Orotate, pyrimidine biosynthesis

The phenoxyquinolines are a recent addition to crop fungicides and only one compound has been described. LY214352, an experimental fungicide, inhibits a novel target, dihydro-orotate dehydrogenase (DHO-DH) in the pyrimidine biosynthesis pathway that catalyses the conversion of dihydro-orotate into orotic acid9 (Figure 4.13). 

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.]... 

Dihydroorotate dehydrogenase, the enzyme catalyzing the dehydrogenation of dihydroorotate to orotate (reaction 4 of the pathway Fig. 15-15), is located on the outer side of the inner mitochondrial membrane. This enzyme has FAD as a prosthetic group and in mammals electrons are passed to ubiquinone. The de novo pyrimidine pathway is thus compartmentalized dihydroorotate synthesized by trifunctional DHO synthetase in the cytosol must pass across the outer mitochondrial membrane to be oxidized to orotate, which in turn passes back to the cytosol to be a substrate for bifunctional UMP synthase. Mammalian cells contain two carbamoyl phosphate synthetases the glutamine-dependent enzyme (CPSase II) which is part of CAD, and an ammonia-dependent enzyme (CPSase /) which is found in the mitochondrial matrix, and which is used for urea and arginine biosynthesis. Under certain conditions (e.g., hyperammonemia), carbamoyl phosphate synthesized in the matrix by CPSase I may enter pyrimidine biosynthesis in the cytosol. 

The metabolic interrelationship between mitochondrial carbamoyl phosphate synthesis to urea formation and to cytosolic carbamoyl phosphate channeled into pyrimidine biosynthesis. In ornithine transcarbamoyiase (OTC) deficiency, mitochondrial carbamoyl phosphate diffuses into the cytosol and stimulates pyrimidine biosynthesis, leading to orotidinuria. Administration of allopurinol augments orotidinuria by increasing the flux in the pyrimidine biosynthetic pathway. CPS = Carbamoyl phosphate synthase, AT = aspartate transcarbamoyiase, D = dihydroorotase, DH = dihydroorotate dehydrogenase, OPRT = orotate phosphoribosyltransferase, XO = xanthine oxida.se,... 

Deficiency of folate or vitamin Bn can cause hematological changes similar to hereditary orotic aciduria. Folate is directly involved in thymidylic acid synthesis and indirectly involved in vitamin Bn synthesis. Orotic aciduria without the characteristic hematological abnormalities occurs in disorders of the urea cycle that lead to accumulation of carbamoyl phosphate in mitochondria (e.g., ornithine transcarbamoylase deficiency see Chapter 17). The carbamoyl phosphate exits from the mitochondria and augments cytosolic pyrimidine biosynthesis. Treatment with allopurinol or 6-azauridine also produces orotic aciduria as a result of inhibition of orotidine-5 phosphate decarboxylase by their metabolic products. 

Although the exact nature of the various enzymes involved in pyrimidine biosynthesis is not fully worked out, it seems that leishmania and trypanosomes possess phosphoribosyl transferase, which is specific for uracil. This makes the protozoal phosphoribosyl transferase distinct from the mammalian orotate phosphoribosyl transferase and, therefore, may be explored in protozoal chemotherapy. 

A 4-year-old girl presents in the clinic with megaloblastic anemia and failure to thrive. Blood chemistries reveal orotic aciduria. Enzyme measurements of white blood cells reveal a deficiency of the pyrimidine biosynthesis enzyme orotate phosphoribosyltransferase and abnormally high activity of the enzyme aspartate transcarbamoylase. Which one of the following treatments will reverse all symptoms if carried out chronically ... 

DHODs are FMN-containing enzymes that convert dihydroorotate (DHO) to orotate (OA) in the only redox step in the wow synthesis of pyrimidines (Scheme 12). DHODs have been grouped into two classes based on sequence. Class 2 DHODs are membrane-bound monomers that are reoxidized by ubiquinone, coupling pyrimidine biosynthesis to the respiratory chain. On the other hand, Class 1 DHODs are cytosolic proteins that have been further divided into two subclasses. Class lA DHODs are homodimers that are reoxidized by fumarate. Class 1B DHODs are azfiz heterotetramers with an FMN-containing subunit very similar to Class 1A enzymes and a second subunit that contains an iron—sulfur cluster and FAD, allowing Class IB DHODs to be reoxidized by NAD. ... 

Pyrimidines play a central role in cellular regulation and metabolism. They are substrates for DNA and RNA biosynthesis, regulators of biosynthesis of some amino acids, and cofactors in the biosynthesis of phospholipids, glycolipids, sugars, and polysaccharides (17B45). Pyrimidine biosynthesis is very complicated and involves formic acid, glutamate, and aspartate as starting materials in a series of enzymatic reactions to eventually form orotic acid. Orotic acid, or uracil-... 

When ornithine transcarbamoylase (OTC) is deficient, the carbamoyl phosphate that normally would enter the urea cycle accumulates and floods the pathway for pyrimidine biosynthesis. Under these conditions, excess orotic acid (orotate), an intermediate in pyrimidine biosynthesis, is excreted in the urine. It produces no ill effects but is indicative of a problem in the urea cycle. 

In the next step of pyrimidine biosynthesis, the entire aspartate molecule adds to carbamoyl phosphate in a reaction catalyzed by aspartate transcarbamoylase. The molecule subsequently closes to produce a ring (catalyzed by dihydroorotase), which is oxidized to form orotic acid (or its anion, orotate) through the actions of dihydroorotate dehydrogenase. The enzyme orotate phosphoribosyl transferase catalyzes the transfer of ribose 5-phosphate from PRPP to orotate, producing orotidine 5 -phosphate, which is decarboxylated by orotidylic acid dehydrogenase to form... 

The reaction of carbamoyl phosphate with aspartate to produce W-carbamo-ylaspartate is the committed step in pyrimidine biosynthesis. The compounds involved in reactions up to this point in the pathway can play other roles in metabolism after this point, A -carbamoylaspartate can be used only to produce pyrimidines—thus the term committed step. This reaction is catalyzed by aspartate transcarbamoylase, which we discussed in detail in Ghapter 7 as a prime example of an allosteric enzyme subject to feedback regulation. The next step, the conversion of A-carbamoylaspartate to dihydroorotate, takes place in a reaction that involves an intramolecular dehydration (loss of water) as well as cyclization. This reaction is catalyzed by dihydroorotase. Dihydroorotate is converted to orotate by dihydroorotate dehydrogenase, with the concomitant conversion of NAD to NADH. A pyrimidine nucleotide is now formed by the reaction of orotate with PRPP to give orotidine-5 -monophosphate (OMP), which is a reaction similar to the one that takes place in purine salvage (Section 23.8). Orotate phosphoribosyltransferase catalyzes this reaction. Finally, orotidine-5 -phosphate decarboxylase catalyzes the conversion of OMP to UMP... 

Orotate condenses with PRPP in a reaction catalyzed by orotate phosphoribosyl transferase to form the nucleotide orotidylate (OMP). Orotidylate decarboxylase converts OMP to the more abundant nucleotide UMP. The reaction occurs during de novo pyrimidine biosynthesis and is therefore not a salvage reaction. 

Orotidine 5 -phosphate decarboxylase (ODCase, E. C. 4.1.1.23) catalyzes the decarboxylation of orotidine 5 -phosphate (OMP) to form uridine 5 -phos-phate in the sixth and final step of pyrimidine biosynthesis (Fig. 1) [1]. The discovery of ODCase in 1954 followed the identification, three years earlier, of orotic acid as the metabolic precursor of nucleic acids [2, 3]. ODCase is a distinct, monofunctional polypeptide in bacteria and fungi, whereas in mammals it combines with orotate phosphoribosyltransferase (OPRTase) to form the bifunctional enzyme UMP synthase. Human deficiencies in either OPRTase or ODCase activity result in an autosomal recessive disorder called hereditary orotic aciduria [4]. The disease is characterized by depleted levels of pyrimidine nucleotides in the blood and by the appearance of crystalline... 

The first step in de novo pyrimidine biosynthesis is the synthesis of carbamoyl phosphate from bicarbonate and ammonia in a multistep process, requiring the cleavage of two molecules of ATP. This reaction is catalyzed by carbamoyl phosphate synthetase (CPS), and the bicarbonate is phosphorylated by ATP to form carboxyphosphate and ADP (adenine dinucleotide phosphate). Ammonia then reacts with carboxyphosphate to form carbamic acid. The latter is phosphorylated by another molecule of ATP with the mediation of CPS to form carbamoyl phosphate, which reacts with aspartate by aspartate transcarbamoy-lase to form A-carbamoylaspartate. The latter cyclizes to form dihydroorotate, which is then oxidized by NAD-1- to generate orotate. Reaction of orotate with 5-phosphoribosyl-l-pyrophosphate (PRPP), catalyzed by pyrimidine PT, forms the pyrimidine nucleotide orotidylate. This reaction is driven by the hydrolysis of pyrophosphate. Decarboxylatin of orotidylate, catalyzed by orotidylate decarboxylase, forms uridylate (uridine-5 -monophosphate, UMP), a major pyrimidine nucleotide that is a precursor of RNA (Figure 6.53). 

Pyrimidine biosynthesis commences with a reaction between carbamyl phosphate and aspartic acid to give carbamyl aspartic acid which then nndergoes ring closure and oxidation to orotic acid. A reaction then occurs between orotic acid and 5-phosphoribosyl pyrophosphate to give orotidine-5-phosphate which on decarboxylation yields uridine-5-phosphate (UMP). By means of two successive reactions with ATP, UMP can then be converted into UTP and this by reaction with ammonia can give rise to cytidine triphosphate, CTP (11.126). 

Nutrition studies in bacteria indicated that carbamoyl aspartate (also known as ureidosuccinate) is an intermediate in pyrimidine biosynthesis. Finally, uridine nucleotides were found as end products of pyrimidine synthesis de novo [73]. The sequence of reactions leading to the synthesis of UMP is designated as the orotate pathway or pyrimidine synthesis de novo in distinction to the salvage pathway. 

Classical orotic aciduria is a rare autosomal recessive disorder which is characterized by retarded growth and excretion of large quantities of orotic acid in the urine [221,222]. The disease was described in 1959 as an inborn error of pyrimidine biosynthesis in patients with crystals of orotic acid in the urine [223]. The urinary excretion of orotic acid by these patients was 1.34 g per day in contrast to approximately 0.014 g per day excreted by normal individuals [222,224]. When the diet of patients was supplemented with uridine, clinical remission and a remarkable reduction in orotic acid excretion took place [221,225,226]. 

B37 Bresnick, E., Mayfield, E. D. and Mosse, H. Increased activity of enzymes for de novo pyrimidine biosynthesis after orotic acid administration. Mol. Pharmacol., 4, 173-180 (1968)... 

Ito, K. and Uchino, H. Control of pyrimidine biosynthesis in human lymphocytes. Induction of glutamine-utilizing car-bamyl phosphate synthetase and operation of orotic acid pathway during blastogenesis. J. Biol. Chem., 246, 4060-4065 (1971)... 

Carbamoyl phosphate HjN-COO PO3H2, an energy-rich phosphoiylated carbamate and an important metabolic intermediate. Carbamic acid, NHjCOOH, is unstable in fi ee form. Carbamate removed hydrolytically fi om carbamyl compounds, e.g. ureidopropionic acid (see Pyrimidine degradation), decomposes immediately into COj and NHj. Cp. is a specific precursor of arginine and urea (see Urea cycle), and of pyrimidines via orotic acid (see Pyrimidine biosynthesis). 

Ororid i -5 -phosphate pyrophosphorylase (EC 2.4.2.10) and orotidme-5 -phosphate decarboxylase (EC 4.1.1.23). Abnormally high urinary orotic add. Severe megaloblastic anemia. Marked retardation of growth and development, and slight mental retardation. [A gross defidency of 2 enzymes of pyrimidine biosynthesis]... 

Orotic acid, Oro uracil 4-carboxylic acid, M, 156.1, m.p. 344 347°C (d.). Oro is an intermediate in Pyrimidine biosynthesis (see), and it is secreted in large quantities by mutants of Neurospora crassa that show a growth requirement for uridine, cytidine or uracil. 

Orotidine S -monophosphate, OMPi a nucleotide of orotic acid. M, 368.2. OMP is an intermediate in Pyrimidine biosynthesis (see). Orotidine 5 -phos-phate pyrophosphorylase catalyses the synthesis of OMP from orotic add and S-phosphoribosyl 1-pyro-phosphate. 

How were the simple precursors of the pyrimidine ring recognized In 1943, Barnes and Schoenheimer showed that heavy nitrogen from N-ammonium citrate was incorporated into animal polynucleotides. It was apparent as early as 1944 that orotate (6-carboxyuracil) was involved in pyrimidine biosynthesis, because this compound would satisfy the pyrimidine requirement for some bacteria [see review (5)]. Between 1949 and 1952 it became apparent that orotate was probably an intermediate in pyrimidine biosynthesis because this compound was utilized efficiently in mammalian tissues as a precursor of both DNA and RNA p uimidines. However, the possibility remained that orotate per se w as not an actual intermediate, but rather was easily converted to one. 

To determine whether orotate was actually an intermediate compound in the process of pyrimidine biosynthesis or, alternatively, entered the sequence by a side reaction, Reichard and Lagerkvist (7) undertook... 

The sequence of reactions which culminates in the formation of uridylate is very widely distributed in nature and is referred to as the orotate pathway, or as the de novo route of pyrimidine biosynthesis. This pathway is summarized in the following diagram segments of it will be discussed next in the historical sequence of their discovery and development, which is given by the order A, B, C. 

Historically, carbamyl aspartate was recognized as a likely intermediate in pyrimidine biosynthesis because (a) this compound is an assembly of two elementary precursors of the pyrimidine ring, (b) carbamyl aspartate would satisfy the nutritional requirement of L. bvlgaricus 09 for orotate, and (c) labeled carbamyl aspartate was incorporated into ribonucleic acid pyrimidines in L. bulgaricus and, as well, served as an orotate precursor in liver slice trapping experiments such as those mentioned above. 

The orotate phosphoribosyltransferase of yeast is specific for orotate and will not accept uracil as a substrate. This enzyme occurs in most animal cells as part of the de novo pathway of pyrimidine biosynthesis, but the specificity of the animal enzyme is unknown. Animal cells have a phosphoribosyltransferase activity capable of accepting pyrimidine substrates other than orotate, but it is not clear whether this is due to a phosphoribosyltransferase distinct from that of the orotate pathway (see Chapter 11). 

Dihydroorotate dehydrogenase (EC 1.3.99.11) catalyses the oxidation of dihydroorotate to orotate in the pyrimidine biosynthesis pathway. Inhibition of cytochrome c oxidase indirectly inhibits dihydroorotate dehydrogenase activity. In digitonin-permeabilized cells, sodium l,l-diethyl-2-hydroxy-2-nitroso-hydrazine, a chemical nitric oxide donor, induced a dramatic decrease in dihydroorotate-dependent O2 consumption (Beuneu et al. 2000). The inhibition was reversible and more pronounced at low O2 concentration it was correlated with a decrease in orotate synthesis. 

As early as 1949, it was demonstrated that injected or " C-labeled orotic acid was readily incorporated into DNA and RNA of mammalian tissue, indicating that orotic acid is a precursor of nucleic acid pyrimidine. The next step in pyrimidine biosynthesis is the formation of the first nucleotide in the sequence. It involves the reaction between ribosyl pyrophosphate and orotic acid to yield 5 -orotidylic acid the reaction is catalyzed by orotidylic pyrophosphorylase. Thus, the first steps of pyrimidine biosynthesis differ from the early steps of purine biosynthesis in at least two ways. Orotic acid, instead of being synthesized atom by atom as is the case for the purine ring, is made from the condensation of rather large molecules, namely, carbamyl phosphate and aspartic acid. Furthermore, all the steps of purine biosynthesis occur at the level of the nucleotide, but the the pyrimidine ring is closed at the level of the base. 

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