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At least 19 recordsLinked to original sources
PubMed1977

Biochemical genetic analysis of pyrimidine biosynthesis in mammalian cells: I. Isolation of a mutant defective in the early steps of de novo pyrimidine synthesis.

The isolation and characterization of a new mutant of Chinese hamster ovary cells is described. This mutant, Urd-A, shows an absolute requirement for exogenously added pyrimidines for growth. Complementation analysis indicates that the lesion in this mutant is recessive. Revertants can be isolated at frequencies suggesting that it is a single gene alteration. Biochemical analysis of cell-free extracts of CHO-K1 (Urd+) and Urd-A revealed that Urd-A possesses no more than 10% of wild-type levels of carbamyl phosphate synthetase (EC 2.7.2.9) activity, no more than 1% of wild-type levels of aspartate transcarbamylase (EC 1.2.3.2) activity, and undetectable levels of dihydroorotase (EC 3.5.2.3) activity. Thus, this mutant appears simultaneously to possess marked or complete deficiencies in the activities of the first three enzymes of pyrimidine biosynthesis. Activities of the other enzymes of the pathway appear normal. The use of this mutant for biochemical-genetic studies of pyrimidine biosynthesis is discussed.

Aspartate Carbamoyltransferase↗
PubMed1979

Alteration in structure of multifunctional protein from Chinese hamster ovary cells defective in pyrimidine biosynthesis.

A combined genetic, biochemical, and immunological approach has clarified structural relationships involving the first three enzymes of de novo pyrimidine biosynthesis. A procedure involving antibody and protein A-Sepharose was used to isolate the enzymes carbamoyl-phosphate synthase [ATP:carbamate phosphotransferase (dephosphorylating, amido-transferring), EC 2.7.2.9], aspartate transcarbamoyltransferase (carbamoylphosphate:L-aspartate carbamoyltransferase, EC 2.1.3.2), and dihydro-orotase (L-5,6-dihydroorotate amidohydrolase, EC 3.5.2.3) from Chinese hamster ovary cell CHO-K1, the uridine-requiring auxotroph Urd(-)A, and selected Urd(-)A revertants. The enzymes of Urd(-)A and the Urd(-)A revertants were significantly altered in activity, native structure, and molecular weight from those of CHO-K1. The results presented permit the conclusion that (i) these three enzymes reside in a single multifunctional 220,000-dalton polypeptide; (ii) the aspartate transcarbamoyltransferase activity is located on a portion ( approximately 20,000 daltons) at one end of the polypeptide; (iii) this portion may also be required for monomers to aggregate into the multimeric from present in mammalian cells; (iv) the mutations in Urd(-)A and the Urd(-)A revertants lie in the structural gene for this multifunctional protein; and (v) increased sensitivity to proteases could account for the alterations in the structure of these enzymes in the mutants.

Amidohydrolases↗
PubMed1979

Effect of 6-azauridine on de novo pyrimidine biosynthesis in cultured Ehrlich ascites cells. Orotate inhibition of dihydroorotase and dihydroorotate dehydrogenase.

The inhibition of dihydro-orotase (E 3.5.2.3) and dihydroorotate (DHO) dehydrogenase (dihydro-orotate oxidase, EC 1.3.3.1) by cellular orotate (OA) in Ehrlich ascites cells was studied by measuring the accumulation of the intermediates of de novo pyrimidine biosynthesis at various times after the addition of 6-azauridine to the culture medium. The addition of 6-azauridine resulted in the accumulation of orotidine, OA, DHO, and carbamyl aspartate (CAA). The use of the observed ratios of [CCA]/[OA] and [DHO]/[OA] and other known constants allowed us to calculate that the increased cellular OA concentration caused primarily an inhibition of DHO dehydrogenase rather than an inhibition of dihydroorotase. A constant ratio of [CAA]/[DHO] was observed which probably indicates that the interconversion of these two intermediates catalyzed by dihydroorotase is near equilibrium in these cells as has been observed in vitro (Christopherson, R.I., Matsuura, T., and Jones, M.E. (1978) Anal. Biochem. 89, 225-234). It is suggested that the probable intracellular accumulation of CAA in patients with oroticaciduria may have significant secondary effects.

Amidohydrolases↗
PubMed1979

Glutamine-dependent carbamoyl-phosphate synthetase and control of pyrimidine biosynthesis in the parasitic helminth Schistosoma mansoni.

1. Carbomoyl-phosphate synthetase in Schistosoma mansoni utilizes L-glutamine as well as ammonia as nitrogen donor but does not require N-acetyl-L-glutamate for the activity. 2. The enzyme activity was inhibited by UDP, UTP, ADP and AMP, among which UDP was the most effective. 3. Aspartate carbamoyltransferase and dihydroorotase were also found and copurified with the synthetase. 4. Relative activities among these three enzymes were 1:30-60:3-8 throughout the purification. 5. These results suggest that the synthetase plays a key role in the control of pyrimidine biosynthesis de novo.

Animals↗
PubMed1979

Biochemical genetic analysis of pyrimidine biosynthesis in mammalian cells: III. Association of carbamyl phosphate synthetase, aspartate transcarbamylase, and dihydroorotase in mutants of cultured Chinese hamster cells.

Carbamyl phosphate synthetase (EC 2.7.2.9), aspartate transcarbamylase (EC 2.1.3.2), and dihydroorotase (EC 3.5.2.3), the first three enzymes in de novo pyrimidine synthesis in Chinese hamster ovary cell strain Kl (CHO-Kl), cose diment through a glycerol gradient. When an extract from Urd- A, a pyrimidine-requiring auxotroph reduced in all three activities, is run on a glycerol gradient, the enzyme activities appear in two peaks higher in the gradient, a peak of aspartate transcarbamylase separated from a peak of carbamyl phosphate synthetase and dihydroorotase. Revertants of Urd- A have increased activity of all three enzymes and give glycerol gradient patterns similar to either CHO-Kl or Urd- A. The gradient pattern for Urd- A and some of its revertants can be mimicked by treating the CHO-Kl cell extract with trypsin. Hybrids made between a CHO-Kl purine-requiring auxotroph (Ade- C) and a Urd- A revertant gave a glycerol gradient pattern which is a composite of the CHO-Kl and revertant patterns. A model is presented for the structure of this multifunctional protein.

Amidohydrolases↗
PubMed1979

Genetical and cytological location of the structural parts coding for the first three steps of pyrimidine biosynthesis in Drosophila melanogaster.

The rudimentary locus (r; X--55.3) of Drosophila melanogaster is shown to contain the structural sequences for the enzymes CPSase, ATCase and DHOase. The enzyme concentration in adult flies is correlated with the number of r+ copies in the genome. The expression of the locus follows the rules of the gene dosage compensation hypothesis when extracts of newly emerged males and females are compared.

Animals↗
PubMed2026

Characterization of carABpyrB operon and role of pyrE in Francisella novicida biofilm.

Pyrimidine biosynthesis is essential for bacterial growth, but its role in regulating biofilm formation in Francisella (F.) novicida remains poorly defined. In this study, we experimentally defined the carABpyrB operon in F. novicida and investigated how disruption of the de novo pyrimidine biosynthesis pathway affects growth and biofilm formation under nutrient-restricted conditions. Reverse transcriptase PCR confirmed co-transcription of carA, carB, and pyrB, and promoter prediction identified two putative σ70-dependent promoter regions upstream of carA. Transposon mutants disrupted in carA, carB, and pyrB exhibited pronounced growth defects in Chamberlain's Defined Medium that were restored by uracil supplementation, confirming pyrimidine auxotrophy and functional disruption of de novo pyrimidine biosynthesis. We then extended this analysis to additional genes in the pyrimidine biosynthetic pathway and assessed biofilm formation in modified Mueller-Hinton broth, a nutrient-restricted condition. In this medium, carA, carB, pyrB, and pyrE mutants exhibited growth deficiencies; however, the pyrE mutant uniquely produced significantly more biofilm than the wild type. This phenotype remained evident even without growth normalization, with the pyrE mutant producing 3.3-fold more biofilm than wild type, despite impaired growth, and increased to 11.8-fold when normalized to growth. Quantitative PCR demonstrated that uracil supplementation represses carA, carB, and pyrB transcription, consistent with feedback regulation of the pathway. Together, these findings indicate that pyrimidine limitation is not simply a growth-limiting condition but can alter biofilm regulation, with pyrE disruption revealing a strong association between de novo pyrimidine biosynthesis and biofilm formation.

Biofilms↗
PubMed1976

Stable mutants of mammalian cells that overproduce the first three enzymes of pyrimidine nucleotide biosynthesis.

Upon exposure to 0.1 mM N-phosphonacetyl-L-aspartate (PALA), a transition state analog inhibitor of aspartate transcarbamylase, most cells of a simian virus 40 (SV40)-transformed Syrian hamster line are killed within a few days, but resistant mutants form spontaneously with frequency 2-5 X 10(-5) in a stochastic process not dependent upon the presence of the inhibitor. The resistant phenotype is stable for many months in the absence of PALA. Other cell lines also give resistant mutants, but with substantially lower frequencies. Serial selection with PALA at concentrations up to 25 mM has yielded clones with more than 100 times the original aspartate transcarbamylase activity. The activities of carbamyl-P synthetase and dihydroorotase, which co-purify with aspartate transcarbamylase as a three-enzyme complex, increase in parallel with aspartate transcarbamylase activity in each resistant clone tested, but there is no substantial change in the activities of the last three enzymes of the de novo pathway, which are not in this complex. In each of the three resistant clones tested, there is an increase in the number of aspartate transcarbamylase active sites, determined by titration with 3H-PALA, which closely parallels the increase in enzyme activity. In one resistant clone tested, there is no change in the Ki for PALA or the Km for carbamyl-P. The only mechanism detected for achieving resistance to PALA is an increase in the steady state amount of the three enzyme complex.

Amidohydrolases↗
PubMedDate not supplied

Sex-dependent differences in the biosynthesis of pyrimidine nucleotides in rat liver after repeated administration of alpha-hexachlorocyclohexane.

Utilization of [2-14C]orotic acid for the synthesis of the pyrimidine components of the free nucleotide pool and RNA from isolated cytoplasmic ribosomes was investigated in the liver of female and male rats. In females, labeled orotic acid is incorporated relatively more into uridine than into cytidine nucleotides; the opposite is true for males. The administration of alpha-hexachlorocyclohexane (alpha-HCH) decreases the specific radioactivity of rRNA cytidylic acid more in males than in females. Simultaneously, the level of liver microsomal cytochrome P-450 is increased after the administration of alpha-HCH more in males than in females. After four days of treatment with alpha-HCH, the concentrations of the uridine and cytidine components of the acid-soluble pool are slightly depressed. Following repeated administration of alpha-HCH, the utilization of labeled uridine for the synthesis of cytidine nucleotides of the acid-soluble pool and RNA is depressed, whereas that of labeled cytidine is enhanced. Repeated administration of alpha-HCH decreases the utilization of [2-14C]orotic acid for the synthesis of cytidine nucleotides of DNA; the specific radioactivity of thymidylic acid is increased. The administration of beta-diethylaminoethyl diallylacetate (CFT-1201) to animals that have repeatedly received alpha-HCH decreases the specific radioactivity of DNA thymine.

Animals↗
PubMed2025

De novo pyrimidine synthesis is a collateral metabolic vulnerability in NF2-deficient mesothelioma.

Pleural mesothelioma (PM) is one of the deadliest cancers, with limited therapeutic options due to its therapeutically intractable genome, which is characterized by the functional inactivation of tumor suppressor genes (TSGs) and high tumor heterogeneity, including diverse metabolic adaptations. However, the molecular mechanisms underlying these metabolic alterations remain poorly understood, particularly how TSG inactivation rewires tumor metabolism to drive tumorigenesis and create metabolic dependencies. Through integrated multi-omics analysis, we identify for the first time that NF2 loss of function defines a distinct PM subtype characterized by enhanced de novo pyrimidine synthesis, which NF2-deficient PM cells are critically dependent on for sustained proliferation in vitro and in vivo. Mechanistically, NF2 loss activates YAP, a downstream proto-oncogenic transcriptional coactivator in the Hippo signalling pathway, which in turn upregulates CAD and DHODH, key enzymes in the de novo pyrimidine biosynthesis pathway. Our findings provide novel insights into metabolic reprogramming in PM, revealing de novo pyrimidine synthesis as a synthetic lethal vulnerability in NF2-deficient tumors. This work highlights a potential therapeutic strategy for targeting NF2-deficient mesothelioma through metabolic intervention.

Pyrimidines↗
PubMed1978

Rudimentary locus of Drosophila melanogaster: partial purification of a carbamylphosphate synthase--aspartate transcarbamylase--dihydroorotase complex.

Glutamine-dependent CPSase, ATCase, and DHOase from Drosophila, the first three enzymes in pyrimidine biosynthesis, show coordinate variation in activity throughout development. The three activities were highest in first instar larvae and decreased as development proceeded. The three activities cosediment in sucrose gradients as a single peak with a relative sedimentation coefficient of approximately 30S. CPSase, ATCase, and DHOase copurify during (NH4)2SO4 fractionation and during DEAE-cellulose and hydroxylapatite chromatography.

Amidohydrolases↗
PubMed2025

Uridine 5'-monophosphate (UMP) synthesis connects nucleotide metabolism to programmed cell death in C. elegans.

Nucleotide metabolism is essential for fundamental cellular functions such as growth, repair and proliferation. Emerging evidence suggests that metabolic pathways also influence programmed cell death (PCD), though the underlying mechanisms remain poorly understood. One model organism that has provided key insights into the regulation of PCD is Caenorhabditis elegans (C. elegans). In this nematode, apoptosis is often initiated through asymmetric cell division (ACD), a process that unequally distributes fate determinants between daughter cells to produce a larger surviving cell and a smaller cell destined for apoptosis. Here, we demonstrate that the simultaneous disruption of PCD and ACD leads to aberrant cell survival and the formation of extra hypodermal cells. Through a genetic screen in the grp-1 ACD mutant background, we identified pyr-1 as a regulator of PCD. pyr-1 encodes the C. elegans carbamoyl-phosphate synthetase/aspartate transcarbamoylase/dihydroorotase (CAD) enzyme which catalyzes the rate-limiting step of de novo pyrimidine biosynthesis, producing uridine 5'-monophosphate (UMP). UMP is a critical metabolite for the synthesis of nucleotides, lipids and carbohydrates. Genetic analysis of UMP metabolic pathways, combined with exogenous nucleoside supplementation, confirms that UMP availability is essential for PYR-1-mediated PCD. Loss of grp-1 induces cellular stress by disrupting fate determinant partitioning during ACD, whereas pyr-1 mutations cause metabolic stress through UMP depletion. While both mutations independently activate autophagy, they function redundantly to upregulate the mitochondrial chaperone hsp-6. Knockdown of autophagy-related genes and hsp-6 reveals that these pathways serve as compensatory mechanisms to protect against cell death in the pyr-1; grp-1 double mutants. Collectively, our findings establish a direct link between metabolism and cell death, demonstrating how UMP availability and proper ACD coordinate apoptotic regulation and developmental outcomes. This study highlights the intricate interplay between metabolic homeostasis and PCD, providing new insights into the metabolic control of cell fate decisions.

Caenorhabditis elegans↗