Pyrimidine Nucleotide Metabolism and Pathways of Thymidine Triphosphate Biosynthesis in Salmonella typhimurium
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The nucleoside triphosphate pools of two cytidine auxotrophic mutants of Salmonella typhimurium LT-2 were studied under different conditions of pyrimidine starvation. Both mutants, DP-45 and DP-55, are defective in cytidine deaminase and cytidine triphosphate (CTP) synthase. In addition, DP-55 has a requirement for uracil (uridine). Cytidine starvation of the mutants results in accumulation of high concentrations of uridine triphosphate (UTP) in the cells, while the pools of CTP and deoxy-CTP drop to undetectable levels within a few minutes. Addition of deoxycytidine to such cells does not restore the dCTP pool, indicating that S. typhimurium has no deoxycytidine kinase. From the kinetics of UTP accumulation during cytidine starvation, it is concluded that only cytidine nucleotides participate in the feedback regulation of de novo synthesis of UTP; both uridine and cytidine nucleotides participate in the regulation of UTP synthesis from exogenously supplied uracil or uridine. Uracil starvation of DP-55 in presence of cytidine results in extensive accumulation of CTP, suggesting that CTP does not regulate its own synthesis from exogenous cytidine. Analysis of the thymidine triphosphate (dTTP) pool of DP-55 labeled for several generations with 32 P-orthophosphate and 3 H-uracil in presence of 12 C-cytidine shows that only 20% of the dTTP pool is derived from uracil (via the methylation of deoxyuridine monophosphate); 80% is apparently synthesized from a cytidine nucleotide.Keywords:
Cytidine
Uracil
Nucleotide salvage
Uridine triphosphate
Thymidine
Pyrimidine metabolism
Deoxyuridine
Pyrimidine nucleotide biosynthesis in humans is a promising chemotherapeutic target for infectious diseases caused by RNA viruses. Because mammalian cells derive pyrimidine ribonucleotides through a combination of de novo biosynthesis and salvage, combined inhibition of dihydroorotate dehydrogenase (DHODH; the first committed step in de novo pyrimidine nucleotide biosynthesis) and uridine/cytidine kinase 2 (UCK2; the first step in salvage of exogenous nucleosides) strongly attenuates viral replication in infected cells. However, while several pharmacologically promising inhibitors of human DHODH are known, to date there are no reports of medicinally viable leads against UCK2. Here, we use structure-based drug prototyping to identify two classes of promising leads that noncompetitively inhibit UCK2 activity. In the process, we have identified a hitherto unknown allosteric site at the intersubunit interface of this homotetrameric enzyme. By reducing the kcat of human UCK2 without altering its KM, these new inhibitors have the potential to enable systematic dialing of the fractional inhibition of pyrimidine salvage to achieve the desired antiviral effect with minimal host toxicity.
Pyrimidine metabolism
Nucleotide salvage
Dihydroorotate Dehydrogenase
Aspartate carbamoyltransferase
Cytidine
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The role of uridine and cytidine compounds in regulating pyrimidine nucleotide biosynthesis de novo was studied with cytidine deaminase-negative mutants of Bacillus subtilis. In the wild type strain, the formation of six enzymes for uridine S'-monophosphate (UMP) biosynthesis was severely repressed by exogenous cytidine or uracil, whereas the formation of the enzymes in a cytidine deaminase-negative mutant was repressed only by uracil. On the other hand, the formation of cytidine 5'-triphosphate (CTP) synthetase was not affected by uracil. This enzynme was repressed only when a cytidine deaminase-negative mutant was grown in the presence of excess cytidine. Studies on feedback inhibition also showed that the activity of CTP synthetase was inhibited by cytidine nucleotides, but not by uridine nucleotides.
Cytidine
Uracil
Nucleotide salvage
Activation-induced (cytidine) deaminase
Pyrimidine metabolism
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Uridine triphosphate
Nucleotide salvage
Cytidine
Nucleoside-diphosphate kinase
Nucleoside triphosphate
Ribonucleotide
Adenosine triphosphate
Pyrimidine metabolism
Uridine diphosphate
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Effect of inhibitors of the de novo pyrimidine biosynthetic pathway on serum uridine levels in mice.
Since C57BL X DBA F1 (hereafter called BDF1) mice possess a relatively constant concentration of serum uridine [9.7 +/- 1.3 (S.D.) nmol/ml], circulating uridine is available to cells with an intact pyrimidine salvage pathway and thus could influence the effectiveness of certain antitumor agents which inhibit de novo pyrimidine biosynthesis and whose cytotoxic properties are reversed by uridine. Three inhibitors of the de novo pyrimidine biosynthetic pathway were studied to determine their effects on circulating uridine concentration in BDF1 mice. Pyrazofurin and 6-azauridine were found to have no significant effect on serum uridine levels when administered as a single dose or on 4 consecutive days. In contrast, N-(phosphonacetyl)-L-aspartate reduced serum uridine levels by 55% when administered either as a single dose or on 4 consecutive days. This reduction could contribute to the antitumor effectiveness of N-(phosphonacetyl)-L-aspartate by limiting the rescue of cells possessing a salvage pathway. D-Galactosamine, a stimulator of the de novo pyrimidine pathway, was also studied and found to increase total liver uridine (uridine plus uracil nucleotides and uridine diphosphate esters) by 4-fold at 8 hr, returning to normal by 24 to 48 hr. However, these large effects were not reflected in the serum.
Uracil
Pyrimidine metabolism
Nucleotide salvage
De novo synthesis
Uridine triphosphate
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Orotate (OA) is well-known as a precursor in biosynthesis of pyrimidines; in mammals it is released from the mitochondrial dihydroorotate dehydrogenase (DHODH) for conversion to UMP by the cytoplasmic UMP synthase enzyme. OA is also a normal part of the diet, being found in milk and dairy products, and it is converted to uridine for use in the pyrimidine salvage pathway predominantly in liver, kidney and erythrocytes. Early research into nutrition identified orotate as "vitamin B13," and its use as a complex with organic cations or metal ions was promulgated in body-building, and in assisting therapies of metabolic syndromes. It has recently been established that the amelioration of gout by dairy products arises from the competition of orotate and urate at the hURAT1 transporter. The orotic aciduria that arises in children with defective UMP synthase can be rescued by oral uridine therapy, since UMP is the end-product and also a feedback inhibitor of the de novo pathway. In contrast, Miller (dysmorphology) syndrome is connected with defects in DHODH, and hence in the supply of OA, and cannot be helped by uridine. Other models of dysmorphisms are connected with enzymes early in the pyrimidine de novo pathway. We conclude that the OA molecule is itself required for the regulation of genes that are important in the development of cells, tissues and organisms.
Orotic acid
Pyrimidine metabolism
Dihydroorotate Dehydrogenase
Nucleotide salvage
Uridine triphosphate
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Abstract In order to gain a better understanding of how a defect in pyrimidine biosynthesis leads to the specific set of morphological aberrations seen in Drosophila bearing the mutant allele of rudimentary , we made the following measurements of enzyme activity in wild type flies: (1) during ovarian development – the first three enzymes of the de novo pathway for pyrimidine biosynthesis (aspartate transcarbamylase, dihydroorotase, and carbamyl phosphate synthetase) and two enzymes in the salvage pathway for pyrimidine biosynthesis – uridine kinase and uridine phosphorylase; (2) during embryonic development – uridine kinase and uridine phosphorylase in embryos taken at hourly intervals; and (3) during postembryonic development – uridine kinase and uridine phosphorylase. Our results suggest: (1) that the de novo pyrimidine enzymes are synthesized during the early stages of oogenesis, while the pattern for the salvage pathway enzymes is somewhat different; (2) that a lowering of uridine kinase activity at 13 hours of embryonic development may be responsible for bringing on the developmental crisis in r embryos; and (3) that the postembryonic pattern of activity for salvage pathway enzymes, with one exception, is quite similar to that for the de novo pathway enzymes.
Pyrimidine metabolism
Nucleotide salvage
De novo synthesis
Aspartate carbamoyltransferase
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Abstract The biosynthesis of purine and pyrimidine nucleotides takes place over de novo synthetic pathways from small molecules and by salvage pathways from preformed purine or pyrimidine bases or nucleosides. The pathways of de novo synthesis are the same in animals and microorganisms. Salvage pathways are considerably more energy‐efficient than de novo pathways, which require 5 (pyrimidine) or 6 (purine) moles of ATP for each mole of nucleotide produced. Salvage pathways are integral to the cause or treatment of a number of human diseases of purine or pyrimidine metabolism. Among disorders of purine metabolism, the Lesch–Nyhan disease is characterised by overproduction of uric acid, clinical gout, nephropathy, neurologic disease, and unusual self‐injurious behaviours. Key Concepts: Salvage of purines is catalysed by adenine phosphoribosyltransferase (APRT) and hypoxanthine guanine phosphoribosyltransferase (HGPRT). Pyrimidine salvage is catalysed by thymidine kinase. Pyrimidine salvage is effective in the treatment of orotic aciduria, a disorder of pyrimidine nucleotide synthesis. Deficiency of APRT leads to renal calculi. Deficiency of HGPRT is the cause of Lesch–Nyhan disease.
Nucleotide salvage
Adenine phosphoribosyltransferase
Phosphoribosyltransferase
Pyrimidine metabolism
Purine metabolism
De novo synthesis
Hypoxanthine Phosphoribosyltransferase
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Citations (19)
Cytidine
Nucleotide salvage
Deoxyuridine
Activation-induced (cytidine) deaminase
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Citations (8)
The role of uridine and cytidine compounds in regulating pyrimidine nucleotide biosynthesis de novo was studied with cytidine deaminase-negative mutants of Bacillus subtilis. In the wild type strain, the formation of six enzymes for uridine 5′-monophosphate (UMP) biosynthesis was severely repressed by exogenous cytidine or uracil, whereas the formation of the enzymes in a cytidine deaminase-negative mutant was repressed only by uracil. On the other hand, the formation of cytidine 5′-triphosphate (CTP) synthetase was not affected by uracil. This enzynme was repressed only when a cytidine deaminase-negative mutant was grown in the presence of excess cytidine. Studies on feedback inhibition also showed that the activity of CTP synthetase was inhibited by cytidine nucleotides, but not by uridine nucleotides.
Cytidine
Uracil
Nucleotide salvage
Activation-induced (cytidine) deaminase
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Nucleotide salvage
Pyrimidine metabolism
Cytidine
De novo synthesis
Metabolic pathway
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Citations (26)