Uracil salvage is necessary for early Arabidopsis development

Uridine nucleotides can be formed by energy-consuming de novo synthesis or by the energy-saving recycling of nucleobases resulting from nucleotide catabolism. Uracil phosphoribosyltransferases (UPRTs; EC 2.4.2.9) are involved in the salvage of pyrimidines by catalyzing the formation of uridine monophosphate (UMP) from uracil and phosphoribosylpyrophosphate. To date, UPRTs are described as non-essential, energy-saving enzymes. In the present work, the six genes annotated as UPRTs in the Arabidopsis genome are examined through phylogenetic and functional complementation approaches and the available T-DNA insertion mutants are characterized. We show that a single nuclear gene encoding a protein targeted to plastids, UPP , is responsible for almost all UPRT activity in Arabidopsis. The inability to salvage uracil caused a light-dependent dramatic pale-green to albino phenotype, dwarfism and the inability to produce viable progeny in loss-of-function mutants. Plastid biogenesis and starch accumulation were affected in all analysed tissues, with the exception of stomata. Therefore we propose that uracil salvage is of major importance for plant development.     

Comparative studies on pyrimidine metabolism in excised cotyledons of Pinus radiata during shoot formation in vitro

Changes in the pattern of pyrimidine nucleotide metabolism were investigated in Pinus radiata cotyledons cultured under shoot-forming (SF; +N(6)-benzyladenine) and non-shoot-forming (NSF, -N(6)-benzyladenine) conditions, as well as in cotyledons unresponsive (OLD) to N(6)-benzyladenine. This was carried out by following the metabolic fate of externally supplied (14)C-labeled orotic acid, intermediate of the de novo pathway, and (14)C-labeled uridine and uracil, substrates of the salvage pathway. Nucleic acid synthesis was also investigated by following the metabolic fate of (14)C-labeled thymidine during shoot bud formation and development. The de novo synthesis of pyrimidine nucleotides was operative under both SF and NSF conditions, and the activity of orotate phosphoribosyltransferase (OPRT), a key enzyme of the de novo pathway, was higher in SF tissue. Utilization of both uridine and uracil for nucleotide and nucleic acid synthesis clearly indicated that the salvage pathway of pyrimidine metabolism is also operative during shoot organogenesis. In general, uridine was a better substrate for the synthesis of salvage products than uracil, possibly due to the higher activity of uridine kinase (UK), compared to uracil phosphoribosyltransferase (UPRT). Incorporation of uridine into the nucleic acid fraction of OLD cotyledons was lower than that observed for their responsive (day 0) counterparts. Similarly, uracil utilization for nucleic acid synthesis was lower in NSF cotyledons, compared to that observed for SF tissue after 10 days in culture. This difference was ascribed to higher UPRT activity measured in the latter. Thus, there was an apparent difference in the utilization of nucleotides derived from uracil and uridine for nucleotide synthesis. The increased ability to produce pyrimidine nucleotides via the salvage pathway during shoot bud formation may be required in support of nucleic acid synthesis occurring during the process. Studies on thymidine metabolism confirmed this notion.     

Effect of plasma concentrations of uridine on pyrimidine biosynthesis in cultured L1210 cells

The concentration of uridine in the media of cultured L1210 cells was maintained within the concentration range found in plasma (1 to 10 microM) to determine if such concentrations are sufficient to satisfy the pyrimidine requirements of a population of dividing cells and to determine if cells utilize de novo and/or salvage pathways when exposed to plasma concentrations of uridine. When cells were incubated in the presence of N-(phosphonacetyl)-L-aspartate to block de novo biosynthesis, plasma concentrations of uridine maintained normal cell growth. De novo pyrimidine biosynthesis, as determined by [14C]sodium bicarbonate incorporation into uracil nucleotides, was affected by the low concentrations of uridine found in the plasma. Below 1 microM uridine, de novo biosynthesis was not affected; between 3 and 5 microM uridine, de novo biosynthesis was inhibited by approximately 50%; and above 12 microM uridine, de novo biosynthesis was inhibited by greater than 95%. Inhibition of de novo biosynthesis correlated with an increase in the uracil nucleotide pool. The de novo pathway was much more sensitive to the uracil nucleotide pool size than was the salvage pathway, such that when de novo biosynthesis was inhibited by greater than 95% the uracil nucleotide pool continued to expand and the cells continued to take up [14C]uridine. Thus, the pyrimidine requirements of cultured L1210 cells can be met by concentrations of uridine found in the plasma and, when exposed to such physiologic concentrations, L1210 cells decrease their dependency on de novo biosynthesis and utilize their salvage pathway. Circulating uridine, therefore, may be of physiologic importance and could be an important determinant in anti-pyrimidine chemotherapy.     

Pyrimidine biosynthesis in Pseudomonas stutzeri ATCC 17588

The de novo pyrimidine biosynthetic enzymes in the denitrifying bacterium Pseudomonas stutzeri ATCC 17588 were assayed and their activities were lower in glucose-grown cells than in succinate-grown cells. When P. stutzeri was grown in the presence of uracil, the de novo enzyme activities in succinate-grown cells were lowered while they remained largely unchanged in glucose-grown cells. A uracil auxotroph of P. stutzeri, deficient for aspartate transcarbamoylase activity, was isolated and its auxotrophic requirement was met by only uracil and cytosine. The inability of pyrimidine ribonucleosides to meet the auxotrophic requirement was related to the limited ability of P. stutzeri to transport uridine and cytidine. Pyrimidine limitation of the auxotroph elevated the de novo enzyme activities indicating that this pathway may be repressible by a uracil-related compound in succinate-grown P. stutzeri cells. Regulation of pyrimidine synthesis in P. stutzeri was similar to that observed for other pseudomonads classified within rRNA homology group I.     

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.     

Pyrimidine synthesis in Burkholderia cepacia ATCC 25416

K. LI AND T.P. WEST. 1995. Pyrimidine synthesis in the food spoilage agent Burkholderia cepacia ATCC 25416 was investigated. The five de novo pathway enzymes of pyrimidine biosynthesis were found to be active in B. cepacia ATCC 25416 and growth of this strain on uracil had an effect on the de novo enzyme activities. The in vitro regulation of aspartate transcarbamoylase activity in B. cepacia ATCC 25416 was studied and its activity was inhibited by PP_i, ATP, GTP, CTP and UTP. The enzymes cytidine deaminase, uridine phosphorylase and cytosine deaminase were found to be active in the salvage of pyrimidines in ATCC 25416. Overall, de novo pyrimidine synthesis in B. cepacia ATCC 25416 was regulated at the level of enzyme activity and its pyrimidine salvage enzymes differed from those found in B. cepacia ATCC 17759.     

A 13C tracer method for quantitating de novo pyrimidine biosynthesis in vitro and in vivo

Gas chromatographic/mass spectrometric methods for the measurement of the flux through the de novo pyrimidine biosynthetic pathway by quantitating the incorporation of [13C]bicarbonate and 13CO2 into the uracil nucleotide pool in L1210 tumors are reported. Simultaneous measurements of the incorporation of [13C]bicarbonate and the more commonly used [14C]bicarbonate into uridine of L1210 cells in vitro showed that the two methods were comparable. A modification of the method was applied to in vivo studies where the incorporation of 13CO2 into the uracil nucleotide pool of L1210 tumors in mice was quantitated. The measurements were used to determine changes in the flux through the de novo pyrimidine pathway in animals pretreated with known inhibitors of the pathway. A comparison of control animals with those pretreated with 6-azauridine, acivicin, and pyrazofurin resulted in mean percentage inhibitions of 87, 95, and 94%, respectively. This technique should allow investigation of the respective contributions of salvage and de novo synthesis in the formation of pyrimidines in vivo and the effects of agents designed as enzyme inhibitors of the de novo pathway.     

Characterization of pyrimidine metabolism in the cellular slime mold, Dictyostelium discoideum

The arginine-independent, de novo biosynthetic pathway of pyrimidines in Dictyostelium discoideum is initiated by a class II carbamoyl-phosphate synthetase (EC 6.3.5.5) specific for pyrimidine biosynthesis which utilized L-glutamine as its N donor and was partially inhibited by both UTP and CTP. The second step in the de novo pathway was provided by an unregulated aspartate transcarbamoylase (EC 2.1.3.2) which primarily appeared as a multimeric enzyme of 105 kilodaltons. The next enzyme, dihydroorotase (EC 3.5.2.3), was approximately 90-100 kilodaltons. Although the early enzymatic activities of the pyrimidine pathway appeared to reside in independent protein complexes, various unstable molecular species were observed. These structural variants may represent proteolytic fragments of a multienzyme complex. In addition to de novo synthesis, the amoeba demonstrated the capacity for salvage utilization of uracil, uridine, and cytidine. Upon starvation on a solid substratum, axenically grown amoebas began a concerted developmental program accompanied by a restructuring of nucleotide metabolism. The absolute levels of the ribonucleotide pools droppedby 98% within 30 h; however, both the adenylate energy charge and the GTP/ATP ratios were maintained for 50 h after the initiation of development. The maintenance of these metabolic energy parameters required the tight cell-cell contact necessary for development, and the capacity for pyrimidine metabolism was maintained throughout developmental morphogenesis.     

De novo pyrimidine biosynthesis in isolated rat hepatocytes. Quantitative aspects of the regulation by UTP

Quantitative aspects of de novo pyrimidine biosynthesis in rat hepatocytes were monitored. A reduction of intracellular UTP contents by different concentrations of D-galactosamine led to a dose-dependent increase of 14CO2 incorporation into the sum of all acid-soluble uracil nucleotides. In controls the rate of de novo synthesis which was calculated from the incorporation rate of 14CO2 into the sum of all acid-soluble uracil nucleotides was 0.014 mumol X h-1 X g-1 compared to 0.056 mumol X h-1 X g-1 wet weight of liver in situations of a maximally stimulated de novo synthesis. Incubation of hepatocytes with uridine led to a dose-dependent reduction of 14CO2 incorporation to less than 25% of the amount incorporated in the controls. Alterations of the CTP content had no influence on the 14CO2 incorporation. In the presence of high D-galactosamine concentrations the increase of the total amount of acid-soluble uracil nucleotides exceeded the rate of the de novo synthesis derived from the incorporation of 14CO2 into the sum of the acid-soluble uracil nucleotide pool. It was also greater than the increase of the total amount of intra- and extracellular orotate after acidic hydrolysis--even in the presence of 6-azauridine, which stimulated de novo pyrimidine biosynthesis by itself.     

The effect of various concentrations of nucleobases, nucleosides or glutamine on the incorporation of [3H]thymidine into DNA in rat mesenteric-lymph-node lymphocytes stimulated by phytohaemagglutinin.

1. The rate of [3H]thymidine incorporation into DNA was measured in phytohaemagglutinin-stimulated lymph-node lymphocytes of the rat. 2. Addition of nucleobases or nucleosides to culture medium that already contained 0.2 mM-glutamine had a small stimulatory effect on incorporation. At lower concentrations of glutamine, adenosine (even at 1 microM) caused a marked increase in the rate of incorporation. 3. In the absence of added glutamine, addition of nucleosides or nucleobases markedly increased the rate of incorporation: nucleosides were more effective than nucleobases; and the rate of proliferation in the presence of 10 microM-adenosine plus 10 microM-uridine was similar to that in the presence of optimal concentrations of glutamine. 4. The rate of incorporation was dramatically decreased by an inhibitor of the pathway of pyrimidine nucleotide synthesis de novo. Addition of the pyrimidine nucleosides completely overcame the inhibition; addition of the pyrimidine nucleobases was much less effective. 5. These results indicate that, for proliferation of lymphocytes, glutamine is not essential and can be partially or totally replaced by nucleosides and, to some extent, by nucleobases.     

Isolation and initial characterization of a uracil auxotroph of the blue-green alga Anacystis nidulans.

A uracil-requiring auxotroph of Anacystis nidulans was isolated after treatment with N-methyl-N'-nitrosoguanidine. Neither precursors in the de novo pyrimidine pathway nor compounds of "salvage" or degradative pathways could replace the uracil requirement. The reversion frequency for mutation to a nonuracil requirement for growth was 2.0 times 10(-8). The calculated average rate of uracil utilization was 1.1 times 10(-17) mol of uracil per unit cell mass/h. The amount of uracil required for the synthesis of a unit cell mass was 3.8 times 10(-17) mol of uracil.     

Pyrimidine biosynthesis in Pseudomonas oleovorans

AIMS: To investigate the regulation of de novo pyrimidine biosynthesis in the polyhydroxyalkanoate-producing bacterium Pseudomonas oleovorans at the level of enzyme synthesis and at the level of aspartate transcarbamoylase activity. METHODS AND RESULTS: The effect of pyrimidine supplementation on the pyrimidine biosynthetic pathway enzyme activities was analysed relative to carbon source. Two uracil auxotrophs of P. oleovorans were isolated that were deficient for aspartate transcarbamoylase or dihydroorotase activity. Pyrimidine limitation of these auxotrophs increased the de novo pathway activities to varying degrees depending on the pathway mutation and the carbon source utilized. At the level of aspartate transcarbamoylase activity, pyrophosphate and uridine ribonucleotides were found to be strongly inhibitory of the Ps. oleovorans enzyme. CONCLUSIONS: Pyrimidine biosynthesis is regulated in Ps. oleovorans. Taxonomically, the regulation of the pyrimidine biosynthetic pathway appeared dissimilar from previously studied Pseudomonas species. SIGNIFICANCE AND IMPACT OF THE STUDY: New insights regarding the regulation of nucleic acid metabolism are provided that could prove significant during the genetic manipulation of Ps. oleovorans to increase the synthesis of polyhydroxyalkanoates.     

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.     

Genetic dissection of pyrimidine biosynthesis and salvage in Leishmania donovani

Protozoan parasites of the Leishmania genus express the metabolic machinery to synthesize pyrimidine nucleotides via both de novo and salvage pathways. To evaluate the relative contributions of pyrimidine biosynthesis and salvage to pyrimidine homeostasis in both life cycle stages of Leishmania donovani, individual mutant lines deficient in either carbamoyl phosphate synthetase (CPS), the first enzyme in pyrimidine biosynthesis, uracil phosphoribosyltransferase (UPRT), a salvage enzyme, or both CPS and UPRT were constructed. The Δcps lesion conferred pyrimidine auxotrophy and a growth requirement for medium supplementation with one of a plethora of pyrimidine nucleosides or nucleobases, although only dihydroorotate or orotate could circumvent the pyrimidine auxotrophy of the Δcps/Δuprt double knockout. The Δuprt null mutant was prototrophic for pyrimidines but could not salvage uracil or any pyrimidine nucleoside. The capability of the Δcps parasites to infect mice was somewhat diminished but still robust, indicating active pyrimidine salvage by the amastigote form of the parasite, but the Δcps/Δuprt mutant was completely attenuated with no persistent parasites detected after a 4-week infection. Complementation of the Δcps/Δuprt clone with either CPS or UPRT restored infectivity. These data establish that an intact pyrimidine biosynthesis pathway is essential for the growth of the promastigote form of L. donovani in culture, that all uracil and pyrimidine nucleoside salvage in the parasite is mediated by UPRT, and that both the biosynthetic and salvage pathways contribute to a robust infection of the mammalian host by the amastigote. These findings impact potential therapeutic design and vaccine strategies for visceral leishmaniasis.     

De novo synthesis of pyrimidine nucleotides by Helicobacter pylori

The incorporation of pyrimidine nucleotide precursors into Helicobacter pylori and the activities of enzymes involved in their synthetic pathways were investigated by radioactive tracer analysis and ³¹P nuclear magnetic resonance spectroscopy. The bacterium was found to take up aspartate and bicarbonate and to incorporate carbon atoms from these precursors into its genomic DNA. Orotate, an intermediate of de novo pyrimidine biosynthesis, and uracil and uridine, precursors for pyrimidine pathways, were also incorporated by the micro-organism. Radiolabelled substrates were used to assess the activities of aspartate transcarbamoylase, orotate phosphoribosyltransferase, orotidylate decarboxylase, CTP synthetase, uracil phosphoribosyltransferase, thymidine kinase and deoxycytidine kinase in bacterial lysates. The study provided evidence for the presence in H. pylori of an operational de novo pathway, and a less active salvage pathway for the biosynthesis of pyrimidine nucleotides.