Orotic acids synthesis

Orotic acid is an intermediate in pyrimidine synthesis. It is synthesized from the transcar-bamylation of aspartic acid and subsequent intramolecular condensation. Any defect in ureagenesis causing accumulation of intracellular carbamoyl phosphate provides substrate for orotic acid synthesis. Therefore, a defect of OTC, or any defect distal to this step, can cause orotic aciduria. The detection of elevated orotic acid in the urine is most useful in differentiating between patients with OTC deficiency and either CPSI- or NAGS-deficient patients in whom orotic aciduria is not present. 

The reduction in urinary excretion of both compounds following uridine therapy reflects the utilization of uridine for the formation of UMP by the salvage pathway. A similar phenomenon was observed in hereditary orotic aciduria following uridine replacement therapy which bypasses the congenital enzyme defect (Chapter 5). The reversal of 6-azauridine-induced orotic aciduria by hydroxyurea, methotrexate and cyclophosphamide [251] (i.e. by the drugs affecting the synthesis of DNA without any effect on orotic acid synthesis) suggests that the control of pyrimidine synthesis de novo is linked to DNA synthesis. 

While mammahan cells reutilize few free pyrimidines, salvage reactions convert the ribonucleosides uridine and cytidine and the deoxyribonucleosides thymidine and deoxycytidine to their respective nucleotides. ATP-dependent phosphoryltransferases (kinases) catalyze the phosphorylation of the nucleoside diphosphates 2 "-de-oxycytidine, 2 -deoxyguanosine, and 2 -deoxyadenosine to their corresponding nucleoside triphosphates. In addition, orotate phosphoribosyltransferase (reaction 5, Figure 34-7), an enzyme of pyrimidine nucleotide synthesis, salvages orotic acid by converting it to orotidine monophosphate (OMP). 

Lieberman 1, A Komberg (1953) Enzymatic synthesis and breakdown of a pyrimidine, orotic acid I. Dihydro-orotic dehydrogenase. Biochim Biophys Acta 12 223-234. 

Urinary orotic acid generally is very elevated in babies with OTC deficiency and normal or even low in the infant with CPS deficiency. Patients with OTC deficiency have orotic aciduria because carbamyl phosphate spills into the cytoplasm, where it enters the pathway of pyrimidine synthesis. 

The underlying biochemical defect is a failure of mitochondrial uptake of ornithine. This results in a failure of citrulline synthesis and a consequent hyperammonemia. Urinary orotic acid is high, presumably because of underutilization of carbamyl phosphate. In contrast, excretion of creatine is low, reflecting the inhibition of glycine trans-amidinase by excessive levels of ornithine. 

A rigorous structural proof of the insecticidal exotoxin (34) from Bacillus thurin-giensis has now been published,107 confirming the a-configuration of the glucosidic bond. The total synthesis of (34) is further confirmation of the correctness of the structural assignment.108 The exotoxin inhibits RNA synthesis in insects and animals and affects the incorporation of orotic acid into nuclear RNA.109... 

The two conditions can be distinguished by an increase in orotic add and uracil, which occurs in ornithine transcarbamoylase deficiency, but not in the defldency of carbamoyl phosphate synthetase. Orotic acid and uracil are intermediates in pyrimidine synthrais (see Chapter 18). This pathway is stimulated by the accumulation of carbamoyl phosphate, the substrate for ornithine transcarbamoylase in the urea cycle and for aspartate transcarbamoylase in pyrimidine synthesis. 

Answen B. Accumulation of orotic acid indicates megaloblastic anemia arises since pyrimidines are required for DNA synthesis. 

Carbamoyl phosphate synthetase formation in liver taken from tadpoles treated with thyroxine is enhanced by the addition of orotic acid, uracil or uridine (cytosine and adenosine had no effect). The synthesis of this enzyme is not affected by these pyrimidines in untreated animals. This indicates that there is a relative pyrimidine deficiency during thyroxine-induced metamorphosis [140]. 

Orotic acid in the diet (usually at a concentration of 1 per cent) can induce a deficiency of adenine and pyridine nucleotides in rat liver (but not in mouse or chick liver). The consequence is to inhibit secretion of lipoprotein into the blood, followed by the depression of plasma lipids, then in the accumulation of triglycerides and cholesterol in the liver (fatty liver) [141 — 161], This effect is not prevented by folic acid, vitamin B12, choline, methionine or inositol [141, 144], but can be prevented or rapidly reversed by the addition of a small amount of adenine to the diets [146, 147, 149, 152, 162]. The action of orotic acid can also be inhibited by calcium lactate in combination with lactose [163]. It was originally believed that the adenine deficiency produced by orotic acid was caused by an inhibition of the reaction of PRPP with glutamine in the de novo purine synthesis, since large amounts of PRPP are utilized for the conversion of orotic acid to uridine-5 -phosphate. However, incorporation studies of glycine-1- C in livers of orotic acid-fed rats revealed that the inhibition is caused rather by a depletion of the PRPP available for reaction with glutamine than by an effect on the condensation itself [160]. 

These three compounds exert many similar effects in nucleotide metabolism of chicks and rats [167]. They cause an increase of the liver RNA content and of the nucleotide content of the acid-soluble fraction in chicks [168], as well as an increase in rate of turnover of these polynucleotide structures [169,170]. Further experiments in chicks indicate that orotic acid, vitamin B12 and methionine exert a certain action on the activity of liver deoxyribonuclease, but have no effect on ribonuclease. Their effect is believed to be on the biosynthetic process rather than on catabolism [171]. Both orotic acid and vitamin Bu increase the levels of dihydrofolate reductase (EC 1.5.1.4), formyltetrahydrofolate synthetase and serine hydroxymethyl transferase in the chicken liver when added in diet. It is believed that orotic acid may act directly on the enzymes involved in the synthesis and interconversion of one-carbon folic acid derivatives [172]. The protein incorporation of serine, but not of leucine or methionine, is increased in the presence of either orotic acid or vitamin B12 [173]. In addition, these two compounds also exert a similar effect on the increased formate incorporation into the RNA of liver cell fractions in chicks [174—176]. It is therefore postulated that there may be a common role of orotic acid and vitamin Bj2 at the level of the transcription process in m-RNA biosynthesis [174—176]. 

The fact that many agents which interrupt the synthesis of pyrimidine nucleotides from orotic acid in animals can also inhibit the growth of experimental neoplasms suggests a search for additional antimetabolites whose locus of action is in this metabolic sequence. Two in vitro biological screening systems were developed for this purpose [202—207]. From a study of systems with oxidative energy sources, 5-bromo-[208—209] (Villa), 5-chloro-[210] (Vlllb) and 5-diazo-orotic acid [211] (IX) were found to inhibit the conversion of orotic acid to the uridine nucleotides by 40—100 per cent [202]. 

A common intermediate for all the nucleotides is 5-phosphoribosyl-l-diphosphate (PRPP), produced by successive ATP-dependent phosphorylations of ribose. This has an a-diphosphate leaving group that can be displaced in Sn2 reactions. Similar Sn2 reactions have been seen in glycoside synthesis (see Section 12.4) and biosynthesis (see Box 12.4), and for the synthesis of aminosugars (see Section 12.9). For pyrimidine nucleotide biosynthesis, the nucleophile is the 1-nitrogen of uracil-6-carboxylic acid, usually called orotic acid. The product is the nucleotide orotidylic acid, which is subsequently decarboxylated to the now recognizable uridylic acid (UMP). 

The chlorine adduct to the tetrasubstituted exocyclic double bond in 108 is quantitatively obtained by gas-solid reaction [27]. Conversely, related trisub-stituted double bonds lose HX after the gas-solid halogen addition such as in the reactions of 110 and 112 that give 111 and 113,respectively [28] (Scheme 12). The completion of these solid-state eliminations (Sect. 11) is faster at 100 °C. The product 113 is an interesting substrate for the synthesis of orotic acids. Furthermore, the production of 116 from solid 114 and chlorine gas proceeds with 100% yield via the intermediate adduct 115 [58,60-61] (Scheme 12). 

Mutation of one of the two enzyme activities of UMP synthase leads to orotic aciduria, characterized by accumulation of its first substrate orotic acid and insufficient levels of the product UMP, which reduces availability of uridine triphosphate (UTP) and cytidine triphosphate (CTP) for use in nucleic acid synthesis. 

The second step in pyrimidine synthesis is the formation of car-bamoylaspartate, catalyzed by aspartate transcarbamoylase. The pyrimidine ring is then closed hydrolytically by dihydroorotase. Thi resulting dihydroorotate is oxidized to produce orotic acid (onotate, Figure 22.21). The enzyme that produces orotate, dihydroorotate dehydrogenase, is located inside the mitochondria. All other reactions in pyrimidine biosynthesis are cytosolic. [Note The first three enzymes in this pathway (CPS II, aspartate transcarbamoylase, and dihydroorotase) are all domains of the same polypeptide chain. (See k p. 19 for a discussion of domains.) This is an example of a multifunctional or multicatalytic polypeptide that facilitates the ordered synthesis of an important compound.]... 

The end-product of pyrimidine base synthesis is orotic acid, which is converted to the nucleotide OMP by the addition of ribose 6-phosphate (donated by PRPP). OMP is then converted to UMP, which is phosphorylated to UTP. UTP is then aminated to form CTP. A deficiency of the enzyme complex (UMP synthase) that converts orotic acid to UMP causes orotic aciduria. 

Vitamins and Minerals. Milk is a rich source of vitamins and other organic substances that stimulate microbial growth. Niacin, biotin, and pantothenic acid are required for growth by lactic streptococci (Reiter and Oram 1962). Thus the presence of an ample quantity of B-complex vitamins makes milk an excellent growth medium for these and other lactic acid bacteria. Milk is also a good source of orotic acid, a metabolic precursor of the pyrimidines required for nucleic acid synthesis. Fermentation can either increase or decrease the vitamin content of milk products (Deeth and Tamime 1981 Reddy et al. 1976). The folic acid and vitamin Bi2 content of cultured milk depends on the species and strain of culture used and the incubation conditions (Rao et al. 1984). When mixed cultures are used, excretion of B-complex vita-... 

The stimulatory effect of l-(chloromethyl)silatrane on the DNA synthesis in the cells of the regenerating liver is shown in Table 10 and Fig. 3. In a similar way (according to incorporation of 3H-thymidine, 14C-orotic acid and 3H-leucine) it has been found that l-(chloromethyl)silatrane intensifies the DNA, RNA and protein biosynthesis in other developing cells (by 20—60%). This has shown the importance of further investigation of this preparation as a stimulator of cell division and biosynthesis of nucleic acids and proteins. 

Eukaryotic organisms contain a multifunctional enzyme with carbamoylphosphate synthetase, aspartate transcarbamoylase, and dihydroorotase activities. Two mechanisms control this enzyme. First, control at the level of enzyme synthesis exists the transcription of the gene for the enzyme is reduced if an excess of pyrimidines is present. Secondly, control exists at the level of feedback inhibition by pyrimidine nucleotides. This enzyme is also an example of the phenomenon of metabolic channeling aspartate, ammonia, and carbon dioxide enter the enzyme and come out as orotic acid. 

A second, cytosolic CPS activity (CPSII) occurs in mammals as part of the CAD trifunctional protein that catalyzes the first three steps of pyrimidine synthesis (CPSII, asparate tran-scarbamoylase, and dihydroorotase). The activities of these three enzymes—CPSII, aspartate transcarbamoylase, and dihydroorotase—result in the production of orotic acid from ammonium, bicarbonate, and ATP. CPSII has no role in ureagenesis, but orotic aciduria results from hepatocellular accumulation of carbamyl phosphate and helps distinguish CPSI deficiency from other UCDs. Defects in CPSI classically present with neonatal acute hyperammonemic encephalopathy. The plasma citrulline and urine orotic acid concentrations are both low. A definitive diagnosis can be established by enzyme assay of biopsied liver tissue or by mutation analysis. 

The fourth step in the de novo synthesis of pyrimidine nucleotides—the conversion of dihydroorotic acid to orotic acid—is catalyzed by dihydroorotic acid dehydrogenase. The enzyme, located on the cytosolic side of the inner membrane of mitochondria, is a target for antitumor agents. 

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