Functional analysis of the pyrimidine de novo synthesis pathway in solanaceous species.

Pyrimidines are particularly important in dividing tissues as building blocks for nucleic acids, but they are equally important for many biochemical processes, including sucrose and cell wall polysaccharide metabolism. In recent years, the molecular organization of nucleotide biosynthesis in plants has been analyzed. Here, we present a functional analysis of the pyrimidine de novo synthesis pathway. Each step in the pathway was investigated using transgenic plants with reduced expression of the corresponding gene to identify controlling steps and gain insights into the phenotypic and metabolic consequences. Inhibition of expression of 80% based on steady-state mRNA level did not lead to visible phenotypes. Stepwise reduction of protein abundance of Asp transcarbamoylase or dihydro orotase resulted in a corresponding inhibition of growth. This was not accompanied by pleiotropic effects or by changes in the developmental program. A more detailed metabolite analysis revealed slightly different responses in roots and shoots of plants with decreased abundance of proteins involved in pyrimidine de novo synthesis. Whereas in leaves the nucleotide and amino acid levels were changed only in the very strong inhibited plants, the roots show a transient increase of these metabolites in intermediate plants followed by a decrease in the strong inhibited plants. Growth analysis revealed that elongation rates and number of organs per plant were reduced, without large changes in the average cell size. It is concluded that reduced pyrimidine de novo synthesis is compensated for by reduction in growth rates, and the remaining nucleotide pools are sufficient for running basic metabolic processes.

Characterization of the dihydroorotate dehydrogenase as a soluble fumarate reductase in Trypanosoma cruzi.

Trypanosoma cruzi, a protozoan causing Chagas' disease, excretes a considerable amount of succinate even though it uses the TCA cycle and the aerobic respiratory chain. For this reason, it was believed that unknown metabolic pathways participate in succinate production in this parasite. In the present study, we examined the molecular properties of dihydroorotate dehydrogenase (DHOD), the fourth enzyme of de novo pyrimidine biosynthetic pathway, as a soluble fumarate reductase (FRD) because our sequence analysis of pyr genes cluster showed that the amino acid sequence of T. cruzi DHOD is quite similar to that of type 1A DHOD of Saccharomyces cerevisiae, an enzyme that uses fumarate as an electron acceptor and produces succinate. Biochemical analyses of the cytosolic enzyme purified from the parasite and of the recombinant enzyme revealed that T. cruzi DHOD has methylviologen-fumarate reductase (MV-FRD) activity. In addition, T. cruzi DHOD was found to catalyze electron transfer from dihydroorotate to fumarate by a ping-pong Bi-Bi mechanism. The recombinant enzyme contained FMN as a prosthetic group. Dynamic light scattering analysis indicated that T. cruzi DHOD is a homodimer. These results clearly indicated that the cytosolic MV-FRD is attributable to T. cruzi DHOD. The DHOD may play an important role in succinate/fumarate metabolism as well as de novo pyrimidine biosynthesis in T. cruzi.

Leflunomide: mode of action in the treatment of rheumatoid arthritis.

Leflunomide is a selective inhibitor of de novo pyrimidine synthesis. In phase II and III clinical trials of active rheumatoid arthritis, leflunomide was shown to improve primary and secondary outcome measures with a satisfactory safety profile. The active metabolite of leflunomide, A77 1726, at low, therapeutically applicable doses, reversibly inhibits dihydroorotate dehydrogenase (DHODH), the rate limiting step in the de novo synthesis of pyrimidines. Unlike other cells, activated lymphocytes expand their pyrimidine pool by approximately eightfold during proliferation; purine pools are increased only twofold. To meet this demand, lymphocytes must use both salvage and de novo synthesis pathways. Thus the inhibition of DHODH by A77 1726 prevents lymphocytes from accumulating sufficient pyrimidines to support DNA synthesis. At higher doses, A77 1726 inhibits tyrosine kinases responsible for early T cell and B cell signalling in the G(0)/G(1) phase of the cell cycle. Because the immunoregulatory effects of A77 1726 occur at doses that inhibit DHODH but not tyrosine kinases, the interruption of de novo pyrimidine synthesis may be the primary mode of action. Recent evidence suggests that the observed anti-inflammatory effects of A77 1726 may relate to its ability to suppress interleukin 1 and tumour necrosis factor alpha selectively over their inhibitors in T lymphocyte/monocyte contact activation. A77 1726 has also been shown to suppress the activation of nuclear factor kappaB, a potent mediator of inflammation when stimulated by inflammatory agents. Continuing research indicates that A77 1726 may downregulate the glycosylation of adhesion molecules, effectively reducing cell-cell contact activation during inflammation.

Malarial dihydroorotate dehydrogenase mediates superoxide radical production.

Dihydroorotate dehydrogenase purified from mitochondria of Plasmodium berghei, a rodent malaria parasite, mediates production of superoxide radical during oxidation of dihydroorotate to orotate. Reduction of dichlorophenolindophenol or cytochrome c or nitroblue tetrazolium was significantly inhibited by superoxide dismutase or theonyltrifluoroacetone, a specific iron chelator of the enzyme. These results, together with the recent evidence of manganese-superoxide dismutase activity in malarial mitochondria [Ranz, A., and Meshnick, S.R. (1989) Exp. Parasitol. 69, 125-128], suggest that the production of superoxide radical may occur in vivo.

Purification and characterization of dihydroorotate dehydrogenase from the rodent malaria parasite Plasmodium berghei.

Dihydroorotate dehydrogenase (DHODase) has been purified 400-fold from the rodent malaria parasite Plasmodium berghei to apparent homogeneity by Triton X-100 solubilization followed by anion-exchange, Cibacron Blue F3GA-agarose affinity, and gel filtration chromatography. The purified enzyme has a molecular mass of 52 +/- 2 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and of 55 +/- 6 kDa by gel filtration chromatography, and it has a pI of 8.2. It is active in monomeric form, contains 2.022 mol of iron and 1.602 acid-labile sulfurs per mole of enzyme, and does not contain a flavin cofactor. The purified DHODase exhibits optimal activity at pH 8.0 in the presence of the ubiquinone coenzyme CoQ6, CoQ7, CoQ9, or CoQ10. The Km values for L-DHO and CoQ6 are 7.9 +/- 2.5 microM and 21.6 +/- 5.5 microM, respectively. The kcat values for both substrates are 11.44 min-1 and 11.70 min-1, respectively. The reaction product orotate and an orotate analogue, 5-fluoroorotate, are competitive inhibitors of the enzyme-catalyzed reaction with Ki values of 30.5 microM and 34.9 microM, respectively. The requirement of the long-chain ubiquinones for activity supports the hypothesis of the linkage of pyrimidine biosynthesis to the electron transport system and oxygen utilization in malaria by DHODase via ubiquinones [Gutteridge, W. E., Dave, D., & Richards, W. H. G. (1979) Biochim. Biophys. Acta 582, 390-401].

Synthesis and antiproliferative activity of threo-5-fluoro-L-dihydroorotate.

Fluorinated compounds play an important role in enzymology as well as clinical medicine. Based on the stereochemical preferences of dihydroorotate oxidase and enzymes that use fluoroaspartate, it was anticipated that threo-5-fluoro-L-dihydroorotate (t-FDHO) would have the properties of an antimetabolite. Thus, t-FDHO was synthesized via the reduction of 5-fluoroorotate using NADH and dihydroorotate dehydrogenase that was free of dihydroorotase. When the product was purified and studied by high field proton and carbon 13 NMR, the fluorine, the five carbons, and all the nonexchangeable protons were readily observed. Confirmation of threo configuration was obtained by examining the vicinal coupling constants between the substituents on carbon 5 and carbon 6 of the newly synthesized compound. Additionally, t-FDHO could be reoxidized to 5-fluoroorotate in the presence of dihydroorotate dehydrogenase and NAD+. Treatment of t-FDHO with dihydroorotase generated N-carbamyl-threo-3-fluoro-L-aspartate (CTF-ASP) which was also purified and characterized by NMR. The antiproliferative activity of t-FDHO was determined against a diploid human fibrosarcoma cell line (HT-1080). Fifty microM t-FDHO caused 50% inhibition of HT-1080 cell proliferation. During the 48-h toxicity study, extracellular t-FDHO underwent significant hydrolysis to CTF-ASP. Further extracellular degradation to fluoroaspartate was not seen. The antiproliferative activity of t-FDHO was not due to extracellular degradation since CTF-ASP itself was essentially nontoxic.

Glutamine-dependent carbamoyl-phosphate synthetase and other enzyme activities related to the pyrimidine pathway in spleen of Squalus acanthias (spiny dogfish).

The first two steps of urea synthesis in liver of marine elasmobranchs involve formation of glutamine from ammonia and of carbamoyl phosphate from glutamine, catalysed by glutamine synthetase and carbamoyl-phosphate synthetase, respectively [Anderson & Casey (1984) J. Biol. Chem. 259, 456-462]; both of these enzymes are localized exclusively in the mitochondrial matrix. The objective of this study was to establish the enzymology of carbamoyl phosphate formation and utilization for pyrimidine nucleotide biosynthesis in Squalus acanthias (spiny dogfish), a representative elasmobranch. Aspartate carbamoyltransferase could not be detected in liver of dogfish. Spleen extracts, however, had glutamine-dependent carbamoyl-phosphate synthetase, aspartate carbamoyltransferase, dihydro-orotase, and glutamine synthetase activities, all localized in the cytosol; dihydro-orotate dehydrogenase, orotate phosphoribosyltransferase, and orotidine-5'-decarboxylase activities were also present. Except for glutamine synthetase, the levels of all activities were very low. The carbamoyl-phosphate synthetase activity is inhibited by UTP and is activated by 5-phosphoribosyl 1-pyrophosphate. The first three enzyme activities of the pyrimidine pathway were eluted in distinctly different positions during gel filtration chromatography under a number of different conditions; although complete proteolysis of inter-domain regions of a multifunctional complex during extraction cannot be excluded, the evidence suggests that in dogfish, in contrast to mammalian species, these three enzymes of the pyrimidine pathway exist as individual polypeptide chains. These results: (1) establish that dogfish express two different glutamine-dependent carbamoyl-phosphate synthetase activities, (2) confirm the report [Smith, Ritter & Campbell (1987) J. Biol. Chem. 262, 198-202] that dogfish express two different glutamine synthetases, and (3) provide indirect evidence that glutamine may not be available in liver for biosynthetic reactions other than urea formation.

Analysis of the kinetic mechanism of the bovine liver mitochondrial dihydroorotate dehydrogenase.

The steady-state kinetic mechanism of highly purified bovine liver mitochondrial dihydroorotate dehydrogenase has been investigated. Initial velocity analysis using S-dihydroorotate and coenzyme Q6 revealed parallel-line, double-reciprocal plots, indicative of a ping-pong mechanism. The dead-end inhibition pattern with barbituric acid and the reactions with alternate cosubstrates methyl-S-dihydroorotate and menadione also point to a ping-pong mechanism. However, product orotate was found to be competitive with dihydroorotate and uncompetitive with Q6. These findings suggest that dihydroorotate dehydrogenase may follow a nonclassical, two-site ping-pong mechanism, typical of an enzyme that contains two non-overlapping and kinetically isolated substrate binding sites. That these two sites communicate by an intramolecular electron-transfer system involving FMN and perhaps an iron-sulfur center is also suggested by the kinetic behavior of the enzyme.

Mechanistic studies on the bovine liver mitochondrial dihydroorotate dehydrogenase using kinetic deuterium isotope effects.

Dihydroorotates deuteriated at both C5 and C6 have been prepared and used to probe the mechanism of the bovine liver mitochondrial dihydroorotate dehydrogenase. Primary deuterium isotope effects on kcat are observed with both (6RS)-[5(S)-2H]- and (6RS)-[6-2H] dihydroorotates (3 and 6, respectively); these effects are maximal at low pH. At pH 6.6, DV = 3.4 for the C5-deuteriated dihydroorotate (3), and DV = 2.3 for the C6-deuteriated compound (6). The isotope effects approach unity at pH 8.8. Analysis of the pH dependence of the isotope effects on kcat reveals a shift in the rate-determining step of the enzyme mechanism as a function of pH. Dihydroorotate oxidation appears to require general base catalysis (pKB = 8.26); this step is completely rate-determining at low pH and isotopically sensitive. Reduction of the cosubstrate, coenzyme Q6, is rate-limiting at high pH and is isotopically insensitive; this step appears to require general acid catalysis (pKA = 8.42). The results of double isotope substitution studies and analysis for substrate isotope exchange with solvent point toward a concerted mechanism for oxidation of dihydroorotate. This finding serves to distinguish further the mammalian dehydrogenase from its parasitic cognate, which catalyzes a stepwise oxidation reaction [Pascal, R., & Walsh, C.T. (1984) Biochemistry 23, 2745].

Cellular pharmacology of DUP-785, a new anticancer agent.

DUP-785, a new inhibitor of dihydroorotate dehydrogenase, is currently undergoing clinical evaluation for anticancer activity. We developed a GC/MS method to quantitate dihydroorotate that accumulates in cultures of L1210 cells exposed to growth inhibitory concentrations of DUP-785. This method was used to follow the onset, extent, and duration of inhibition of de novo pyrimidine synthesis in intact L1210 cells and to compare this inhibition with cell proliferation and cellular concentrations of pyrimidine nucleotides. There were direct relations between inhibition of de novo pyrimidine synthesis, changes in pyrimidine nucleotide concentrations, and cell proliferation following short (less than 24 hr) drug exposures; with prolonged exposures (greater than 24 hr), however, there was a departure from these relationships in that restoration of pyrimidine nucleotide pools and de novo pyrimidine pathway activity did not restore cell proliferation. Exposure of L1210 cells to 15 microM DUP-785 produced a maximum cell kill (99.9% as determined by cloning efficiency) at 24 hr, and no increase in cell kill was observed with drug exposure up to 96 hr.

Development and characterization of continuous avian cell lines depleted of mitochondrial DNA.

Populations of quail and chicken cells were treated with ethidium bromide, an inhibitor of mitochondrial DNA replication. After long-term exposure to the drug, the cell populations were transferred to ethidium bromide (EtdBr)-free medium, and cloned. Clones HCF7 (quail) and DUS-3 (chicken) were propagated for more than a year, and then characterized. Analysis of total cellular DNA extracted from these cells revealed no characteristic mitochondrial DNA molecule by Southern blot hybridization of HindIII- or AvaI-digested total cellular DNA probed with cloned mitochondrial DNA fragments. Reconstruction experiments, where a small number of parental cells was mixed with HCF7 cells and DUS-3 cells before extraction of total cellular DNA, further strengthen the notion that the drug-treated cells are devoid of mitochondrial DNA molecules. The cell populations were found to proliferate at a moderately reduced growth rate as compared to their respective parents, to be auxotrophic for uridine, and to be stably resistant to the growth inhibitory effect of EtdBr and chloramphenicol. At the ultrastructural level, mitochondria were considerably enlarged and there was a severe reduction in the number of cristae within the organelles and loss of cristae orientation. Morphometric analysis revealed a fourfold increase of the mitochondrial profile area along with a twofold decrease of the numerical mitochondrial profiles. Analysis of biochemical parameters indicated that the cells grew with mitochondria devoid of a functional respiratory chain. The activity of the mitochondrial enzyme dihydroorotate dehydrogenase was decreased by 95% and presumably accounted for uridine auxotrophy.

Dihydroorotic acid dehydrogenase activity in actinomycin-D-treated and normal chick embryos.

The effect of actinomycin D on chick embryos cultivated in vitro by New's culturing method was studied. Exposure of chick embryos to actinomycin D (0.05 micrograms/ml) at the primitive streak stage (stage 4; Hamburger and Hamilton) for 6 h showed interference in orotic acid formation. The assay of the enzyme dihydroorotic acid dehydrogenase was carried out in both treated and control embryos. No enzymic activity was observed in actinomycin-D-treated embryos in contrast to the considerable activity in the controls. These observations suggest an interference by actinomycin D in the biogenesis of the enzyme dihydroorotic acid dehydrogenase.

Human spleen dihydroorotate dehydrogenase: properties and partial purification.

Human spleen dihydroorotate dehydrogenase is associated with the mitochondrial membrane and is linked to the respiratory chain via ubiquinone. The enzyme activity was unaffected by pyridine nucleotides. The product of the reaction, orotate, was a potent inhibitor. However, a range of other naturally occurring pyrimidines or purines had no significant effect on the activity. No evidence for the involvement of a complexed metal ion or for an active sulfhydryl group was obtained. Purification of the enzyme was achieved by preparation of an acetone powder and extraction with Triton X-100, followed by preparative polyacrylamide gel electrophoresis. Activity was observed by the addition of the artificial electron acceptors, ubiquinone 50 or PMS. Purification resulted in alteration of the pH optimum and of other kinetic characteristics. Two molecular-weight species, of molecular weight 88,000 and 98,000, were consistently observed. The properties of the human spleen enzyme were similar in principle to those for the rat liver enzyme. Differences in the mode of linkage to the respiratory chain for the mitochondrially bound enzyme, and in the characteristics of the purified enzyme, were observed.

Detection of dihydroorotate dehydrogenase activity as a marker of de novo nucleic acid synthesis in germinal centers of spleens following antigen administration.

A histochemical technique, developed for the localization of DHO-D activity in tissue, has been utilized to study the distribution of this activity in rabbit spleen. This enzyme is involved in the de novo synthesis of pyrimidines of nucleic acids via orotic acid as an intermediate. The enzyme was found to be most active at the periphery of follicles in the resting spleen. Following antigenic challenge, enzyme activity became prominent for several days in macrophages within germinal centers, but not in the intensely pyroninophilic cells which are the dominant cell type of these structures. Antigenicity of the injected compound appeared to be necessary for the production of this effect. The pattern of localization of DHO-D is similar to the pattern of distribution of antigen within splenic tissue.

Yeast regulatory gene PPR1. II. Chromosomal localization, meiotic map, suppressibility, dominance/recessivity and dosage effect.

The Saccharomyces cerevisiae gene PPR1 encodes a positive regulator of the expression of the two unlinked structural genes URA1 and URA3. The gene has been mapped to a position 6.5 cM from the centromere of chromosome XII. Uninducible alleles have been selected and used to establish a meiotic map. Suppressible alleles have been identified. The sequencing of a suppressible allele confirms the nonsense nature of the mutation as well as the reading frame deduced from the nucleotide sequence. No evidence of intracistronic complementation was found, and enzymatic analysis of leaky mutants did not reveal any mutations dissociating regulation of URA1 from that of URA3. Three in vitro-constructed deletions of PPR1 have been integrated at the chromosomal locus, giving strains with a completely negative phenotype. These deletion mutants display the wild-type basal level of URA1 and URA3 expression and show a semi-dominant phenotype in heteroallelic ppr1+/ppr1-delta diploids. Amplifying PPR1 by introduction into yeast on a multicopy vector increases the induction factor of URA1 and URA3 expression. These results show that the extent of regulation of the two structural genes is dependent on the concentration of the active PPR1 protein.

Lesch-Nyhan syndrome, caffeine model: increase of purine and pyrimidine enzymes in rat brain.

Rats ingesting high doses of caffeine reproduce the self-destructive behaviour observed in the Lesch-Nyhan syndrome. This syndrome includes a deficit in hypoxanthine-guanine phosphoribosyltransferase. We have observed, however, that the activity of hypoxanthine-guanine phosphoribosyltransferase increases in direct proportion to the concentration of caffeine found in rat brain. It appears, therefore, that the caffeine model is not a true model for the Lesch-Nyhan syndrome, or alternatively, that the deficit in hypoxanthine-guanine phosphoribosyltransferase is coincidental and not a main key to the multifarious aspects of the syndrome, particularly the self-mutilation. The possibility that levels of dopamine are increased in the caffeine model are discussed as a basis for the destructive behaviour. We have found also that ingestion of large amounts of caffeine increases the activities in rat brain of adenosine deaminase, purine nucleoside phosphorylase, aspartate carbamoyl-transferase, dihydroorotase, and dihydroorotate oxidase.

Pyrimidine de novo synthesis during the life cycle of the intraerythrocytic stage of Plasmodium falciparum.

The 6 enzymes involved in de novo synthesis of pyrimidines were measured in Plasmodium falciparum isolated by saponin lysis from RBC's nonsynchronized and synchronized in vitro cultures. The total activities were found to be dependent on the stage of the P. falciparum cycle. In parasites isolated from synchronized cultures, the highest activities for all enzymes were found at about 27 hr after synchronization in the late trophozoite stage, or just before schizont formation. Merozoites and ring forms contained little de novo activity. The first enzyme of the pathway, carbamyl phosphate synthetase (CPS-II) preferentially utilized glutamine. Ammonia was a poor substrate. CPS-II was unstable in the absence of the cryoprotectants, dimethylsulfoxide and glycerol. The apparent Km for MgATP--was 3.8 +/- 0.7 mM and the enzyme in all morphological forms of P. falciparum (ring, mature trophozoites and schizonts) was inhibited by UTP. The activity of the fourth enzyme of the pathway, dihydroorotate dehydrogenase, appeared to be linked to the cell's respiratory chain; inhibitors of mammalian electron transport such as cyanide, amytal, antimycin A, thenoyltrifluoroacetone and ubiquinone analogs also inhibited the P. falciparum enzyme. The demonstration of the variation of activity of the pyrimidine enzymes correlates with the increased synthesis of nucleic acids in the late trophozoite stage. These observations provide a basis for the testing of the effectiveness of pyrimidine analogs as potential antimetabolites against various forms of the parasite.

Cyclic modulation of enzymes of pyrimidine nucleotide biosynthesis precedes sialoglycoconjugate changes during 2-acetylaminofluorene-induced hepatocarcinogenesis in the rat.

Three enzymatic activities associated with pyrimidine nucleotide biosynthesis were monitored at weekly or bi-weekly intervals during 2-acetylaminofluorene- (0.025% in a Farber Basal Carcinogenic diet) induced hepatocarcinogenesis in the rat. Dihydroorotate dehydrogenase, the fourth of six enzymes in de novo pyrimidine biosynthesis, declined in activity while UDP kinase and CTP synthetase showed sequential increases in activity. The alterations in activity appeared to be cyclic, followed by a full or partial return to control values. Three full cycles were monitored. The first cycle preceded nodule formation. The second cycle accompanied nodule formation and preceded sialoglycoconjugate changes reported previously. The third cycle accompanied the early glycoconjugate changes. The cyclic pattern was reproducible in three separate experiments. In each cycle, the order of events was as follows: decrease in dihydroorotate dehydrogenase, sequential increases in UDP kinase, CTP synthetase and CMPsialic acid synthase, and finally increases in the enzyme lactosylceramide: CMPsialic acid sialyltransferase, lipid-soluble sialic acid and total sialic acid. In livers of animals fed 1.87% of the hepatotoxin, 4-acetamidophenol, no biochemical alterations resembling those induced by 2-acetylaminofluorene were obtained, despite acute centrilobular necrosis of the livers. The findings point to a biochemical cascade beginning with administration of carcinogen and continuing through the development of hyperplastic nodules and of frank carcinomas resulting not from hepatotoxicity but as events associated with the hepatocarcinogenic progression.