Critical importance of the de novo pyrimidine biosynthesis pathway for Trypanosoma cruzi growth in the mammalian host cell cytoplasm

The intracellular parasitic protist Trypanosoma cruzi is the causative agent of Chagas disease in Latin America. In general, pyrimidine nucleotides are supplied by both de novo biosynthesis and salvage pathways. While epimastigotes-an insect form-possess both activities, amastigotes-an intracellular replicating form of T. cruzi-are unable to mediate the uptake of pyrimidine. However, the requirement of de novo pyrimidine biosynthesis for parasite growth and survival has not yet been elucidated. Carbamoyl-phosphate synthetase II (CPSII) is the first and rate-limiting enzyme of the de novo biosynthetic pathway, and increased CPSII activity is associated with the rapid proliferation of tumor cells. In the present study, we showed that disruption of the T. cruzi cpsII gene significantly reduced parasite growth. In particular, the growth of amastigotes lacking the cpsII gene was severely suppressed. Thus, the de novo pyrimidine pathway is important for proliferation of T. cruzi in the host cell cytoplasm and represents a promising target for chemotherapy against Chagas disease.     

The dihydroorotase domain of the multifunctional protein CAD. Subunit structure, zinc content, and kinetics

Dihydroorotase (DHOase) catalyzes the third step in eukaryotic de novo pyrimidine biosynthesis. In mammalian cells, this enzyme activity is carried by a large chimeric protein, CAD, that also catalyzes the first two steps in the pathway: glutamine-dependent carbamyl phosphate synthetase (CPSase) and aspartate transcarbamylase (ATCase). Controlled elastase cleavage of CAD released a 44,000 +/- 2,000-dalton proteolytic fragment which catalyzed only the dihydroorotase reaction. We have devised a rapid and simple method for the isolation of the DHO domain from elastase digests. The domain, which was obtained in 36% yield, was found to be homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing. The domain was also characterized by amino acid analysis and analytical high pressure liquid chromatography peptide mapping. The amino terminus of both the DHO domain and intact CAD was blocked suggesting that this domain is located at the extreme amino terminus of the CAD polypeptide, a result consistent with the suspected juxtaposition of domains as DHO-CPS-ATC. The isoelectric point of the DHO domain was 5.1, while that of the ATC domain was 9.4, so that the ends of the CAD polypeptide are oppositely charged at physiological pH. Immunoblotting with DHO domain-specific antibodies showed that a 47-kDa species was generated in the early stages of controlled proteolysis of CAD. Thus there are two elastase cleavage sites within a 3-kDa connecting region that links the DHO and CPS domains. The domain was shown by atomic absorption spectrophotometry and by isolating a 65Zn-containing DHO domain from mammalian cells grown in the presence of the radionuclide to contain 1 g eq of tightly bound zinc in each polypeptide chain. Zinc was not found in any other CAD domain. Chelating agents inhibit dihydroorotase activity of the isolated domain supporting the conclusion, based on studies of intact CAD by others, that zinc participates in catalysis. At moderate protein concentrations the DHO domain was a 88,000 dimer with a Stokes radius of 37.6 A, a S20,w = 5.1 X 10(-13) s, a diffusion coefficient of 3.17 X 10(-7) cm2 s-1, and a frictional ratio of 1.26. On dilution the dimer dissociated and was in rapid concentration-dependent equilibrium with a 43,500 monomer. The hydrodynamic parameters of the monomer have also been estimated (Stokes radius of 29.8 A, D20,w = 4.11 X 10(-7) cm2 s-1, and f/f0 1.21).(ABSTRACT TRUNCATED AT 400 WORDS)     

Intersubunit communication in the dihydroorotase–aspartate transcarbamoylase complex of Aquifex aeolicus

Aspartate transcarbamoylase and dihydroorotase, enzymes that catalyze the second and third step in de novo pyrimidine biosynthesis, are associated in dodecameric complexes in Aquifex aeolicus and many other organisms. The architecture of the dodecamer is ideally suited to channel the intermediate, carbamoyl aspartate from its site of synthesis on the ATC subunit to the active site of DHO, which catalyzes the next step in the pathway, because both reactions occur within a large, internal solvent‐filled cavity. Channeling usually requires that the reactions of the enzymes are coordinated so that the rate of synthesis of the intermediate matches its rate of utilization. The linkage between the ATC and DHO subunits was demonstrated by showing that the binding of the bisubstrate analog, N‐phosphonacetyl‐L ‐aspartate to the ATC subunit inhibits the activity of the distal DHO subunit. Structural studies identified a DHO loop, loop A, interdigitating between the ATC domains that would be expected to interfere with domain closure essential for ATC catalysis. Mutation of the DHO residues in loop A that penetrate deeply between the two ATC domains inhibits the ATC activity by interfering with the normal reciprocal linkage between the two enzymes. Moreover, a synthetic peptide that mimics that part of the DHO loop that binds between the two ATC domains was found to be an allosteric or noncompletive ATC inhibitor (K_i = 22 μM). A model is proposed suggesting that loop A is an important component of the functional linkage between the enzymes.     

The evolutionary history of the first three enzymes in pyrimidine biosynthesis

Some metabolic pathways are nearly ubiquitous among organisms: the genes encoding the enzymes for such pathways must therefore be ancient and essential. De novo pyrimidine biosynthesis is an example of one such metabolic pathway. In animals a single protein called CAD

1 Abbreviations: CAD, trifunctional protein catalyzing the first three steps of de novo pyrimidine biosynthesis in higher eukaryotes; CPS, carbamyl phosphate synthetase domain; CPSase, carbamyl phosphate synthetase activity; ATC, aspartate transcarbamylase domain; ATCase, aspartate transcarbamylase activity; DHO, dihydroorotase domain; DHOase, dihydroorotase activity; GLN, glutaminase subdomain or subunit of carbamyl phosphate synthetase, GL Nase, glutaminase activity; SYN, synthetase subdomain or subunit of carbamyl phosphate synthetase; SYNase, synthetase activity.

carries the first three steps of this pathway. The same three enzymes in prokaryotes are associated with separate proteins. The CAD gene appears to have evolved through a process of gene duplication and DNA rearrangement, leading to an in-frame gene fusion encoding a chimeric protein. A driving force for the creation of eukaryotic genes encoding multienzymatic proteins such as CAD may be the advantage of coordinate expression of enzymes catalyzing steps in a biosynthetic pathway. The analogous structure in bacteria is the operon. Differences in the translational mechanisms of eukaryotes and prokaryotes may have dictated the different strategies used by organisms to evolve coordinately regulated genes.     

PHYLOGENETIC IMPLICATIONS OF THE CAD COMPLEX FROM THE PRIMITIVE RED ALGA CYANIDIOSCHYZON MEROLAE (CYANIDIALES,RHODOPHYTA)1

The de novo pyrimidine biosynthetic pathway consists of six enzymes: carbamoyl‐phosphate synthetase II (CPS II), aspartate carbamoyltransferase (ACT), dihydroorotase (DHO), dihydroorotate dehydrogenase, orotate phosphoribosyltransferase, and orotidine‐5′‐monophosphate decarboxylase. The origin and organization of the first three enzymes differ markedly between Opisthokonta (Metazoa and Fungi) and the Amoebozoa and green plants. However, no information has been available regarding the characteristics of such genes in other photosynthetic eukaryotes. In this study, we examined the pyrimidine biosynthetic cluster in the primitive red alga Cyanidioschyzon merolae P. DeLuca et al. isolate 10D. Unlike the situation in green plants, the CPS II, ACT, and DHO of C. merolae were fused to form a single open reading frame (the CAD complex), as in the Opisthokonta and Amoebozoa. Phylogenetic analysis of the CPS domain sequences suggested that this red algal CAD complex did not result from a recent lateral gene transfer from Metazoa or Fungi but that the fusion of the three genes occurred before the divergence of Opisthokonta, Amoebozoa, and the red algae. These results cast doubt on the recent hypothesis that the Opisthokonta and Amoebozoa form a monophyletic group, based on the presence in both of the CAD complex.     

Biochemical genetic analysis of pyrimidine biosynthesis in mammalian cells. II. Isolation and characterization of a mutant of Chinese hamster ovary cells with defective dihydroorotate dehydrogenase (E.C. 1.3.3.1) activity.

A mutant (A204) of Chinese hamster ovary cells (CHO-K1), which is deficient in dihydroorotate (DHO) dehydrogenase (E.C. 1.3,3.1) activity, has been isolated by a replica plating procedure. The mutant does not show a requirement for exogenously added pyrimidines. Examination of intact cells shows that the mutant accumulates a large amount of carbamyl aspartate and is markedly but not totally deficient in biosynthesis of orotate from earlier precursors of pyrimidine biosynthesis, including aspartate and dihydroorotic acid, when compared to wild-type cells. Analysis of cell-free extracts of mutant and wild-type cells shows that the mutant is deficient in DHO dehydrogenase activity, possessing ca. 5% of the wild-type activity. this evidence leads to the conclusion that this mutant, A204, is in fact partially deficient in DHO dehydrogenase, and that in these cells it is this enzyme which carries out the fourth step of de novo pyrimidine biosynthesis.     

Immunochemical analysis of the domain structure of CAD, the multifunctional protein that initiates pyrimidine biosynthesis in mammalian cells

CAD, is a multidomain polypeptide, with a molecular weight of over 200,000, that has glutamine-dependent carbamyl-phosphate synthetase, aspartate transcarbamylase, and dihydroorotase activity as well as regulatory sites that bind UTP and 5-phosphoribosyl 1-pyrophosphate. The protein thus catalyzes the first three steps of de novo pyrimidine biosynthesis and controls the activity of the pathway in higher eukaryotes. Controlled proteolysis of CAD isolated from Syrian hamster cells, cleaves the molecule into seven major proteolytic fragments that contain one or more of the functional domains. The two smallest fragments, which had molecular weights of 44,000 and 40,000, corresponded to the fully active dihydroorotase (DHO) and aspartate transcarbamylase (ATC) domains, respectively, but the larger fragments have not been previously characterized. In this study, enzymatic assays of partially fractionated digests and immunoblotting with antibodies specifically directed against the purified ATC domain, the purified dihydroorotase domain and an 80-kDa fragment of the putative carbamyl-phosphate synthetase domain established the precursor-product relationships among all of the major proteolytic fragments of CAD. These results indicate that 1) only the intact molecule had all of the functional domains, 2) a species with a molecular weight of 200,000 was produced in the first step of proteolysis which had glutamine-dependent carbamyl-phosphate synthetase and dihydroorotase activity, but neither aspartate transcarbamylase activity nor the antigenic determinants present on the isolated ATC domain, and 3) cleavage of the 200-kDa species produced a species, with a molecular mass of 150,000 which lacked both aspartate transcarbamylase and dihydroorotase domains. This 150-kDa species, containing the postulated carbamyl-phosphate synthetase, glutamine, and regulatory (UTP, 5-phosphoribosyl 1-pyrophosphate) domains, had two elastase-sensitive sites that divided this region of the polypeptide chain into 10-, 65-, and 80-kDa segments. The location of the functional sites on these segments has not yet been established. The immunochemical analysis also revealed the existence of possible precursors of the stable aspartate transcarbamylase and dihydroorotase domains, suggesting that the chain segments connecting the functional domains of CAD are extensive and that the overall size of the intact polypeptide chain has been underestimated. On the basis of these studies we have proposed a model of the domain structure of CAD.     

Evolutionary implications of the mosaic pyrimidine-biosynthetic pathway in eukaryotes

The de-novo pyrimidine biosynthetic pathway involves six enzymes, in order from the first to the sixth step, carbamoyl-phosphate synthetase II (CPS II) comprising glutamine amidotransferase (GAT) and carbamoyl-phosphate synthetase (CPS) domains or subunits, aspartate carbamoyltransferase (ACT), dihydroorotase (DHO), dihydroorotate dehydrogenase (DHOD), orotate phosphoribosyltransferase (OPRT), and orotidine-5'-monophosphate decarboxylase (OMPDC). In contrast with reports on molecular evolution of the individual enzymes, we attempted to draw an evolutionary picture of the whole pathway using the protein phylogeny. We demonstrate highly mosaic organizations of the pyrimidine biosynthetic pathway in eukaryotes. During evolution of the eukaryotic pathway, plants and fungi (or their ancestors) in particular may have secondarily acquired the characteristic enzymes. This is consistent with the fact that the organization of plant enzymes is highly chimeric: (1) two subunits of CPS II, GAT and CPS, cluster with a clade including cyanobacteria and red algal chloroplasts, (2) ACT not with a cyanobacterium, Synechocystis spp., irrespective of its putative signal sequence targeting into chloroplasts, and (3) DHO with a clade of proteobacteria. In fungi, DHO and OPRT cluster respectively with the corresponding proteobacterial counterparts. The phylogenetic analyses of DHOD and OMPDC also support the implications of the mosaic pyrimidine biosynthetic pathway in eukaryotes. The potential importance of the horizontal gene transfer(s) and endosymbiosis in establishing the mosaic pathway is discussed.     

Contribution of the bacterial endosymbiont to the biosynthesis of pyrimidine nucleotides in the deep-sea tube worm Riftia pachyptila

The deep-sea tube worm Riftia pachyptila (Vestimentifera) from hydrothermal vents lives in an intimate symbiosis with a sulfur-oxidizing bacterium. That involves specific interactions and obligatory metabolic exchanges between the two organisms. In this work, we analyzed the contribution of the two partners to the biosynthesis of pyrimidine nucleotides through both the "de novo" and "salvage" pathways. The first three enzymes of the de novo pathway, carbamyl-phosphate synthetase, aspartate transcarbamylase, and dihydroorotase, were present only in the trophosome, the symbiont-containing tissue. The study of these enzymes in terms of their catalytic and regulatory properties in both the trophosome and the isolated symbiotic bacteria provided a clear indication of the microbial origin of these enzymes. In contrast, the succeeding enzymes of this de novo pathway, dihydroorotate dehydrogenase and orotate phosphoribosyltransferase, were present in all body parts of the worm. This finding indicates that the animal is fully dependent on the symbiont for the de novo biosynthesis of pyrimidines. In addition, it suggests that the synthesis of pyrimidines in other tissues is possible from the intermediary metabolites provided by the trophosomal tissue and from nucleic acid degradation products since the enzymes of the salvage pathway appear to be present in all tissues of the worm. Analysis of these salvage pathway enzymes in the trophosome strongly suggested that these enzymes belong to the worm. In accordance with this conclusion, none of these enzyme activities was found in the isolated bacteria. The enzymes involved in the production of the precursors of carbamyl phosphate and nitrogen assimilation, glutamine synthetase and nitrate reductase, were also investigated, and it appears that these two enzymes are present in the bacteria.     

Pyrimidine nucleotide synthesis in the rat kidney in early diabetes.

Early renal hypertrophy of diabetes is associated with increases in the tissue content of RNA, DNA, and sugar nucleotides involved in the formation of carbohydrate-containing macromolecules. We have previously reported an increase in the activity of enzymes of the de novo and salvage pathways of purine synthesis in early diabetes; the present communication explores the changes in the pathways of pyrimidine synthesis. Measurements have been made of key enzymes of the de novo and salvage pathways at 3, 5, and 14 days after induction of diabetes with streptozotocin (STZ), phosphoribosyl pyrophosphate (PPRibP), and some purine and pyrimidine bases. Carbamoyl-phosphate synthetase II, the rate-limiting enzyme of the de novo route, did not increase in the first 5 days after STZ treatment, the period of most rapid renal growth; a significant rise was seen at 14 days (+38%). Dihydroorotate dehydrogenase, a mitochondrial enzyme, showed the most marked rise (+147%) at 14 days. The conversion of orotate to UMP, catalyzed by the enzymes of complex II, was increased at 3 days (+42%), a rise sustained to 14 days. The salvage route enzyme, uracil phosphoribosyltransferase (UPRTase), showed a pattern of change similar to complex II. The effect of the decreased concentration of PPRibP on the activities of CPSII, for which it is an allosteric activator, and on activities of OPRTase and UPRTase, for which it is an essential substrate, is discussed with respect to the relative Ka and Km values for PPRibP and the possibility of metabolite channeling.     

Transcriptional repression of human cad gene by hypoxia inducible factor-1alpha

De novo biosynthesis of pyrimidine nucleotides provides essential precursors for DNA synthesis and cell proliferation. The first three steps of de novo pyrimidine biosynthesis are catalyzed by a multifunctional enzyme known as CAD (carbamoyl phosphate synthetase-aspartate carbamoyltransferase-dihydroorotase). In this work, a decrease in CAD expression is detected in numerous cell lines and primary culture human stromal cells incubated under hypoxia or desferrioxamine (DFO)-induced HIF-1alpha accumulation. A putative hypoxia response element (HRE) binding matrix is identified by analyzing human cad-gene promoter using a bioinformatic approach. Promoter activity assays, using constructs harboring the cad promoter (-710/+122) and the -67/HRE fragment (25-bases), respectively, demonstrate the suppression of reporter-gene expression under hypoxia. Suppression of cad-promoter activity is substantiated by forced expression of wild-type HIF-1alpha but abolished by overexpression of dominant-negative HIF-1alpha. A chromatin immunoprecipitation assay provides further evidence that HIF-1alpha binds to the cad promoter in vivo. These data demonstrate that the cad-gene expression is repressed by HIF-1alpha, which represents a functional link between hypoxia and cell-cycle arrest.     

Pyrimidine metabolism of Bdellovibrio bacteriovorus grown intraperiplasmically and axenically.

Bdellovibrio bacteriovorus grown axenically or intraperiplasmically on Escherichia coli has pathways for the interconversion of pyrimidines and the synthesis of pyrimidine nucleoside 5'-triphosphates similar to those found in the enteric bacteria. Minimal differences in enzyme activities were observed for axenically and intraperiplasmically grown cells. As might be expected for an organism which takes up deoxyribonucleoside 5'-monophosphates per se, high levels of enzymes which catalyze the generation of deoxyribonucleoside triphosphates from monophosphates were found. In addition, all enzymes of the thymine salvage pathway, except for thymidine kinase, were directly demonstrated in wild-type strains. It was possible to demonstrate this activity only indirectly owing to an inhibitor in wild-type extracts. Investigations with inhibitors of pyrimidine interconversion reactions showed that essentially all B. bacteriovorus deoxyribonucleic acid not synthesized from units derived from E. coli deoxyribonucleic acid is made from components of the substrate organism's ribonucleic acid. Evidence for de novo pyrimidine synthesis from the amino acid level was not found for B. bacteriovorus grown on E. coli that had a high protein/deoxyribonucleic acid ratio or on normal E. coli. The potential for de novo pyrimidine synthesis by intraperiplasmically grown B. bacteriovorus, however, cannot be totally ruled out on the basis of these investigations.     

Regulation of pyrimidine nucleotide biosynthesis in Pseudomonas synxantha

Regulation of pyrimidine nucleotide biosynthesis in Pseudomonas synxantha ATCC 9890 was investigated and the pyrimidine biosynthetic pathway enzyme activities were affected by pyrimidine supplementation in cells grown on glucose or succinate as a carbon source. In pyrimidine-grown ATCC 9890 cells, the activities of four de novo enzymes could be depressed which indicated possible repression of enzyme synthesis. To learn whether the pathway was repressible, pyrimidine limitation experiments were conducted using an orotate phosphoribosyltransferase (pyrE) mutant strain identified in this study. Compared to excess uracil growth conditions for the succinate-grown mutant strain cells, pyrimidine limitation of this strain caused dihydroorotase activity to increase about 3-fold while dihydroorotate dehydrogenase and orotidine 5'-monophosphate decarboxylase activities rose about 2-fold. Regulation of de novo pathway enzyme synthesis by pyrimidines appeared to be occurring. At the level of enzyme activity, aspartate transcarbamoylase activity in P. synxantha ATCC 9890 was strongly inhibited in vitro by pyrophosphate, UTP, ADP, ATP, CTP and GTP under saturating substrate concentrations.     

Intracellular location of the multidomain protein CAD in mammalian cells

The first three steps of mammalian de novo pyrimidine biosynthesis are catalyzed by the multifunctional protein CAD, consisting of glutamine-dependent carbamylphosphate synthetase, aspartate transcarbamylase, and dihydroorotase. The intracellular distribution of CAD in two hamster cell lines, BHK 21 and BHK 165-23 (a strain in which the CAD gene was selectively amplified), was determined by differential centrifugation and by two different cytochemical immunolocalization methods. Ammonia-dependent carbamylphosphate synthetase I was found in both cell types at a concentration of 0.01% of the total cell protein, so its distribution was also determined as a control for possible cross-reactivity of the CAD antibody probes and as a mitochondrial marker. CAD was localized in the cytoplasmic compartment and almost completely excluded from the nucleus. A punctate staining pattern suggested that it was not uniformly dispersed throughout the cytosol (unlike typical soluble proteins) but was associated with subcellular organelles. Although there was a slight tendency for CAD to be localized in the vicinity of the nuclear envelope, the amount of staining was much less than expected from differential centrifugation, which showed that 30% of the protein was found in the nuclear fraction. No interactions with other subcellular components could be detected by centrifugation. It is possible, however, that CAD is associated with subcellular structures that cosediment with the nuclei. Despite a 150-fold increase in CAD concentration in the over-producing cells, the distribution of the protein was unaltered. CAD was not concentrated near the mitochondria where the next enzyme of the de novo pathway, dihydroorotate dehydrogenase, is localized, which indicates that the intermediate dihydroorotate is not channeled, but rather dissociates from CAD and diffuses through the bulk cellular fluid.     

Localization of the multifunctional protein CAD in astrocytes of rodent brain.

The CAD multidomain protein, which includes active sites of carbamyl phosphate synthetase II (CPS II, glutamine-dependent), aspartate transcarbamylase, and dihydroorotase, was immunostained in normal rat brains, the gliotic brains of myelin-deficient mutant rats, and brains from normal weanling hamsters. In each of these tissues CAD was observed in cells resembling astrocytes. In hamster brain, CAD immunofluorescence was also found in cells closely related to astrocytes, i.e., the Bergmann glia in cerebellum and the tanycytes surrounding the third ventricle. The astrocytic identity of the CAD-positive cells in rat brain was confirmed by double immunofluorescence staining with antibodies against glial fibrillary acidic protein (GFAP). The two enzymes carbonic anhydrase and glutamine synthetase occur in the cytoplasm of normal astrocytes in gray matter and of reactive astrocytes during gliosis. Products of each enzyme, i.e., bicarbonate and glutamine, are required for the CPS II reaction, which is the first step in the biosynthesis of pyrimidines. Therefore, the present results suggest roles for carbonic anhydrase and glutamine synthetase, as well as CAD, in pyrimidine biosynthesis in brain and a role for the astrocytes in the de novo synthesis of pyrimidines.     

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.