Pyrimidine Nucleotide Precursors Research Articles

Engineering Escherichia colinative metabolism for efficient biosynthesis of orotate.

Orotate (OA) is a precursor of pyrimidine nucleotides and is widely used in food, pharmaceutical, and cosmetic industries. Although various microorganisms have been used for OA production, the production efficiency needs to be further improved for industrial application. In this study, we engineered Escherichia coli native metabolism for efficient OA production. The entire pathway was divided into the downstream OA synthesis, the midstream aspartate/glutamine supply, and the upstream glycolysis modules. First, the downstream module was optimized by disrupting pyrE to block OA consumption and release the feedback inhibition, and tuning expression of the biosynthetic genes. Second, the midstream pathway was enhanced by increasing the supply of the precursors and the cofactor nicotinamide adenine dinucleotide phosphate (NADPH). More importantly, we observed that pyrE disruption may lead to metabolic disorder as indicated by the accumulation of large amount of acetate. This problem was solved by reducing the flux of glycolysis. With these efforts, the final strain produced 80.3 g/L OA with a yield of 0.56 g/g glucose in fed-batch fermentation, which are the highest titer and yield reported so far. This work paves the way for industrial production of OA and represents as a good example of modulating cell metabolism for efficient chemical production.

Filament assembly regulates the catalytic activity of phosphoribosyl pyrophosphate synthetase 1

The enzyme phosphoribosyl pyrophosphate synthetase 1 (PRPS1) regulates nucleotide biosynthesis, as it generates phosphoribosyl pyrophosphate, a precursor of both purine and pyrimidine nucleotides. Mutations in human PRPS1 lead to a spectrum of human diseases associated with increased or decreased activity. PRPS1 has long been known for form large, heterogeneous oligomers in vitro. Recently, the protein was shown to assemble large, filamentous structures in vertebrates and budding yeast, suggesting evolutionary conservation of these cellular structures.

Overexpression of Phosphoribosyl Pyrophosphate Synthase Enhances Resistance of Chlamydomonas to Ionizing Radiation.

The enzyme phosphoribosyl pyrophosphate synthase (PRPS) catalyzes the conversion of ribose 5-phosphate into phosphoribosyl diphosphate; the latter is a precursor of purine and pyrimidine nucleotides. Here, we investigated the function of PRPS from the single-celled green alga Chlamydomonas reinhardtii in its response to DNA damage from gamma radiation or the alkylating agent LiCl. CrPRPS transcripts were upregulated in cells treated with these agents. We generated CrPRPS-overexpressing transgenic lines to study the function of CrPRPS. When grown in culture with LiCl or exposed to gamma radiation, the transgenic cells grew faster and had a greater survival rate than wild-type cells. CrPRPS overexpression enhanced expression of genes associated with DNA damage response, namely RAD51, RAD1, and LIG1. We observed, from transcriptome analysis, upregulation of genes that code for key enzymes in purine metabolism, namely ribonucleoside-diphosphate pyrophosphokinase subunit M1, adenylate kinase, and nucleoside-diphosphate kinase. We conclude that CrPRPS may affect DNA repair process via regulation of de novo nucleotide synthesis.

A biochemical basis for the myeloid:lymphoid cell ratio, increases in which are associated with poor prognoses in numerous pathological conditions

Background Elevated myeloid:lymphoid cell ratios in peripheral blood, which are indicative of inflammation, have been associated with poor prognoses in a wide variety of disease states. The biochemical and physiological basis of this phenomenon has not previously been investigated. Hypothesis Proliferation of lymphocytes is absolutely dependent on an adequate supply of pyrimidine nucleotides. Increases in the activities of thymidine phosphorylase (TP) and cytidine deaminase (CDA), two key enzymes of pyrimidine metabolism that are found in myeloid cells, are frequently associated with inflammation. Neutrophils and platelets do not proliferate but have high rates of metabolism as well as the capacity to control nucleotide supply by catabolizing essential precursors including pyrimidine nucleosides and glutamine, which is required for de novo nucleotide synthesis. Aim To investigate the relationship between CDA and TP activities and myeloid:lymphoid cell ratios in peripheral blood. Methods Activities of CDA and TP in whole blood samples were simultaneously quantified using a simple HPLC-based assay. Metabolic activity was assessed by resazurin reduction. Results and conclusion Elevated neutrophil:lymphocyte ratios correlated closely with increased CDA and TP activities and increased metabolic flux, supporting the hypothesis that competition for pyrimidine nucleotide precursors is an important influence on the peripheral blood myeloid:lymphoid cell ratio.

Prevention of Neural Tube Defects By Nucleotide Precursors In the Curly Tail Mouse

Evaluation of: Leung KY, De Castro SC, Savery D et al. Nucleotide precursors prevent folic acid-resistant neural tube defects in the mouse. Brain 136(Pt 9), 2836–2841 (2013). In this study, the authors examined the effects of treating curly tail mutant mice, a folate-resistant mouse model, with specific combinations of pyrimidine and purine nucleotide precursors in an effort to prevent neural tube defects. These investigators found that the prevalence of neural tube defects in the group treated with combinations of thymidine and purine nucleotide precursors was significantly lower than that observed in the control groups. Before combination of nucleotide precursors can be used as a new neural tube defect intervention strategy, the beneficial effects, the side effects, as well as other physiological consequences have to be carefully evaluated and compared with other folate-resistant mice and, ultimately, investigated in human cohorts.

Nucleotide precursors prevent folic acid-resistant neural tube defects in the mouse

Closure of the neural tube during embryogenesis is a crucial step in development of the central nervous system. Failure of this process results in neural tube defects, including spina bifida and anencephaly, which are among the most common birth defects worldwide. Maternal use of folic acid supplements reduces risk of neural tube defects but a proportion of cases are not preventable. Folic acid is thought to act through folate one-carbon metabolism, which transfers one-carbon units for methylation reactions and nucleotide biosynthesis. Hence suboptimal performance of the intervening reactions could limit the efficacy of folic acid. We hypothesized that direct supplementation with nucleotides, downstream of folate metabolism, has the potential to support neural tube closure. Therefore, in a mouse model that exhibits folic acid-resistant neural tube defects, we tested the effect of specific combinations of pyrimidine and purine nucleotide precursors and observed a significant protective effect. Labelling in whole embryo culture showed that nucleotides are taken up by the neurulating embryo and incorporated into genomic DNA. Furthermore, the mitotic index was elevated in neural folds and hindgut of treated embryos, consistent with a proposed mechanism of neural tube defect prevention through stimulation of cellular proliferation. These findings may provide an impetus for future investigations of supplemental nucleotides as a means to prevent a greater proportion of human neural tube defects than can be achieved by folic acid alone.

Uridine supplementation antagonizes zidovudine‐induced mitochondrial myopathy and hyperlactatemia in mice

Zidovudine is an antiretroviral nucleoside analog reverse transcriptase inhibitor that induces mitochondrial myopathy by interfering with the replication of mitochondrial DNA (mtDNA). Because zidovudine inhibits thymidine kinases, the mechanism of mtDNA depletion may be related to an impairment of the de novo synthesis of pyrimidine nucleotides, which are required building blocks of mtDNA. This study was undertaken to determine whether mitochondrial myopathy is a class effect of antiretroviral nucleoside analogs, and whether the muscle disease can be prevented by treatment with uridine as a pyrimidine nucleotide precursor. BALB/c mice were treated with zidovudine or zalcitabine. Some of the mice were cotreated with mitocnol, a dietary supplement with high uridine bioavailability. Mice hind limb muscles were examined after 10 weeks. Zidovudine induced muscle fiber thinning, myocellular fat deposition, and abnormalities of mitochondrial ultrastructure. In mice treated with zidovudine, organelles contained low mtDNA copy numbers and reduced cytochrome c oxidase activity. The expression of the mtDNA-encoded cytochrome c oxidase I subunit, but not of nucleus-encoded mitochondrial proteins, was impaired. Zidovudine also increased the levels of myocellular reactive oxygen species and blood lactate. Uridine supplementation attenuated or normalized all pathologic abnormalities and had no intrinsic effects. Zalcitabine did not elicit muscle toxicity. Our findings indicate that zidovudine, but not zalcitabine, induces mitochondrial myopathy, which is substantially antagonized by uridine supplementation. These results provide proof of the importance of pyrimidine pools in the pathogenesis of zidovudine myopathy. Since uridine supplementation is tolerated well by humans, this treatment strategy should be investigated in clinical trials.

Effect of orotic acid on the metabolism of cerebral cortical astrocytes during hypoxia and reoxygenation: an NMR spectroscopy study.

Astrocytes were incubated under normoxic or hypoxic conditions in Dulbecco's minimum essential medium containing [2-13C]acetate, unlabeled glucose and in some cases orotic acid, an intermediate in pyrimidine biosynthesis. After 12 hr the medium was replaced by fresh medium without drug and incubation was continued for 17 hr in a normal oxygen atmosphere (reoxygenation). Thereafter, medium was removed, cell extracts were prepared, and metabolism in the treatment group was compared to the untreated hypoxia group and to control. 13C and 1H NMR spectra revealed that 13C enrichment in citrate and glutamine C-4 in the initial medium were increased in the presence of orotic acid, compared to the untreated hypoxia group but lower than control. The drug increased acetate utilization during hypoxia to normoxic levels. Thus it appears that the treatment group had a more active mitochondrial metabolism, which was also reflected in higher intracellular uridine diphosphoryl sugars and ADP concentrations. Glutamine labeling was increased in the cell extracts in the presence of orotic acid. Thus it appears that, in the presence of the pyrimidine nucleotide precursor, astrocytes are capable of normal metabolism during hypoxia which might have implications for neuronal survival during low oxygen insults, since neurons are dependent on astrocyte produced precursors for their neurotransmitter synthesis.

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.

Effect of dietary nucleotides and orotate on the blood levels of prostacyclin (PGI 2) and thromboxane (TXA 2) in the weanling rat

Dietary nucleotides affect the maintenance of immune responses, tissue repair and polyunsaturated fatty acid metabolism. Orotate, a pyrimidine nucleotide precursor, induces fatty livers by impairing VLDL hepatic secretion. The aim of this study was to evaluate the changes in the blood levels of fatty acids and prostacyclin (PGI 2) and thromboxane (TXA 2) in the weanling rat caused by the dietary intake of nucleotides and orotate. Three groups of rats at weaning were fed a control diet, an orotate supplemented diet (O-50) and a nucleotide supplemented diet (N-50) during 4 weeks, respectively. Absolute values of plasma polyunsaturated fatty acids greater than 18 carbon atoms of the n-6 and n-3 series were increased in the N-50 group and decreased in O-50 with regard to the control. However, the relative fatty acid composition of plasma lipid fractions was mostly unaffected. Plasma 6-keto-PGF 1α showed a trend to be increased in N-50 and serum TXB 2 was significantly increased in that group. Both eicosanoids were unchanged by dietary orotate intake. These results may be explained because of the increased plasma 20:4n-6 found in rats fed a supplemented nucleotide diet. Thus, nucleotides present in foods appear to modulate PUFA conversion and eicosanoids synthesis in early life.

Inhibition of orotidylate decarboxylase by [formula omitted] pyrimidine-3-thiocarboxamide (APR-TC) in B lymphoblasts : Activation by adenosine kinase

The nucleoside allopurinol riboside-3-thiocarboxamide (APR-TC; 4-(5 H)oxo-1-β- d-ribo-furanosylpyrazolo[3,4,d]pyrimidine-3-thiocarboxamide) demonstrates potent in vitro antiviral activity against various DNA and RNA viruses and cytostatic activity against a variety of cell lines in culture. The IC 50 for APR-TC in the splenic derived B lymphoblast cell line, WI-L2, was 0.3 μM. Adenosine kinase-deficient WI-L2 cells were resistant to growth inhibition by APR-TC, indicating that adenosine kinase (EC 2.7.1.20) is responsible for phosphorylation of APR-TC to form the monophosphate derivative (APR-TC-5'P). A 4-hr incubation of cells with 50 μM APR-TC resulted in severe depletion of intracellular pyrimidine nucleotide pools and the accumulation of 3μM APR-TC-5'P. The cytotoxicity of APR-TC was reversed by uridine, indicating that the active form of this compound inhibits the de novo pyrimidine biosynthetic pathway. Further, APR-TC-treated cells could not utilize the pyrimidine nucleotide precursor [6- 14Clorotic acid, suggesting that the UMP synthase complex is the major cellular site of inhibition. In studies utilizing cell-free lysates of WI-L2, chemically prepared APR-TC-5'P provided potent inhibition of the orotidylate decarboxylase activity (ODCase, EC 4.1.1.23) of the UMP synthase complex. APR-TC-5'P was competitive with OMP, and a K d value of 0.35 nM was determined.

Glucocorticoid control of rat growth hormone gene expression: effect on cytoplasmic messenger ribonucleic acid production and degradation.

The effect of the glucocorticoid dexamethasone on the production and degradation of rat GH (rGH) cytoplasmic mRNA was studied in cultured rat pituitary tumor (GC) cells. The incorporation of [3H]uridine into both rGH cytoplasmic mRNA and the pyrimidine nucleotide precursor pool was determined in hormone-treated and control cells. From these measurements glucocorticoid effects on absolute production rates of rGH cytoplasmic mRNA were determined and compared to effects on rGH mRNA accumulation. Rat GH mRNA half-life was then calculated based on a first-order decay model. Rat GH mRNA half-life was also directly assayed by: 1) pulse-chase studies and 2) measuring the kinetics of decay of rGH mRNA in cells after transfer from serum-containing to hormone-deficient media. From these independent analyses rGH mRNA half-life estimates ranged from 28-55 h in different experiments. Within individual experiments there was little variability of rGH mRNA decay rates; glucocorticoids were found not to alter the stability of rGH cytoplasmic mRNA. Glucocorticoid induction of rGH cytoplasmic mRNA accumulation was accounted for solely on the basis of increased mRNA production.

Dietary and metabolic manipulations of the carcinogenic process: role of nucleotide pool imbalances in carcinogenesis.

Perturbations in DNA and/or membranes are considered to be important for the carcinogenic process. A search for nutritional and metabolic means of disturbing the homeostasis of DNA and membranes revealed that nucleotide pools offer an exciting possibility. An imbalance in nucleotide pools can exert a two-pronged attack on both DNA and membranes. When given to rats, orotic acid, a precursor of pyrimidine nucleotides, results in an imbalance in nucleotide pools (an increase in uridine nucleotides and a decrease in inosine/adenine nucleotides), alterations in both DNA and membranes, and promotion of carcinogenesis in the liver initiated by chemical carcinogens. Agents such as adenine and allopurinol, which inhibit the metabolism of orotic acid and thereby decrease the formation of uridine nucleotides, and galactosamine, which traps uridine nucleotides, inhibited the promotional effects of orotic acid in the liver. These results suggested that orotic acid needs to be metabolized to uridine nucleotides and the creation of a subsequent imbalance in nucleotide pools is important for the promotional effects of orotic acid. To determine whether the creation of a nucleotide pool imbalance is a more general mechanism of tumor promotion, two lines of approach were investigated. One was to determine the effect of orotic acid on promotion of carcinogenesis in other organs, and the second approach was to determine how to induce nucleotide pool imbalances by means other than orotic acid administration. It is interesting to note that orotic acid promotes carcinogenesis in duodenum initiated by azoxymethane. Regarding the second approach, it became apparent that several metabolic disturbances result in increased orotic acid synthesis and alterations in nucleotide pools.(ABSTRACT TRUNCATED AT 250 WORDS)

SOURCE AND FATE OF CIRCULATING PYRIMIDINES: 81

Experiments were undertaken to investigate the working hypothesis that the liver is a source of pyrimidine nucleotide precursors for utilization by peripheral tissues, that the precursor is orotate, and that the erythrocytes act as an intermediary buffer, and convert the orotate to uridine, which is more readily utilised by peripheral tissues than orotate. Differentiated human hepatoma cells were labelled with 14C aspartic acid and labelled orotate was removed from the medium by selective precipitation techniques. Acid precipitable radioactivity was low in lymphoblasts, fibroblasts or erythrocytes labelled alone with 14C orotate but were enhanced tenfold if erythrocytes were co-cultured with the nucleated cells. Factors influencing orotate metabolism in the erythrocyte include phosphate, bicarbonate and other factors operating in a time dependent manner. Orotate and many pyrimidine nucleotides are directly quantifiable on high pressure liquid chromatography of extracts from lymphoblasts, fibroblasts and erythrocytes and their measurement provide a ready means for diagnosis of inherited disorders of pyrimidine metabolisms such as orotic aciduria and pyrimidine 5′ nucleotidase deficiency.

Orotate uptake and metabolism by human erythrocytes.

The uptake of the pyrimidine nucleotide precursor, orotate, by non-nucleated human erythrocytes is not immediately explicable, (1). During the course of an investigation into the origin of the increased erythrocyte nucleotides seen in hereditary ythrocyte pyrimidine 5′nucleotidase deficiency, rapid uptake of 14C orotate and its conversion to uridine nucleotides by normal red cells was observed (Figure 1(a) A). Moreover, free uridine was present within the cells and accumulated in significant quantities in the medium (Figure 2).

Effect of an exogenous energy source and amino acids on DNA synthesis in regenerating rat liver

Rats on a protein-free diet synthesized less DNA after partial hepatectomy than rats on a normal diet. In regenerating livers of animals on the protein-free diet, induction of several enzymes involved in the DNA precursor synthetic pathway, and especially ribonucleotide reductase, were depressed. When young rats were maintained solely by parenteral nutrition after partial hepatectomy, exogenous amino acids were more important than the exogenous energy source for induction of enzymes involved in synthesis of DNA and its pyrimidine nucleotide precursors. In particular, induction of ribonucleotide reductase appeared to be controlled by exogenous amino acids. Tryptophan, methionine, phenylalanine, leucine, valine, isoleucine and threonine seemed to stimulate the induction of this enzyme most after partial hepatectomy.

Anabolic and catabolic pathways of pyrimidine metabolism in rat liver

1. 1. Use of pyrimidine nucleotide precursors labelled in various positions on the ring shows that in rat liver, pyrimidine nucleotides formed from orotate follow anabolic pathways almost exclusively, whereas trace quantities of uridine are mostly degraded to β-alanine and its metabolites. 2. 2. Annomalies in the ratios of [ 14C] and [ 3H] in various common nucleotide products of orotate and uridine can be accounted for on the basis of metabolic compartmentation and recycling of CO 2.

Limitations in the use of [3H]thymidine incorporation into DNA as an indicator of epidermal keratinocyte proliferation in vitro.

The validity of using the incorporation of [3H]thymidine into DNA as an indicator of epidermal keratinocyte proliferation in vitro has been investigated. Other parameters of cell proliferation, direct count of cell number and measurement of DNA content, consistently fail to correlate with changes in [3H]thymidine incorporation into DNA in primary and first passage cultures of rabbit and human epidermal keratinocytes. Maximum incorporation of [3H]thymidine precedes the active growth period by three days. Incorporation declines markedly during the proliferative period. Thymidine kinase activity decreases during the proliferative growth phase. Incorporation of another pyrimidine nucleotide precursor, [14C]aspartic acid, suggests that in epidermal keratinocytes in vitro the extent of utilization of the salvage and the de novo pathways may be inversely related. In such cases [3H]thymidine incorporation into TCA precipitable material fails to reflect accurately cell proliferation.

Response of the Pyrimidine Pathway of Escherichia coli K12 to Exogenous Adenine and Uracil

The effect of exogenous adenine or uracil upon the de novo pathway for synthesis of pyrimidine nucleotides in Escherichia coli K12 was investigated. Parameters studied were levels of the enzymes carbamoyl phosphate synthase (EC 2.7.2.9), aspartate carbamoyltransferase (EC 2.1.3.2) and orotate phosphoribosyltransferase (EC 2.4.2.10) and the intermediates carbamoyl phosphate, aspartate and orotate, together with the contributions of exogenous uracil and aspartate to intracellular pyrimidine nucleotide. Taken with earlier data [Bagnara, A.S. & Finch, L. R. (1974) Eur. J. Biochem- 41, 421--430] on contents of UTP, CTP and 5-phosphoribosyl 1-diphosphate in cultures of this strain after the addition of adenine or uracil, the results obtained provide new insights into the regulatory mechanisms operating on the pathway in vivo. These insights enable evaluation of the contributions of such factors as limitation for a substrate, feed-back allosteric control by end products and enzyme repression/depression mechanisms. The evidence presented indicates that depressed levels of orotate phosphoribosyltransferase in E. coli K12 result in the wasteful ultilization of asparatate for excess synthesis of pyrimidine nucleotide precursors during balanced growth of the strain in minimal medium. Exogenous adenine increases the excessive accumulation of these precursors by lowering the intracellular content of 5-phosphoribosyl 1-diphosphate (Bagnara and Finch, 1974). This causes a decrease in the conversion of orotate to orotidine 5'-monophosphate, thus lowering the utilization or orotate and its precursors for synthesis of pyrimidine nucleotides. Further, since the contents of these nucleotide end products are thereby decreased (Bagnara nad Finch, 1974), theri feed-back on the early steps in the pathway is diminished and the production of the precursors is increased. It is postulated that growth of E. coli K12 under these conditions is limited by a compound that is metabolically related to precursors to aspartate.

Changes in activities of the de novo and salvage pathways of pyrimidine nucleotide biosynthesis during germination of black gram (Phaseolus mungo) seeds

Summary The metabolism of 14C- labelled pyrimidine nucleotide precursors in cotyledons and embryonic axes of black gram (Phaseolus mungo L.) seedlings was studied. The incorporation of 14C into the RNA, UTP, UDPG, and UMP fractions from [2-14C]-uridine was greater than that from [2-14C]uracil and [6-14C]orotate throughout the germination period. In the first 24 hr of germination, especially in cotyledons, incorporation of 14C into the RNA and nucleotide fractions from [2-14C]uracil was higher than that from [6-14C]orotate. In contrast, at the later stage of germination, [6-14C]orotate became a much better precursor for RNA and nucleotide biosynthesis than [2-14C]uracil. Considerable degradation of [2-14C]uracil to 14CO2 was found in both cotyledons and embryonic axes in every stage of germination examined. The metabolism of [2-14C]uracil, [2-14C]uridine, and [6-14C]orotate in 15-day-old primary leaves of light grown black gram seedlings was also studied. The metabolic patterns were similar to those in embryonic axes in the latter stages of germination. These results suggest that the uracil salvage pathway to pyrimidine nucleotides plays a role only in cotyledons in the early stage of germination, whereas the uridine salvage pathway is active in cotyledons and embryonic axes in every stage. On the other hand, the de novo pathway of pyrimidine nucleotide synthesis may have an important role in embryonic axes of the actively growing seedlings.