Localization of the gene for uridine monophosphate synthase to human chromosome region 3q13 by in situ hybridization

The bifunctional enzyme uridine monophosphate (UMP) synthase catalyzes the last two steps in de novo pyrimidine biosynthesis. A genetic deficiency in the activity of this enzyme causes the inherited human disease orotic aciduria. We used a human cDNA probe to localize the gene for UMP synthase to human chromosome region 3q13 by the technique of in situ hybridization.     

Orotate (orotic acid): An essential and versatile molecule

ABSTRACT

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.     

Orotate phosphoribosyltransferase and orotidine 5'-monophosphate decarboxylase exist as multienzyme complex in human malaria parasite Plasmodium falciparum

Plasmodium falciparum, the causative agent of the most lethal form of human malaria, totally depends on de novo pyrimidine biosynthetic pathway. Orotate phosphoribosyltransferase (OPRT) and orotidine 5'-monophosphate decarboxylase (OMPDC), the fifth and sixth enzymes in the pathway catalyzing formation of uridine 5'-monophosphate (UMP), remain largely uncharacterized in the protozoan parasite. In this study, we achieved purification of OPRT and OMPDC to near homogeneity from P. falciparum cultivated in vitro. The OPRT and OMPDC activities were co-eluted in all chromatographic columns during purification, suggesting the purified proteins exist as a multienzyme complex with a molecular mass of 140+/-8 kDa and contain two subunits each of OPRT and OMPDC. Monomeric forms of OPRT and OMPDC had molecular masses of 32+/-3 and 38+/-3 kDa, respectively, in agreement with those of proteins predicted from P. falciparum genome database. Interestingly, kinetic parameters and inhibitory constants of both OPRT and OMPDC activities were found to be different to those of the bifunctional human red cell UMP synthase. Our evidence provides the first example of OPRT and OMPDC existing as a multienzyme complex.     

Alteration in de novo pyrimidine biosynthesis during uridine reversal of pyrazofurin-inhibited DNA synthesis

Pyrazofurin, a pyrimidine nucleoside analogue with antineoplastic activity, inhibits cell proliferation and DNA synthesis in cells by inhibiting uridine 5'-phosphate (UMP) synthase. It has been previously shown in concanavalin A (con A)-stimulated guinea pig lymphocytes (23) that pyrazofurin-inhibited DNA synthesis could be selectively reversed by exogenous uridine (Urd). In this report, we have examined possible mechanisms for the Urd reversal with experiments that determine the ability of exogenous Urd to (a) interfere with either the intracellular transport of pyrazofurin, or the conversion of pyrazofurin to its intracellularly active form, pyrazofurin-5'-phosphate; (b) reverse the pyrazofurin block of [14C]orotic acid incorporation into DNA; and (c) alter the pattern of exogenous [3H]Urd incorporation into DNA-thymine (DNA-Thy) and DNA-cytosine (DNA-Cyt) during pyrazofurin inhibition of pyrimidine de novo biosynthesis. The results of these experiments showed that Urd reversal does not occur through altered pyrazofurin transport or intracellular conversion to pyrazofurin-5'-phosphate, nor does it alter the distribution of [3H]Urd in DNA-Thy and DNA-Cyt. Instead, these findings indicate that the primary mechanism for exogenous Urd reversal of pyrazofurin inhibition of DNA synthesis involves the reversal of pyrazofurin inhibition of UMP synthase, thus restoring orotic acid incorporation into lymphocyte DNA through the pyrimidine de novo pathway.     

Introduction of a Fluorine Atom at C3 of 3-Deazauridine Shifts Its Antimetabolic Activity from Inhibition of CTP Synthetase to Inhibition of Orotidylate Decarboxylase, an Early Event in the de Novo Pyrimidine Nucleotide Biosynthesis Pathway

The antimetabolite prodrug 3-deazauridine (3DUrd) inhibits CTP synthetase upon intracellular conversion to its triphosphate, which selectively depletes the intracellular CTP pools. Introduction of a fluorine atom at C3 of 3DUrd shifts its antimetabolic action to inhibition of the orotidylate decarboxylase (ODC) activity of the UMP synthase enzyme complex that catalyzes an early event in pyrimidine nucleotide biosynthesis. This results in concomitant depletion of the intracellular UTP and CTP pools. The new prodrug (designated 3F-3DUrd) exerts its inhibitory activity because its monophosphate is not further converted intracellularly to its triphosphate derivative to a detectable extent. Combinations with hypoxanthine and adenine markedly potentiate the cytostatic activity of 3F-3DUrd. This is likely because of depletion of 5-phosphoribosyl-1-pyrophosphate (consumed in the hypoxanthine phosphoribosyl transferase/adenine phosphoribosyl transferase reaction) and subsequent slowing of the 5-phosphoribosyl-1-pyrophosphate-dependent orotate phosphoribosyl transferase reaction, which depletes orotidylate, the substrate for ODC. Further efficient anabolism by nucleotide kinases is compromised apparently because of the decrease in pK(a) brought about by the fluorine atom, which affects the ionization state of the new prodrug. The 3F-3DUrd monophosphate exhibits new inhibitory properties against a different enzyme of the pyrimidine nucleotide metabolism, namely the ODC activity of UMP synthase.     

Automated screening system for purine and pyrimidine metabolism disorders using high-performance liquid chromatography

An automated screening system for purine and pyrimidine metabolism disorders using high-performance liquid chromatography (HPLC) with column switching is described. The system consists of a reversed-phase column, a cation-exchange column, a column switch, four sets of ultraviolet absorbance detectors, a microcomputer and other conventional equipment. As this system permits the simultaneous determination of urinary orotic acid, uracil, dihydrouracil, pseudouridine, xanthine, 2,8-dihydroxyadenine and succinyladenosine, it offers a useful method for the detection of orotic aciduria, dihydropyrimidine dehydrogenase deficiency, diphydropyrimidinuria, xanthinuria, adenine phosphoribosyltransferase deficiency and adenylosuccinase deficiency.     

The conversion of orotic acid into uridine 5′-monophosphate by isolated perfused normal and regenerating rat livers

The release of (14)CO(2) from [7-(14)C]orotic acid was measured in isolated perfused normal and regenerating rat livers. With some limitations, the release of (14)CO(2) from [7-(14)C]orotic acid can be used to estimate UMP synthesis in perfused livers. Isolated perfused livers rapidly pick up labelled orotic acid added to perfusate and convert most of it into UMP. Perfused regenerating livers produce approx. 2.5 times as much UMP/g of liver as do perfused normal livers. However, the absolute amount of orotic acid converted into UMP is higher in perfused normal livers than in perfused regenerating livers. Perfused regenerating livers do not differ in their orotic acid uptake and UMP synthesis from livers of comparable size in which regeneration is not taking place. The total amount of orotic acid taken up by the liver (rather than the rate of uptake) and the size of the liver appear to be the determining factors in UMP production. The results suggest that the decrease in liver size caused by partial hepatectomy may be in itself sufficient to account for an increase in the flow of metabolites in the pyrimidine pathway at the early stages of liver regeneration.     

Increased levels of UMP synthase protein and mRNA in pyrazofurin-resistant rat hepatoma cells

Rat hepatoma cells that have undergone stepwise selection in increasing concentrations of pyrazofurin have coordinately increased levels of both orotate phosphoribosyltransferase (EC 2.4.2.10) and orotidine-5'-phosphate decarboxylase (EC 4.1.1.23) activity. These two activities catalyze the conversion of orotic acid to UMP in de novo pyrimidine biosynthesis. Cells selected in 50 microM pyrazofurin have over 40 times the wild type level for both activities. A single polypeptide of approximately 55,000 daltons is increased in the resistant cells in amounts corresponding to the increase in the two activities. Resistant cell lines that are grown for extended periods in the absence of pyrazofurin are unstable, losing their elevated levels of both enzyme activities and the increased specific protein. Antibody prepared against a purified protein containing both enzyme activities binds specifically to this increased protein. These results corroborate other evidence indicating the two enzyme activities are contained within a single polypeptide called UMP synthase. Poly(A+) mRNA isolated from wild type and resistant lines was analyzed by in vitro translation for production of UMP synthase protein. Immunoprecipitation of the translation products shows the resistant cells have a 17-fold increase in translatable mRNA activity coding for UMP synthase. The synthase accounts for 0.24% of the total in vitro translation products synthesized with poly(A+) mRNA from the pyrazofurin-resistant cells as opposed to 0.014% with wild type mRNA. A cloned UMP synthase cDNA sequence hybridizes strongly to a 1.8-kilobase mRNA in the resistant cells. This mRNA is only barely detectable in equivalent preparations from wild type cells. Quantitation of the mRNA by dot hybridization techniques indicates a 16-fold increase in UMP synthase mRNA in the resistant cells. Although this increase in mRNA for UMP synthase could explain most of the increased protein, it is not sufficient to totally account for the 40-fold increase in UMP synthase.     

Orotic acid biosynthesis in arginine-deficient rats.

Arginine deficiency is associated with a mild orotic aciduria. Liver slices from rats fed a purified l-amino acid diet with (control) and without arginine supplementation were used for studies of [¹⁴C]bicarbonate incorporation into orotic acid. The nanomoles of orotic acid synthesized in isolated liver slices from both control and arginine-deficient animals increased linearly with time. Orotic acid biosynthesis was significantly greater in liver slices than slices of heart, muscle, kidney, and minced spleen. The order of orotate biosynthesis from [¹⁴C]bicarbonate was liver > spleen = kidney > muscle > heart. Arginine deficiency resulted in a significant stimulation of liver orotic acid biosynthesis. This stimulation in pyrimidine biosynthesis can account for a major portion of the orotic aciduria. Orotic acid synthesis from spleens isolated from arginine-deficient rats was also enhanced compared with controls. Although the rate of orotic acid biosynthesis is small relative to liver production, the spleen may contribute slightly to increased orotic aciduria in the arginine-deficient rat. Arginine supplementation in vitro to livers from rats fed either the control of arginine-deficient diet resulted in a significant reduction in synthesis of orotic acid. Dietary arginine may play a key role in regulating mitochondrial carbamoyl phosphate utilization into both pyrimidine and urea biosynthesis.     

Niemann-Pick type C disease: mutations of NPC1 gene and evidence of abnormal expression of some mutant alleles in fibroblasts

We analyzed Niemann-Pick type C disease 1 (NPC1) gene in 12 patients with Niemann-Pick type C disease by sequencing both cDNA obtained from fibroblasts and genomic DNA. All the patients were compound heterozygotes. We found 15 mutations, eight of which previously unreported. The comparison of cDNA and genomic DNA revealed discrepancies in some subjects. In two unrelated patients carrying the same mutations (P474L and nt 2972del2) only one mutant allele (P474L), was expressed in fibroblasts. The mRNA corresponding to the other allele was not detected even in cells incubated with cycloheximide. The promoter variants (-1026T/G and -1186T/C or -238 C/G), found to be in linkage with 2972del2 allele do not explain the lack of expression of this allele, as they were also found in control subjects. In another patient, (N1156S/Q922X) the N1156S allele was expressed in fibroblasts while the expression of the other allele was hardly detectable. In a fourth patient cDNA analysis revealed a point mutation in exon 20 (P1007A) and a 56 nt deletion in exon 22 leading to a frameshift and a premature stop codon. The first mutation was confirmed in genomic DNA; the second turned out to be a T-->G transversion in exon 22, predicted to cause a missense mutation (V1141G). In fact, this transversion generates a donor splice site in exon 22, which causes an abnormal pre-mRNA splicing leading to a partial deletion of this exon. In some NPC patients, therefore, the comparison between cDNA and genomic DNA may reveal an unexpected expression of some mutant alleles of NPC1 gene.     

Novel mutations in the 3' region of the polycystic kidney disease 1 (PKD1) gene

Mutation screening in 90 unrelated ADPKD1 patients was carried out on some of the exons in the single copy area (37, 38, 39, 44, 45) using genomic PCR and SSCP. Four novel mutations were found: a 15 bp in-frame deletion in exon 39 [nt11449 (del 15)], a 2 bp deletion in exon 44 [nt12252 (del 2)], a G insertion in exon 44 [nt12290 (Ins G)], and a GTT in-frame deletion in exon 45 [nt12601 (del 3)].     

Biosynthesis of pyrimidine nucleotides in cultured root callus ofCucurbita pepo

Summary Callus cultures derived from roots of summer squash (Cucurbita pepo L. c.v. Early Prolific Straightneck) grown in the dark at 27° C on Murashige and Skoog medium supplemented per liter with 30 g sucrose, 100 mg myo-inositol, 10 mg indole-butyric acid, 2 mg glycine, 1 mg thiamin, 0.5 mg nicotinic acid, 0.5 mg pyridoxine, and 2 g Gelrite were capable of synthesizing pyrimidine nucleotides both de novo and through salvage of existing pyrimidine nucleotides and bases. Evidence that the de novo biosynthesis of pyrimidine nucleotides proceeded via the orotate pathway in this tissue included: (a) demonstration of the incorporation of NaH¹⁴CO₃ and [¹⁴C₆]orotic acid into uridine nucleotides (ΣUMP), and (b) demonstration that the addition of 6-azauridine blocked the incorporation of these two precursors into ΣUMP. The synthesis of pyrimidine nucleotides through the salvage of existing pyrimidine bases and ribosides was demonstrated by measuring the incorporation of [¹⁴C₂]uracil and [¹⁴C₂]uridine into ΣUMP. Salvage of both [¹⁴C₂]uracil and [¹⁴C₂]uridine was sensitive to inhibition by 6-azauridine or one of its metabolites. The orotic acid pathway for the de novo biosynthesis of pyrimidine nucleotides was demonstrated to be sensitive to end-product inhibition. Uridine, or one of its metabolites, inhibited the incorporation of NaH¹⁴CO₃, but not [¹⁴C₆]orotic acid, into ΣUMP. Evidence is presented suggesting that Aspartate carbomoyltransferase is the site of feedback control. This work was supported by the Citrus Research Center and Agricultural Experiment Station of the University of California, Riverside, CA. Submitted in partial fulfillment of the requirements of the University of California for the Master of Science degree in botany (F-F.L.)     

Influence of age, sex, lactational state and exogenous growth hormone on erythrocyte UMP synthase in dairy cattle

UMP synthase activity was determined in erythrocytes of Holstein cattle to assess differences attributable to age, sex, lactational state, and exogenous growth hormone. Newborns had 80% more UMP synthase activity per ml erythrocytes than mature cattle. Males had 10% more UMP synthase activity than females of the same age. Lactating and non-lactating mature females did not differ in UMP synthase activity. Over the first six weeks of life, the decrease in UMP synthase activity was less pronounced in calves partially deficient for the enzyme. Ten daily injections of growth hormone altered neither UMP synthase activity in erythrocytes nor orotic acid concentrations in milk and urine.     

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.     

Study on the Optical Rotatory Dispersion of Di-Peptides and its Application to the Recognition of Di-Peptides from Higher Peptides and Amino Acids

During the cource of the investigation of ribotidation of purine and pyrimidine bases by Brevibacterium ammoniagenes ATCC 6872, it was found that a large amount of uridine 5′-monophosphate (UMP) was accumulated in the culture broth when the organism was incubated in a medium containing uracil or orotic acid. The yields of UMP were 83% (4.8 mg/ml) from uracil and 100% (4.3 mg/ml) from orotic acid when each substrate was added at the concentration of 2 mg/ml.

Addition of 6-azauracil or 5-hydroxyuracil to the culture of the organism during cultivation led to the accumulation of both orotidine 5′-monophosphate (OMP) and UMP. The accumulation of OMP seemed to be due to the inhibition of OMP decarboxylase (E. C. 4.1.1.23) by the ribotide formed from each base. The OMP accumulation was enhanced by the addition of orotic acid in addition to 6-azauracil. When 6-azauracil was added to the medium before inoculation, UMP was predominantly accumulated, and when it was added after one day incubation, OMP was predominantly accumulated. A largest accumulation (3.6 mg/ml) of OMP was obtained when 6-azauracil was added on the 1st day and orotic acid was added on the 3rd day.

UMP and OMP accumulated in the medium were isolated from the cultured broth and identified by usual methods.     

Regulation of pyrimidine biosynthesis by purine and pyrimidine nucleosides in slices of rat tissue

Evidence of the primary sites for the regulation of de novo pyrimidine biosynthesis by purine and pyrimidine nucleosides has been obtained in tissue slices through measurements of the incorporation of radiolabeled precursors into an intermediate and end product of the pathway. Both purine and pyrimidine nucleosides inhibited the incorporation of [¹⁴C]-NaHCO₃ into orotic acid and uridine nucleotides, and the inhibition was found to be reversible upon transferring the tissue slices to a medium lacking nucleoside. The ammonia-stimulated incorporation of [¹⁴C]NaHCO₃ into orotic acid, which is unique to liver slices, was sensitive to inhibition by pyrimidine nucleosides at physiological levels of ammonia, but this regulatory mechanism was lost at toxic levels of ammonia. Adenosine, but not uridine, was found to have the additional effects of inhibiting the conversion of [¹⁴C]orotic acid to UMP and depleting the tissue slices of PRPP. Since PRPP is required as an activator of the first enzyme of the de novo pathway, CPSase II, and a substrate of the fifth enzyme, OPRTase, these results indicate that adenosine inhibits the incorporation of [¹⁴C]NaHCO₃ into orotic acid and the incorporation of [¹⁴C]orotic acid into UMP by depriving CPSase II and OPRTase, respectively, of PRPP. Uridine or its metabolites, on the other hand, appear to control the de novo biosynthesis of pyrimidines through end product inhibition of an early enzyme, most likely CPSase II. We found no evidence of end product inhibition of the conversion of orotic acid to UMP in tissue slices.