(m) is 0.65 micrometer, 1/10 that of SHIP (5.95 micrometer), another 5-phosphatase that hydrolyzes phosphatidylinositol 3,4,5-trisphosphate. The activity of 5-phosphatase type IV is sensitive to the presence of detergents in the in vitro assay. Thus the enzyme hydrolyzes lipid substrates in the absence of detergents or in the presence of n-octyl beta-glucopyranoside or Triton X-100, but not in the presence of cetyltriethylammonium bromide, the detergent that has been used in other studies of the hydrolysis of phosphatidylinositol 4,5-bisphosphate. Remarkably SHIP, a 5-phosphatase previously characterized as hydrolyzing only substrates with d-3 phosphates, also readily hydrolyzed phosphatidylinositol 4,5-bisphosphate in the presence of n-octyl beta-glucopyranoside but not cetyltriethylammonium bromide. We used antibodies prepared against a peptide predicted by the cDNA to identify the 5-phosphatase type IV enzyme in human tissues and find that it is highly expressed in the brain as determined by Western blotting. We also performed Western blotting of mouse tissues and found high levels of expression in the brain, testes, and heart with lower levels of expression in other tissues. mRNA was detected in many tissues and cell lines as determined by Northern blotting.     

Dihydroorotate dehydrogenase in oxidative phosphorylation and cancer

Dihydroorotate dehydrogenase (DHODH) is an enzyme of the de novo pyrimidine synthesis pathway that provides nucleotides for RNA/DNA synthesis essential for proliferation. In mammalian cells, DHODH is localized in mitochondria, linked to the respiratory chain via the coenzyme Q pool. Here we discuss the role of DHODH in the oxidative phosphorylation system and in the initiation and progression of cancer. We summarize recent findings on DHODH biology, the progress made in the development of new, specific inhibitors of DHODH intended for cancer therapy, and the mechanistic insights into the consequences of DHODH inhibition.     

Malarial dihydroorotate dehydrogenase. Substrate and inhibitor specificity

The malarial parasite relies on de novo pyrimidine biosynthesis to maintain its pyrimidine pools, and unlike the human host cell it is unable to scavenge preformed pyrimidines. Dihydroorotate dehydrogenase (DHODH) catalyzes the oxidation of dihydroorotate (DHO) to produce orotate, a key step in pyrimidine biosynthesis. The enzyme is located in the outer membrane of the mitochondria of the malarial parasite. To characterize the biochemical properties of the malarial enzyme, an N-terminally truncated version of P. falciparum DHODH has been expressed as a soluble, active enzyme in E. coli. The recombinant enzyme binds 0.9 molar equivalents of the cofactor FMN and it has a pH maximum of 8.0 (k(cat) 8 s(-1), K(m)(app) DHO (40-80 microm)). The substrate specificity of the ubiquinone cofactor (CoQ(n)) that is required for the oxidation of FMN in the second step of the reaction was also determined. The isoprenoid (n) length of CoQ(n) was a determinant of reaction efficiency; CoQ(4), CoQ(6) and decylubiquinone (CoQ(D)) were efficiently utilized in the reaction, however cofactors lacking an isoprenoid tail (CoQ(0) and vitamin K(3)) showed decreased catalytic efficiency resulting from a 4 to 7-fold increase in K(m)(app). Five potent inhibitors of mammalian DHODH, Redoxal, dichloroallyl lawsone (DCL), and three analogs of A77 1726 were tested as inhibitors of the malarial enzyme. All five compounds were poor inhibitors of the malarial enzyme, with IC(50)'s ranging from 0.1-1.0 mm. The IC(50) values for inhibition of the malarial enzyme are 10(2)-10(4)-fold higher than the values reported for the mammalian enzyme, demonstrating that inhibitor binding to DHODH is species specific. These studies provide direct evidence that the malarial DHODH active site is different from the host enzyme, and that it is an attractive target for the development of new anti-malarial agents.     

High-throughput screening for potent and selective inhibitors of Plasmodium falciparum dihydroorotate dehydrogenase

Plasmodium falciparum is the causative agent of the most serious and fatal malarial infections, and it has developed resistance to commonly employed chemotherapeutics. The de novo pyrimidine biosynthesis enzymes offer potential as targets for drug design, because, unlike the host, the parasite does not have pyrimidine salvage pathways. Dihydroorotate dehydrogenase (DHODH) is a flavin-dependent mitochondrial enzyme that catalyzes the fourth reaction in this essential pathway. Coenzyme Q (CoQ) is utilized as the oxidant. Potent and species-selective inhibitors of malarial DHODH were identified by high-throughput screening of a chemical library, which contained 220,000 drug-like molecules. These novel inhibitors represent a diverse range of chemical scaffolds, including a series of halogenated phenyl benzamide/naphthamides and urea-based compounds containing napthyl or quinolinyl substituents. Inhibitors in these classes with IC(50) values below 600 nm were purified by high pressure liquid chromatography, characterized by mass spectroscopy, and subjected to kinetic analysis against the parasite and human enzymes. The most active compound is a competitive inhibitor of CoQ with an IC(50) against malarial DHODH of 16 nm, and it is 12,500-fold less active against the human enzyme. Site-directed mutagenesis of residues in the CoQ-binding site significantly reduced inhibitor potency. The structural basis for the species selective enzyme inhibition is explained by the variable amino acid sequence in this binding site, making DHODH a particularly strong candidate for the development of new anti-malarial compounds.     

Cloning, expression, purification, and characterization of Leishmania major dihydroorotate dehydrogenase

Leishmania major Friedlin (LmjF) is a protozoan parasite whose genomic sequence has been recently elucidated. Here we have cloned, overexpressed, purified, and characterized the product of the gene from LmjF chromosome 16: LmjF16.0530, which encodes a protein with putative dihydroorotate dehydrogenase activity. Dihydroorotate dehydrogenase (DHODH) is a flavoprotein that catalyses the oxidation of L-dihydroorotate to orotate, the fourth sequential step in the de novo pyrimidine nucleotide synthesis pathway. The predicted enzyme from L. major was cloned and expressed in Escherichia coli strain BL21(DE3) as a histidine-tag fusion protein and purified to homogeneity using affinity chromatography. The final product was homogeneous in SDS-PAGE gel electrophoresis. The dihydroorotate oxidase activity has been assayed and the steady-state kinetic mechanism has been determined using fumarate as the oxidizing substrate. The catalysis by LmDHODH enzyme proceeds by a Ping-Pong Bi-Bi mechanism and the kinetic parameters Km were calculated to be 90 and 418 microM for dihydroorotate and fumarate, respectively, and Vmax was calculated to be 11 micromol min-1 mg-1. Our results confirmed that the product of the gene LmjF16.0530, whose function has previously been predicted based on homology to known proteins, can therefore be positively assigned as L. major DHODH.     

Expression and characterization of E. coli-produced soluble, functional human dihydroorotate dehydrogenase: a potential target for immunosuppression

Human dihydroorotate dehydrogenase (huDHODH) is essential for de novo biosynthesis of pyrimidines and the target of two immunosuppressive drugs, brequinar and the leflunomide metabolite A77-1726 (Chen et al., 1992; Davis et al., 1996). Using a T7 RNA polymerase expression system, we produced huDHODH as a fusion protein containing an amino-terminal decahistidine tag. Escherichia coli growth and expression conditions were optimized to enhance huDHODH solubility and to permit purification of the enzyme in the absence of detergent. Soluble huDHODH, purified by a simple two-step procedure, was catalytically active, monomeric, and contained a flavin mononucleotide (FMN) cofactor in a 1:1 FMN/protein molar ratio. Kinetic analysis showed that huDHODH uses a two site ping-pong mechanism, where DHO is oxidized at one site and the second substrate, ubiquinone, is reduced at the other. This result is consistent with the mechanism proposed for bovine liver DHODH (Hines and Johnston, 1989).     

Plant dihydroorotate dehydrogenase differs significantly in substrate specificity and inhibition from the animal enzymes

The mitochondrial membrane bound dihydroorotate dehydrogenase (DHODH; EC 1.3.99.11) catalyzes the fourth step of pyrimidine biosynthesis. By the present correction of a known cDNA sequence for Arabidopsis thaliana DHODH we revealed the importance of the very C-terminal part for its catalytic activity and the reason why--in contrast to mammalian and insect species--the recombinant plant flavoenzyme was unaccessible to date for in vitro characterization. Structure-activity relationship studies explained that potent inhibitors of animal DHODH do not significantly affect the plant enzyme. These difference could be exploited for a novel approach to herb or pest growth control by limitation of pyrimidine nucleotide pools.     

Defects in vesicle core induced by escherichia coli dihydroorotate dehydrogenase

Dihydroorotate dehydrogenase (DHODH) catalyzes the oxidation of dihydroorotate to orotate during the fourth step of the de novo pyrimidine synthesis pathway. In rapidly proliferating mammalian cells, pyrimidine salvage pathway is insufficient to overcome deficiencies in that pathway for nucleotide synthesis. Moreover, as certain parasites lack salvage enzymes, relying solely on the de novo pathway, DHODH inhibition has turned out as an efficient way to block pyrimidine biosynthesis. Escherichia coli DHODH (EcDHODH) is a class 2 DHODH, found associated to cytosolic membranes through an N-terminal extension. We used electronic spin resonance (ESR) to study the interaction of EcDHODH with vesicles of 1,2-dioleoyl-sn-glycero-phosphatidylcholine/detergent. Changes in vesicle dynamic structure induced by the enzyme were monitored via spin labels located at different positions of phospholipid derivatives. Two-component ESR spectra are obtained for labels 5- and 10-phosphatidylcholine in presence of EcDHODH, whereas other probes show a single-component spectrum. The appearance of an additional spectral component with features related to fast-motion regime of the probe is attributed to the formation of a defect-like structure in the membrane hydrophobic region. This is probably the mechanism used by the protein to capture quinones used as electron acceptors during catalysis. The use of specific spectral simulation routines allows us to characterize the ESR spectra in terms of changes in polarity and mobility around the spin-labeled phospholipids. We believe this is the first report of direct evidences concerning the binding of class 2 DHODH to membrane systems.     

Monospecific antiserum to rat spermidine synthase and its application to rat tissues and several mammals

Monospecific antiserum to rat spermidine synthase was prepared by immunization of rabbits with purified enzyme protein from rat prostate, and its usefulness for analysis of spermidine synthase protein in not only rat tissues but also several other mammals was demonstrated by Western blotting and immunotitration of the enzyme activity. Application of the antiserum for elucidating the relationship between the enzyme activity and protein in normal rat tissues strongly suggested that marked difference in spermidine synthase activity among rat tissues depends solely on the difference in the amount of enzyme protein. Also, application of the antiserum for analyzing spermidine synthase from liver of mouse, rat, guinea pig, pig, and human, showed that the enzymes had a similar subunit molecular weight of 35,000 and a cross-reactivity with the antiserum, exhibiting almost the same immunoreactivity to mouse enzyme as to rat enzyme. Thus, it was suggested that the antiserum would be useful for further studies of mammalian spermidine synthase from the viewpoints of enzymology and molecular biology.     

Discovery of small molecule inhibitors of human uridine-cytidine kinase 2 by high-throughput screening

Clinically relevant inhibitors of dihydroorotate dehydrogenase (DHODH), a rate-limiting enzyme in mammalian de novo pyrimidine synthesis, have strong antiviral and anticancer activity in vitro. However, they are ineffective in vivo due to efficient uridine salvage by infected or rapidly dividing cells. The pyrimidine salvage enzyme uridine-cytidine kinase 2 (UCK2), a ∼29 kDa protein that forms a tetramer in its active state, is necessary for uridine salvage. Notwithstanding the pharmacological potential of this target, no medicinally tractable inhibitors of the human enzyme have been reported to date. We therefore established and miniaturized an in vitro assay for UCK2 activity and undertook a high-throughput screen against a ∼40,000-compound library to generate drug-like leads. The structures, activities, and modes of inhibition of the most promising hits are described. Notably, our screen yielded non-competitive UCK2 inhibitors which were able to suppress nucleoside salvage in cells both in the presence and absence of DHODH inhibitors.     

The structures of human dihydroorotate dehydrogenase with and without inhibitor reveal conformational flexibility in the inhibitor and substrate binding sites

Inhibitors of dihydroorotate dehydrogenase (DHODH) have been suggested for the treatment of rheumatoid arthritis, psoriasis, autoimmune diseases, Plasmodium, and bacterial and fungal infections. Here we present the structures of N-terminally truncated (residues Met30-Arg396) DHODH in complex with two inhibitors: a brequinar analogue (6) and a novel inhibitor (a fenamic acid derivative) (7), as well as the first structure of the enzyme to be characterized without any bound inhibitor. It is shown that 7 uses the "standard" brequinar binding mode and, in addition, interacts with Tyr356, a residue conserved in most class 2 DHODH proteins. Compared to the inhibitor-free structure, some of the amino acid side chains in the tunnel in which brequinar binds and which was suggested to be the binding site of ubiquinone undergo changes in conformation upon inhibitor binding. Using our data, the loop regions of residues Leu68-Arg72 and Asn212-Leu224, which were disordered in previously studied human DHODH structures, could be built into the electron density. The first of these loops, which is located at the entrance to the inhibitor-binding pocket, shows different conformations in the three structures, suggesting that it may interfere with inhibitor/cofactor binding. The second loop has been suggested to control the access of dihydroorotate to the active site of the enzyme and may be an important player in the enzymatic reaction. These observations provide new insights into the dynamic features of the DHODH reaction and suggest new approaches to the design of inhibitors against DHODH.