Orotate phosphoribosyl transferase MoPyr5 is involved in uridine 5'-phosphate synthesis and pathogenesis of Magnaporthe oryzae.

Orotate phosphoribosyl transferase (OPRTase) plays an important role in de novo and salvage pathways of nucleotide synthesis and is widely used as a screening marker in genetic transformation. However, the function of OPRTase in plant pathogens remains unclear. In this study, we characterized an ortholog of Saccharomyces cerevisiae Ura5, the OPRTase MoPyr5, from the rice blast fungus Magnaporthe oryzae. Targeted gene disruption revealed that MoPyr5 is required for mycelial growth, appressorial turgor pressure and penetration into plant tissues, invasive hyphal growth, and pathogenicity. Interestingly, the ∆Mopyr5 mutant is also involved in mycelial surface hydrophobicity. Exogenous uridine 5'-phosphate (UMP) restored vegetative growth and rescued the defect in pathogenicity on detached barley and rice leaf sheath. Collectively, our results show that MoPyr5 is an OPRTase for UMP biosynthesis in M. oryzae and indicate that UTP biosynthesis is closely linked with vegetative growth, cell wall integrity, and pathogenicity of fungus. Our results also suggest that UMP biosynthesis would be a good target for the development of novel fungicides against M. oryzae.

Redirecting metabolic flux in Saccharomyces cerevisiae through regulation of cofactors in UMP production.

Although it is generally known that cofactors play a major role in the production of different fermentation products, their role has not been thoroughly and systematically studied. To understand the impact of cofactors on physiological functions, a systematic approach was applied, which involved redox state analysis, energy charge analysis, and metabolite analysis. Using uridine 5'-monophosphate metabolism in Saccharomyces cerevisiae as a model, we demonstrated that regulation of intracellular the ratio of NADPH to NADP(+) not only redistributed the carbon flux between the glycolytic and pentose phosphate pathways, but also regulated the redox state of NAD(H), resulting in a significant change of ATP, and a significantly altered spectrum of metabolic products.

ExoY from Pseudomonas aeruginosa is a nucleotidyl cyclase with preference for cGMP and cUMP formation.

In addition to the well known second messengers cAMP and cGMP, mammalian cells contain the cyclic pyrimidine nucleotides cCMP and cUMP. Soluble guanylyl cyclase and soluble adenylyl cyclase produce all four cNMPs. Several bacterial toxins exploit mammalian cyclic nucleotide signaling. The type III secretion protein ExoY from Pseudomonas aeruginosa induces severe lung damage and effectively produces cGMP. Here, we show that transfection of mammalian cells with ExoY or infection with ExoY-expressing P. aeruginosa not only massively increases cGMP but also cUMP levels. In contrast, the structurally related CyaA from Bordetella pertussis and edema factor from Bacillus anthracis exhibit a striking preference for cAMP increases. Thus, ExoY is a nucleotidyl cyclase with preference for cGMP and cUMP production. The differential effects of bacterial toxins on cNMP levels suggest that cUMP plays a distinct second messenger role.

Nucleotidyl cyclase activity of soluble guanylyl cyclase in intact cells.

Soluble guanylyl cyclase (sGC) is activated by nitric oxide (NO) and generates the second messenger cyclic GMP (cGMP). Recently, purified sGC α1ß1 has been shown to additionally generate the cyclic pyrimidine nucleotides cCMP and cUMP. However, since cyclic pyrimidine nucleotide formation occurred only the presence of Mn(2+) but not Mg(2+), the physiological relevance of these in vitro findings remained unclear. Therefore, we studied cyclic nucleotide formation in intact cells. We observed NO-dependent cCMP- and cUMP formation in intact HEK293 cells overexpressing sGC α1ß1 and in RFL-6 rat fibroblasts endogenously expressing sGC, using HPLC-tandem mass spectrometry. The identity of cCMP and cUMP was unambiguously confirmed by HPLC-time-of-flight mass spectrometry. Our data indicate that cCMP and cUMP play second messenger roles and that Mn(2+) is a physiological sGC cofactor.

The Leishmania donovani UMP synthase is essential for promastigote viability and has an unusual tetrameric structure that exhibits substrate-controlled oligomerization.

The final two steps of de novo uridine 5'-monophosphate (UMP) biosynthesis are catalyzed by orotate phosphoribosyltransferase (OPRT) and orotidine 5'-monophosphate decarboxylase (OMPDC). In most prokaryotes and simple eukaryotes these two enzymes are encoded by separate genes, whereas in mammals they are expressed as a bifunctional gene product called UMP synthase (UMPS), with OPRT at the N terminus and OMPDC at the C terminus. Leishmania and some closely related organisms also express a bifunctional enzyme for these two steps, but the domain order is reversed relative to mammalian UMPS. In this work we demonstrate that L. donovani UMPS (LdUMPS) is an essential enzyme in promastigotes and that it is sequestered in the parasite glycosome. We also present the crystal structure of the LdUMPS in complex with its product, UMP. This structure reveals an unusual tetramer with two head to head and two tail to tail interactions, resulting in two dimeric OMPDC and two dimeric OPRT functional domains. In addition, we provide structural and biochemical evidence that oligomerization of LdUMPS is controlled by product binding at the OPRT active site. We propose a model for the assembly of the catalytically relevant LdUMPS tetramer and discuss the implications for the structure of mammalian UMPS.

Enhanced uridine 5'-monophosphate production by whole cell of Saccharomyces cerevisiae through rational redistribution of metabolic flux.

A whole-cell biocatalytic process for uridine 5'-monophosphate (UMP) production from orotic acid by Saccharomyces cerevisiae was developed. To rationally redistribute the metabolic flux between glycolysis and pentose phosphate pathway, statistical methods were employed first to find out the critical factors in the process. NaH(2)PO(4), MgCl(2) and pH were found to be the important factors affecting UMP production significantly. The levels of these three factors required for the maximum production of UMP were determined: NaH(2)PO(4) 22.1 g/L; MgCl(2) 2.55 g/L; pH 8.15. An enhancement of UMP production from 6.12 to 8.13 g/L was achieved. A significant redistribution of metabolic fluxes was observed and the underlying mechanism was discussed.

The mechanisms of citrate on regulating the distribution of carbon flux in the biosynthesis of uridine 5'-monophosphate by Saccharomyces cerevisiae.

A whole cell biocatalytic process for uridine 5'-monophosphate (UMP) production from orotic acid by Saccharomyces cerevisiae was developed. The concentration of UMP was increased by 23% when 1 g l(-1) sodium citrate was fed into the broth. Effects of citrate addition on UMP production were investigated. Glucose-6-phosphate pool was elevated by onefold, while FBP and pyruvate were decreased by 42% and 40%, respectively. Organic acid pools such as acetate and succinate were averagely decreased by 30% and 49%. The results demonstrated that manipulation of citrate levels could be used as a novel tool to regulate the metabolic fluxes distribution among glycolysis, pentose phosphate pathway, and TCA cycle.

Sustainable synthesis of uridine-5'-monophosphate analogues by immobilized uracil phosphoribosyltransferase from Thermus thermophilus.

Nowadays enzymatic synthesis of nucleic acid derivatives is gaining momentum over traditional chemical synthetic processes. Biotransformations catalyzed by whole cells or enzymes offer an ecofriendly and efficient alternative to the traditional multistep chemical methods, avoiding the use of chemical reagents and organic solvents that are expensive and environmentally harmful. Herein we report for the first time the covalent immobilization a uracil phosphoribosyltransferase (UPRT). In this sense, UPRT from Thermus thermophilus HB8 was immobilized onto glutaraldehyde-activated MagReSyn®Amine magnetic iron oxide porous microparticles (MTtUPRT). According to the catalyst load experiments, MTtUPRT3 was selected as optimal biocatalyst for further studies. MTtUPRT3 was active and stable in a broad range of temperature (70-100 °C) and in the pH interval 6-8, displaying maximum activity at 100 °C and pH 7 (activity 968 IU/gsupport, retained activity 100%). In addition, MTtUPRT3 could be reused up to 8 times in the synthesis of uridine-5'-monophosphate (UMP). Finally, MTtUPRT3 was successfully applied in the sustainable synthesis of different 5-modified uridine-5'-monophosphates at short times. Taking into account these results, MTtUPRT3 would emerge as a valuable biocatalyst for the synthesis of nucleoside monophosphates through an efficient and environmentally friendly methodology.

Inhibition of orotidine 5'-monophosphate decarboxylase and its therapeutic potential.

Orotidine 5'-monophosphate decarboxylase (ODCase) is among the most proficient enzymes, and catalyzes the decarboxylation of OMP to UMP. An overview of ODCase and various proposals for its catalytic mechanism of decarboxylation are briefly presented here. A number of inhibitors of ODCase and new developments in the X-ray structures of ODCases from different species are discussed in the context of their therapeutic potential against cancer and infectious diseases. Latest discoveries in the inhibition of ODCase, for example using the novel C6 substitutions on the uridine, open new doors for drug discovery targeting parasitic diseases such as malaria.

Structure of the gene for CAD, the multifunctional protein that initiates UMP synthesis in Syrian hamster cells.

Two adjacent fragments of genomic DNA spanning the gene for CAD, which encodes the first three enzymes of UMP biosynthesis, were cloned from a mutant Syrian hamster cell line containing multiple copies of this gene. The mutant was selected for resistance to N-(phosphonacetyl)-L-aspartate, a potent and specific inhibitor of aspartate transcarbamylase, the second enzyme in the pathway. The sizes and positions of about 37 intervening sequences within the 25-kilobase CAD gene were mapped by electron microscopy, and the locations of the 5' and 3' ends of the 7.9-kilobase CAD mRNA were established by electron microscopy and by other hybridization methods. The coding sequences are small (100 to 400 bases), as are most of the intervening sequences (50 to 300 bases). However, there are also several large intervening sequences of up to 5,000 bases each. Two small cytoplasmic polyadenylated RNAs are transcribed from a region just beyond the 5' end of the CAD gene, and their abundance reflects the degree of gene amplification.

Point mutations in yeast CBF5 can abolish in vivo pseudouridylation of rRNA.

In budding yeast (Saccharomyces cerevisiae), the majority of box H/ACA small nucleolar RNPs (snoRNPs) have been shown to direct site-specific pseudouridylation of rRNA. Among the known protein components of H/ACA snoRNPs, the essential nucleolar protein Cbf5p is the most likely pseudouridine (Psi) synthase. Cbf5p has considerable sequence similarity to Escherichia coli TruBp, a known Psi synthase, and shares the "KP" and "XLD" conserved sequence motifs found in the catalytic domains of three distinct families of known and putative Psi synthases. To gain additional evidence on the role of Cbf5p in rRNA biosynthesis, we have used in vitro mutagenesis techniques to introduce various alanine substitutions into the putative Psi synthase domain of Cbf5p. Yeast strains expressing these mutated cbf5 genes in a cbf5Delta null background are viable at 25 degrees C but display pronounced cold- and heat-sensitive growth phenotypes. Most of the mutants contain reduced levels of Psi in rRNA at extreme temperatures. Substitution of alanine for an aspartic acid residue in the conserved XLD motif of Cbf5p (mutant cbf5D95A) abolishes in vivo pseudouridylation of rRNA. Some of the mutants are temperature sensitive both for growth and for formation of Psi in the rRNA. In most cases, the impaired growth phenotypes are not relieved by transcription of the rRNA from a polymerase II-driven promoter, indicating the absence of polymerase I-related transcriptional defects. There is little or no abnormal accumulation of pre-rRNAs in these mutants, although preferential inhibition of 18S rRNA synthesis is seen in mutant cbf5D95A, which lacks Psi in rRNA. A subset of mutations in the Psi synthase domain impairs association of the altered Cbf5p proteins with selected box H/ACA snoRNAs, suggesting that the functional catalytic domain is essential for that interaction. Our results provide additional evidence that Cbf5p is the Psi synthase component of box H/ACA snoRNPs and suggest that the pseudouridylation of rRNA, although not absolutely required for cell survival, is essential for the formation of fully functional ribosomes.

UMP synthesis in the kinetoplastida.

All six enzymes of pyrimidine biosynthesis de novo have been detected in homogenates of the culture promastigote form of Leishmania mexicana amazonensis, the blood trypomastigote form of Trypanosoma brucei and the culture epimastigote, blood trypomastigote and intracellular amastigote forms of Trypanosoma cruzi. Dihydroorotate dehydrogenase is mitochondrial in mammals, but the isofunctional enzyme, dihydroorotate oxidase was found to be cytoplasmic, whereas orotate phosphoribosyltransferase and orotidine-5'-phosphate decarboxylase, which are cytoplasmic in mammals, were found to be particulate. Analysis by isopycnic sedimentation in sucrose showed that both particulate enzymes co-sedimented with glycosomal-(microbody-)marker enzymes such as hexokinase. Electron microscopy indicated that fractions containing these activities consisted essentially only of microbodies. It is concluded therefore that these enzymes are associated with glycosomes. Kinetic studies with intact glycosomal preparations suggested that there was no membrane barrier between 5-phosphoribose 1-pyrophosphate (P-Rib-PP) and orotate phosphoribosyltransferase, indicating either that the active site of this enzyme is probably on the outside of the glycosome or that the glycosome may have an efficient transport site for P-Rib-PP. Not all the UMP salvage enzymes assayed were detected. No uridine kinase activity was found in any of the species investigated, suggesting that uridine salvage might be routed via a uridine phosphorylase and uracil phosphoribosyltransferase. In agreement with this suggestion, these latter activities were detected in all organisms tested except the intracellular amastigote form of T. cruzi, where uracil phosphoribosyltransferase appeared absent. All the UMP salvage enzymes investigated occurred in cytoplamic fractions.

Effects of phosphate limitation on expression of genes involved in pyrimidine synthesis and salvaging in Arabidopsis.

Arabidopsis seedlings grown for 14 d without phosphate (P) exhibited stunted growth and other visible symptoms associated with P deficiency. RNA contents in shoots decreased nearly 90%, relative to controls. In shoots, expression of Pht1;2, encoding an inducible high-affinity phosphate transporter, increased threefold, compared with controls, and served as a molecular marker for P limitation. Transcript levels for five enzymes (aspartate transcarbamoylase, ATCase, EC 2.1.3.2; carbamoyl phosphate synthetase, CPSase, EC 6.3.5.5); UMP synthase, EC 2.4.1.10, EC 4.1.1.23; uracil phosphoribosyltransferase, UPRTase, EC 2.4.2.9; UMP kinase, EC 2.7.1.14) increased 2-10-fold in response to P starvation in shoots. These enzymes, which utilize phosphorylated intermediates at putative regulated steps in de novo synthesis and salvaging pathways leading to UMP and pyrimidine nucleotide formation, appear to be coordinately regulated, at the level of gene expression. This response may facilitate pyrimidine nucleotide synthesis under P limitation in this plant. Expression of P-dependent and P-independent phosphoribosyl pyrophosphate (PRPP) synthases (PRS2 and PRS3, respectively) which provide PRPP, the phosphoribosyl donor in UMP synthesis via both de novo and salvaging pathways, was differentially regulated in response to P limitation. PRS2 mRNA levels increased twofold in roots and shoots of P-starved plants, while PRS3 was constitutively-expressed. PRS3 may play a novel role in providing PRPP to cellular metabolism under low P availability.

Enzymes of the de novo pyrimidine biosynthetic pathway in Toxoplasma gondii.

All six enzymes of the de novo biosynthetic pathway leading to the biosynthesis of UMP have been characterized in Toxoplasma gondii. The first three enzymes of the pathway, carbamyl phosphate synthetase-II (CPS-II), aspartate transcarbamylase (ATCase) and dihydroorotase (DHOase) could be consistently separated by sucrose gradient centrifugation. Their molecular weights were estimated to be approximately 540 000, 140 000 and 70 000, respectively. The last two enzymes, orotate phosphoribosyltransferase (OPRTase) and orotidylate decarboxylase (ODCase), cosedimented at the same position, corresponding also to a molecular weight of approximately 70 000. The fourth enzyme, dihydroorotate dehydrogenase (DHO-DHase), was associated with the particulate fraction. Apparent Km values for the respective enzymes were: CPS-II, MgATP2- (19.7 1.2 mM), L-glutamine (12.0 +/- 1.7 microM), ammonia (15.5 +/- 2.7 mM); ATCase, carbamyl phosphate (26.2 +/- 3.5 microM), L-aspartate (17.6 +/- 8.5 mM); DHOase (reverse direction) dihydroorotate (1.6 +/- 0.08 microM); ODCase, orotidine 5'-monophosphate (0.41 +/- 0.04 microM). MgUTP2- was found to act as an inhibitor of CPS-II, with an apparent Ki of 0.41 mM. However, 5-phospho-alpha-D-ribosyl-1-diphosphate, dimethyl sulphoxide and glycerol had no effect on the Km value for MgATP2-. The effect of some inhibitors, including pyrimidine and purine nucleotides and analogs and respiratory chain inhibitors, was also determined for the enzymes of the pathway.

Enzymes of uridine 5'-monophosphate biosynthesis in Schistosoma mansoni.

In Schistosoma mansoni, the major product of in vitro orotate metabolism was orotidine 5'-monophosphate (OMP), whereas in mouse liver it was UMP. In contrast to mammalian cells, OMP appeared not to be 'channeled' from orotate phosphoribosyltransferase to OMP decarboxylase in S. mansoni, resulting in substantial degradation of OMP to orotidine. Significant differences were observed in the inhibitor specificity of phosphoribosyltransferase between S. mansoni and mouse liver, indicating that this enzyme may be a potential chemotherapeutic target in S. mansoni. Two distinct phosphoribosyltransferases were found in S. mansoni. One enzyme, having the higher molecular weight, utilized orotate, 5-fluorouracil and uracil as substrates, while the other only orotate. Both enzymes were inhibited by 5-azaorotic acid (oxonic acid) but only the 'orotate-specific' enzyme was inhibited by 4,6-dihydroxypyrimidine. OMP decarboxylase activity co-eluted with both phosphoribosyltransferases from Sephadex G-100 gel chromatography. We suggest that phosphoribosyltransferase in S. mansoni plays a role in both de novo UMP biosynthesis as well as in the salvage of uracil and uridine.

In vitro inhibition of Plasmodium falciparum by pyrazofurin, an inhibitor of pyrimidine biosynthesis de novo.

The effect of pyrazofurin, an inhibitor of UMP synthesis, on Plasmodium falciparum growth in vitro has been studied. ID50 values (concentration of compound causing 50% inhibition of [3H]hypoxanthine incorporation) for the FCQ-27, FCI-1 and K-1 (chloroquine-resistant) isolates were 10 +/- 8.7, 6.4 +/- 5.3 and 6.3 +/- 0.5 microM, respectively. Comparative ID50 values for chloroquine were 13.5 +/- 4.2, 22.8 +/- 7.6 and 343 +/- 114 microM, respectively. Over the 48-h intraerythrocytic cycle of tightly synchronized parasites, pyrazofurin both reduced the parasitemia and retarded the maturation of trophozoites and schizonts. Addition of uracil or uridine to the in vitro culture did not decrease the anti-parasitic activity of pyrazofurin. Chloroquine reduced the parasitemia, but did not retard development of the remaining viable parasites. Pyrazofurin (20 microM) caused a 50% inhibition of parasite orotate phosphoribosyltransferase (E.C. 2.4.2.10) and, in the presence of adenosine kinase and ATP, a 73% inhibition of orotidine-5'-phosphate decarboxylase (E.C. 4.1.1.23).

Inhibition of dihydroorotate dehydrogenase by the immunosuppressive agent leflunomide.

Leflunomide [HWA 486 or RS-34821, 5-methyl-N-(4-trifluoromethylphenyl)-4-isoxazole carboximide] is an immunosuppressive agent effective in the treatment of rheumatoid arthritis. In spite of its clinical potential, its mechanism of action has not been elucidated. Recent studies suggest that leflunomide may interfere with the metabolism of pyrimidine nucleotides. In our studies, the active metabolite of leflunomide, RS-61980 (A77 1726, 2-hydroxyethylidene-cyanoacetic acid-4-trifluoromethyl anilide), was cytostatic towards a human T-lymphoblastoma cell line (A3.01). The inhibition of growth could be overcome completely by uridine. The other nucleosides, cytidine, adenosine and guanosine, did not overcome the effect of the compound. Since uridine is a precursor for the salvage synthesis of UMP, we propose that RS-61980 may be inhibiting the de novo pathway of UMP synthesis. Using human cells, the six enzymes catalyzing de novo UMP biosynthesis were tested for their sensitivity towards RS-61980. Only one of the enzymes, dihydroortate dehydrogenase (DHODH, EC 1.3.3.1) was inhibited by RS-61980 with a Ki value of 2.7 +/- 0.7 microM. The other five enzymes were not affected. The inhibition exhibited mixed-type kinetics towards both substrates, dihydroorotic acid and coenzyme Q. These results suggest that the molecular target of leflunomide action is DHODH. The immunomodulating activity may be related to the inhibition of UMP synthesis in proliferating lymphocytes.

The derepression of enzymes of de novo pyrimidine biosynthesis pathway in Brevibacterium ammoniagenes producing uridine-5-monophosphate and uracil.

A mutant of Brevibacterium ammoniagenes producing large quantities of UMP and uracil is described. The mutations render bacteria braditrophic for arginine, sensitive to adenine, resistant to rifampicin and pyrimidine analogues 5-fluorouracil, 5-fluorouridine, azauracil and thiouracil. The activities of enzymes involved in the UMP biosynthesis, i.e. orotate phosphoribosyltransferase, orotate-5-monophosphate decarboxylase, dihydroorotate oxidase, are 4-, 3.5- and 4.5-fold higher in the mutant than in the parent strain when grown in minimal medium. The synthesis of these enzymes in mutant cells is not repressed in the presence of exogenous Ura. True revertants, which completely restore the wild-type phenotype, occur among the Arg+ clones. The nature of the mutation is discussed.

Incorporation of glucose carbon into ribonucleotides of axenic Entamoeba histolytica.

Axenic Entamoeba histolytica grown with (UL-14C)glucose in TP-S-1 medium were capable of biosynthesizing ribose from labeled glucose. RNA isolated by phenol extraction was hydrolyzed to the ribonucleotide level by alkaline hydrolysis. The hydrolysate, chromatographed on ion exchange resins, yielded AMP, GMP, and UMP, but not CMP containing labeled glucose carbon. The present nucleotide composition of the isolated amebal RNA was, respectively, as follows, CMP, 0.20; GMP, 0.22; AMP, 0.30; UMP, 0.29. The location of all the radiolabel in each ribonucleotide was the ribose moiety. The relative specific incorporation of glucose carbon into AMP, GMP, and UMP was 0.47, 0.05, and 0.10, respectively. These results suggest that the bulk of amebal nucleic acid precursors are obtained as preformed nucleosides and/or nucleotides from TP-S-1 medium. The mean RNA content per milliliter packed cells of amebae was 4.2 +/- 0.2 mg.

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.