De novo pyrimidine synthesis

Nucleotides are the building blocks of nucleic acids, such as DNA and RNA, which store and transmit genetic information in all living cells. Nucleotides consist of three components: a nitrogenous base, a pentose sugar, and one or more phosphate groups. The nitrogenous base can be either a purine (adenine or guanine) or a pyrimidine (cytosine, thymine, or uracil).

Nucleotides have many important functions in the cell, besides being the monomers of nucleic acids. They serve as energy carriers (e.g., ATP and GTP), coenzymes (e.g., NAD and FAD), signaling molecules (e.g., cAMP and cGMP), and precursors of other biomolecules (e.g., amino acids and lipids). Nucleotides also regulate many cellular processes, such as metabolism, transcription, translation, replication, and repair.

Because of their essential roles in the cell, nucleotides must be synthesized and maintained at adequate levels. There are two major pathways for nucleotide synthesis: de novo and salvage. De novo synthesis refers to the formation of nucleotides from simple precursors, such as amino acids, carbon dioxide, and ribose-5-phosphate. Salvage synthesis refers to the recycling of nucleotides from degraded nucleic acids or other sources.

In this article, we will focus on the de novo synthesis of pyrimidine nucleotides, which are essential for DNA and RNA synthesis, as well as for other cellular functions. We will discuss the location, substrates, products, reactions, enzymes, regulation, alternative pathways, diseases, and significance of this pathway in biology and medicine.

Unlike purine synthesis, which occurs in both the cytoplasm and the mitochondria, de novo pyrimidine synthesis occurs exclusively in the cytoplasm of cells in all tissues. The enzymes involved in this pathway are either cytosolic or membrane-bound to the endoplasmic reticulum. The reason for this difference is that the pyrimidine ring is synthesized before it is attached to the ribose-5-phosphate moiety, which is derived from PRPP in the cytoplasm. In contrast, purine synthesis starts with PRPP and builds the purine ring on it.

The location of de novo pyrimidine synthesis has implications for its regulation and coordination with other metabolic pathways. For example, the cytosolic enzyme carbamoyl phosphate synthetase II, which catalyzes the first and rate-limiting step of the pathway, is inhibited by UTP and activated by ATP and PRPP. This ensures that pyrimidine synthesis is balanced with purine synthesis and energy status of the cell. Moreover, the end product of de novo pyrimidine synthesis, UTP, can be converted to CTP by CTP synthetase, which is also located in the cytoplasm. CTP is an essential component of phospholipid biosynthesis, which occurs in the endoplasmic reticulum. Thus, de novo pyrimidine synthesis is linked to membrane biogenesis and cell growth.

In summary, de novo pyrimidine synthesis takes place in the cytoplasm of cells and is regulated by feedback inhibition and allosteric activation. It also provides precursors for other important cellular processes such as RNA and DNA synthesis, phospholipid biosynthesis, and glycogen metabolism.

The de novo pyrimidine synthesis pathway requires several substrates to produce the pyrimidine nucleotides UTP and CTP. These substrates are:

• CO2: Carbon dioxide is a source of carbon atoms for the pyrimidine ring. It reacts with glutamine and ATP to form carbamoyl phosphate in the first step of the pathway.
• Glutamine: Glutamine is a source of nitrogen atoms for the pyrimidine ring. It donates its amide group to CO2 to form carbamoyl phosphate in the first step of the pathway. It also donates its amide group to UTP to form CTP in the last step of the pathway.
• ATP: Adenosine triphosphate is a source of energy and phosphate groups for the pathway. It provides two phosphate groups to CO2 to form carbamoyl phosphate in the first step of the pathway. It also provides a phosphate group to UMP to form UTP in the fifth step of the pathway.
• Aspartate: Aspartate is a source of nitrogen and carbon atoms for the pyrimidine ring. It adds its entire molecule to carbamoyl phosphate to form orotate in the second step of the pathway.
• H2O: Water is involved in several reactions of the pathway. It hydrolyzes carbamoyl aspartate to form dihydroorotate in the third step of the pathway. It also hydrolyzes orotidine 5′-monophosphate (OMP) to form uridine monophosphate (UMP) in the sixth step of the pathway.
• NAD+: Nicotinamide adenine dinucleotide is a coenzyme that acts as an electron acceptor in oxidation reactions. It accepts two electrons from dihydroorotate to form orotate and NADH in the fourth step of the pathway.
• Phosphoribosyl pyrophosphate (PRPP): PRPP is a precursor for nucleotide synthesis. It provides the ribose-5-phosphate ring to orotate to form OMP in the fifth step of the pathway.

These substrates are derived from various sources such as glucose metabolism, amino acid metabolism, and pentose phosphate pathway. They are used in specific amounts and ratios to ensure a balanced production of pyrimidine nucleotides. The availability and concentration of these substrates can also affect the regulation of the pathway by influencing the activity of some key enzymes.

The de novo pyrimidine synthesis pathway produces two major products: uridine triphosphate (UTP) and cytidine triphosphate (CTP). These are the pyrimidine nucleotides that are used in the synthesis of RNA. UTP and CTP can also be converted to their deoxyribonucleotides, deoxyuridine triphosphate (dUTP) and deoxycytidine triphosphate (dCTP), which are used in the synthesis of DNA. Additionally, dUTP can be further modified to deoxythymidine triphosphate (dTTP), which is the only pyrimidine nucleotide that is exclusively found in DNA.

Besides being the building blocks of nucleic acids, pyrimidine nucleotides also have other important roles in cellular metabolism. For example:

• UTP is a precursor for the synthesis of glycogen, a storage form of glucose.
• CTP is a precursor for the synthesis of phosphatidylcholine and phosphatidylethanolamine, two major components of cell membranes.
• dUTP is a substrate for uracil-DNA glycosylase, an enzyme that removes uracil from DNA and prevents mutations.
• dTTP is a substrate for thymidylate synthase, an enzyme that catalyzes the conversion of dUMP to dTMP and maintains the balance of deoxyribonucleotides.

The de novo pyrimidine synthesis pathway also produces some by-products that are either recycled or excreted. These include:

• Glutamate, which is released from glutamine during the formation of carbamoyl phosphate and CTP. Glutamate can be used for protein synthesis or converted to other amino acids or intermediates of the citric acid cycle.
• NADH, which is generated during the oxidation of dihydroorotate to orotate. NADH can be used for energy production in the electron transport chain or for biosynthetic reactions that require reducing power.
• CO2, which is released during the formation of carbamoyl phosphate and the decarboxylation of OMP. CO2 can be used for other biosynthetic pathways such as fatty acid synthesis or excreted as a waste product.

The products and by-products of the de novo pyrimidine synthesis pathway are summarized in the table below:

The de novo pyrimidine synthesis pathway consists of six main steps that convert simple precursors into the pyrimidine nucleotides UTP and CTP. The pathway can be summarized as follows:

1. Carbamoyl phosphate synthesis: CO2 and glutamine are combined to form carbamoyl phosphate. This reaction is catalyzed by carbamoyl phosphate synthetase II, which is the major regulated step for this pathway. This enzyme is inhibited by UTP and activated by ATP and PRPP.
2. Aspartate transcarbamoylation: Carbamoyl phosphate is then combined with aspartate to form N-carbamoylaspartate. This reaction is catalyzed by aspartate transcarbamoylase, which is part of a multifunctional enzyme complex called CAD (carbamoyl phosphate synthetase II, aspartate transcarbamoylase, and dihydroorotase).
3. Dihydroorotate synthesis: N-carbamoylaspartate is cyclized and dehydrated to form dihydroorotate. This reaction is catalyzed by dihydroorotase, which is also part of the CAD complex.
4. Orotate synthesis: Dihydroorotate is oxidized to form orotate. This reaction is catalyzed by dihydroorotate dehydrogenase, which is a mitochondrial enzyme that uses NAD+ as a cofactor.
5. OMP synthesis: Orotate reacts with PRPP to form orotidine 5′-monophosphate (OMP). This reaction is catalyzed by orotate phosphoribosyl transferase, which is part of a bifunctional enzyme complex called UMP synthase (orotate phosphoribosyl transferase and OMP decarboxylase).
6. UMP synthesis: OMP is decarboxylated to form uridine monophosphate (UMP). This reaction is catalyzed by OMP decarboxylase, which is also part of the UMP synthase complex. This enzyme is inhibited by UMP and CMP.

The pathway can be represented by the following diagram:

\begin{align*}
&\text{CO}_2 + \text{glutamine} + 2 \text{ATP} \xrightarrow{\text{carbamoyl phosphate synthetase II}} \text{carbamoyl phosphate} + \text{glutamate} + 2 \text{ADP} + 2 \text{P}_i\\
&\text{carbamoyl phosphate} + \text{aspartate} \xrightarrow{\text{aspartate transcarbamoylase}} \text{N-carbamoylaspartate} + \text{P}_i\\
&\text{N-carbamoylaspartate} \xrightarrow{\text{dihydroorotase}} \text{dihydroorotate} + \text{H}_2\text{O}\\
&\text{dihydroorotate} + \text{NAD}^+ \xrightarrow{\text{dihydroorotate dehydrogenase}} \text{orotate} + \text{NADH}\\
&\text{orotate} + \text{PRPP} \xrightarrow{\text{orotate phosphoribosyl transferase}} \text{OMP} + \text{P}_i\\
&\text{OMP} \xrightarrow{\text{OMP decarboxylase}} \text{UMP} + \text{CO}_2
\end{align*}

The pathway does not end with UMP, as UMP can be further converted into other pyrimidine nucleotides:

• UMP can be phosphorylated to form UDP and UTP by nucleoside monophosphate kinase and nucleoside diphosphate kinase, respectively.
• UTP can be converted to CTP by CTP synthetase, which transfers an amino group from glutamine to the 4-position of the pyrimidine ring. This enzyme is inhibited by CTP.
• UDP and UTP can be reduced to dUDP and dUTP by ribonucleotide reductase, which uses NADPH as a cofactor. This enzyme is regulated by allosteric effectors and feedback inhibition.
• dUTP can be hydrolyzed to dUMP and PPi by dUTPase, which prevents the incorporation of dUTP into DNA.
• dUMP can be methylated to form dTMP by thymidylate synthase, which transfers a methyl group from N5,N10-methylene tetrahydrofolate (THF). This enzyme is inhibited by fluorouracil and methotrexate.
• dTMP can be phosphorylated to form dTDP and dTTP by nucleoside monophosphate kinase and nucleoside diphosphate kinase, respectively.

The following diagram shows the interconversion of pyrimidine nucleotides:

\begin{align*}
&\underset{\downarrow}{\overset{\uparrow}{\underset{\downarrow}{\overset{\uparrow}{\underset{\downarrow}{\overset{\uparrow}{\underset{\downarrow}{\overset{\uparrow}{\underset{\downarrow}{\overset{\uparrow}{\underset{\downarrow}{\overset{\uparrow}{\underset{\downarrow}{\overset{\uparrow}{\underset{\downarrow}{\overset{\uparrow}{\underset{\downarrow}{\overset{\uparrow}{\underset{\downarrow}{\overset{\uparrow}{UMP}}}}}}}}}}}}}}}}}}\\
&||\\
&UDP\\
&||\\
&UTP\\
&||\\
&CTP\\
&||\\
&dUDP\\
&||\\
&dUTP\\
&||\\
&dUMP\\
&||\\
&dTMP\\
&||\\
&dTDP\\
&||\\
&TTP
\end{align*}

The de novo pyrimidine synthesis pathway is essential for the production of DNA and RNA precursors, as well as for other metabolic functions such as glycogen synthesis and phospholipid biosynthesis. The pathway is tightly regulated at multiple levels to ensure a balanced supply of pyrimidine nucleotides in different tissues and under different physiological conditions. Defects in the pathway can lead to various diseases such as orotic aciduria, immunodeficiency, and cancer.

The de novo pyrimidine synthesis pathway can be divided into two phases: the synthesis of the pyrimidine ring and the formation of the nucleotides.

Synthesis of the pyrimidine ring

The first reaction is the formation of carbamoyl phosphate from glutamine, carbon dioxide and ATP. This reaction is catalyzed by carbamoyl phosphate synthetase II (CPS II), which is located in the cytosol and is different from CPS I that is involved in the urea cycle. CPS II is the rate-limiting and regulated enzyme of this pathway. It is inhibited by UTP, the end product of the pathway, and activated by ATP and PRPP, the substrates of the pathway.

The second reaction is the condensation of carbamoyl phosphate with aspartate to form N-carbamoylaspartate. This reaction is catalyzed by aspartate transcarbamoylase (ATC), which is also located in the cytosol.

The third reaction is the cyclization of N-carbamoylaspartate to form dihydroorotate. This reaction is catalyzed by dihydroorotase (DHO), which is a cytosolic enzyme in humans but a mitochondrial enzyme in some other organisms.

The fourth reaction is the oxidation of dihydroorotate to orotate. This reaction is catalyzed by dihydroorotate dehydrogenase (DHODH), which is a mitochondrial enzyme that uses NAD+ as an electron acceptor. This is the only reaction in this pathway that requires oxygen.

Formation of the nucleotides

The fifth reaction is the transfer of a ribose-5-phosphate group from PRPP to orotate to form orotidine 5`-monophosphate (OMP). This reaction is catalyzed by orotate phosphoribosyltransferase (OPRT), which is a cytosolic enzyme.

The sixth reaction is the decarboxylation of OMP to form uridine 5`-monophosphate (UMP). This reaction is catalyzed by OMP decarboxylase (ODC), which is also a cytosolic enzyme. ODC is a highly efficient enzyme that has a very low Km for OMP and a very high kcat. It is also inhibited by UMP and CMP, the products of this pathway.

The seventh reaction is the phosphorylation of UMP to form uridine 5`-diphosphate (UDP). This reaction is catalyzed by uridine monophosphate kinase (UMPK), which is a cytosolic enzyme that uses ATP as a phosphate donor.

The eighth reaction is the phosphorylation of UDP to form uridine 5`-triphosphate (UTP). This reaction is catalyzed by uridine diphosphate kinase (UDPK), which is also a cytosolic enzyme that uses ATP as a phosphate donor.

The ninth reaction is the conversion of UTP to cytidine 5`-triphosphate (CTP) by the addition of an amino group from glutamine. This reaction is catalyzed by CTP synthetase (CTPS), which is also a cytosolic enzyme. CTPS is regulated by feedback inhibition by CTP, as well as allosteric activation by GTP and ATP.

UTP and CTP are the final products of this pathway and are used for RNA synthesis. They can also be converted to deoxyribonucleotides for DNA synthesis by ribonucleotide reductase and other enzymes.

The de novo pyrimidine synthesis pathway involves several enzymes that catalyze the reactions and regulate the flux of substrates and products. The main enzymes and their regulation are:

• Carbamoyl phosphate synthetase II (CPS II): This enzyme catalyzes the first and rate-limiting step of the pathway, which is the formation of carbamoyl phosphate from CO2, glutamine and ATP. CPS II is inhibited by UTP, the end product of the pathway, and activated by ATP and PRPP, the substrates of the pathway. This ensures that the synthesis of pyrimidines is balanced with the demand and availability of precursors.
• Aspartate transcarbamoylase (ATCase): This enzyme catalyzes the second step of the pathway, which is the condensation of carbamoyl phosphate and aspartate to form N-carbamoylaspartate. ATCase is also inhibited by UTP, as well as CTP, another end product of the pathway. This provides a feedback inhibition mechanism to prevent excess production of pyrimidines.
• Dihydroorotase (DHOase): This enzyme catalyzes the third step of the pathway, which is the cyclization of N-carbamoylaspartate to form dihydroorotate. DHOase is not regulated by any allosteric effectors, but it is sensitive to inhibition by some drugs such as leflunomide and brequinar, which are used to treat autoimmune diseases and cancer.
• Dihydroorotate dehydrogenase (DHODH): This enzyme catalyzes the fourth step of the pathway, which is the oxidation of dihydroorotate to orotate. DHODH is located in the inner mitochondrial membrane and uses NAD+ as a cofactor. DHODH is also inhibited by leflunomide and brequinar, as well as by some antiviral agents such as teriflunomide and A771726.
• Orotate phosphoribosyl transferase (OPRTase): This enzyme catalyzes the fifth step of the pathway, which is the transfer of a ribose-5-phosphate group from PRPP to orotate to form orotidine 5`-monophosphate (OMP). OPRTase is inhibited by UMP and CMP, the products of its reaction. This provides another feedback inhibition mechanism to regulate pyrimidine synthesis.
• Orotidine 5`-monophosphate decarboxylase (OMPDC): This enzyme catalyzes the sixth and final step of the pathway, which is the decarboxylation of OMP to form uridine 5`-monophosphate (UMP). OMPDC is also inhibited by UMP and CMP, as well as by some analogs of OMP such as 6-azauridine monophosphate and 5-fluorouridine monophosphate, which are used as anticancer drugs.
• Uridine monophosphate kinase (UMPK): This enzyme catalyzes the phosphorylation of UMP to form uridine 5`-diphosphate (UDP), which can then be further phosphorylated to form uridine 5`-triphosphate (UTP) by nucleoside diphosphate kinase (NDPK). UMPK is not regulated by any allosteric effectors, but it is sensitive to inhibition by some nucleoside analogs such as 5-fluorouracil and 5-fluorouridine, which are also used as anticancer drugs.
• CTP synthetase (CTPS): This enzyme catalyzes the conversion of UTP to CTP by adding an amino group from glutamine. CTPS is inhibited by CTP, its product, and activated by GTP, a purine nucleotide. This provides a cross-regulation mechanism between purine and pyrimidine synthesis.

These enzymes are responsible for controlling the de novo pyrimidine synthesis pathway and ensuring that it meets the cellular needs for DNA and RNA synthesis, as well as other metabolic functions. Any defects or imbalances in these enzymes can lead to diseases or disorders that affect various tissues and organs.

Pyrimidines can also be synthesized from pre-existing bases that are derived from the degradation of nucleic acids or dietary sources. This process is called the salvage pathway and it requires less energy than the de novo pathway. The salvage pathway involves the attachment of a ribose-5-phosphate moiety to a free pyrimidine base by the enzyme pyrimidine phosphoribosyl transferase (PPRT). The resulting nucleotide can then be converted to other pyrimidine nucleotides by kinases and other enzymes.

The salvage pathway is especially important for certain tissues and cells that have a high demand for pyrimidine nucleotides, such as the brain, lymphocytes, and erythrocytes. The salvage pathway also helps to maintain a balance between the levels of purine and pyrimidine nucleotides in the cell.

The main sources of free pyrimidine bases for the salvage pathway are orotic acid, uracil, and thymine. Orotic acid can be salvaged by PPRT to form orotidine 5′-monophosphate (OMP), which can then be converted to UMP by OMP decarboxylase. Uracil can be salvaged by uracil phosphoribosyl transferase (UPRT) to form UMP, which can then be converted to UTP and CTP. Thymine can be salvaged by thymine phosphoribosyl transferase (TPRT) to form thymidine monophosphate (TMP), which can then be converted to TTP and dTTP.

Cytosine cannot be salvaged directly by PPRT because it lacks a keto group at position 2. However, cytosine can be deaminated to uracil by cytidine deaminase and then salvaged by UPRT. Alternatively, cytosine can be converted to cytidine by cytosine deaminase and then salvaged by cytidine kinase to form CMP.

The salvage pathway is regulated by feedback inhibition of PPRT, UPRT, and TPRT by their respective products. For example, PPRT is inhibited by OMP, UPRT is inhibited by UMP, and TPRT is inhibited by TMP.

Defects in the enzymes involved in de novo pyrimidine synthesis can lead to various metabolic disorders that affect different organs and systems. Some of the diseases associated with deficiencies in the pathway are:

• Orotic aciduria: This is a rare autosomal recessive disorder caused by mutations in either orotate phosphoribosyl transferase (OPRT) or OMP decarboxylase (ODC), which are both encoded by the same gene (UMPS). These enzymes catalyze the last two steps of de novo pyrimidine synthesis, converting orotic acid to UMP. Patients with orotic aciduria present with megaloblastic anemia, growth retardation, developmental delay, and urinary excretion of large amounts of orotic acid.
• Dihydropyrimidine dehydrogenase (DPD) deficiency: This is an autosomal recessive disorder caused by mutations in the gene encoding DPD, which is the rate-limiting enzyme in the catabolism of pyrimidines. DPD converts uracil and thymine to dihydrouracil and dihydrothymine, respectively. Patients with DPD deficiency have neurological symptoms such as seizures, intellectual disability, microcephaly, and ataxia. They also have increased levels of uracil and thymine in plasma and urine.
• Dihydropyrimidinase (DHP) deficiency: This is an autosomal recessive disorder caused by mutations in the gene encoding DHP, which is the second enzyme in the catabolism of pyrimidines. DHP converts dihydrouracil and dihydrothymine to N-carbamoyl-beta-alanine and N-carbamoyl-beta-aminoisobutyrate, respectively. Patients with DHP deficiency have similar neurological symptoms as those with DPD deficiency, but they also have increased levels of dihydrouracil and dihydrothymine in plasma and urine.
• Beta-ureidopropionase (BUP) deficiency: This is an autosomal recessive disorder caused by mutations in the gene encoding BUP, which is the third and final enzyme in the catabolism of pyrimidines. BUP converts N-carbamoyl-beta-alanine and N-carbamoyl-beta-aminoisobutyrate to beta-alanine and beta-aminoisobutyrate, respectively. Patients with BUP deficiency have neurological symptoms such as seizures, intellectual disability, hypotonia, and spasticity. They also have increased levels of N-carbamoyl-beta-alanine and N-carbamoyl-beta-aminoisobutyrate in plasma and urine.
• Mitochondrial DNA-dependent inflammation: This is a novel condition that has been recently discovered to be linked to de novo pyrimidine synthesis. It is caused by mutations in the gene encoding YME1L1, which is a mitochondrial protease that regulates pyrimidine pools by supporting de novo nucleotide synthesis and by proteolysis of the pyrimidine nucleotide carrier SLC25A33 . Patients with YME1L1 deficiency have chronic inflammation in various tissues, such as retina, liver, muscle, and brain. They also have mitochondrial dysfunction and increased levels of mitochondrial DNA (mtDNA) fragments that activate innate immune receptors .

These diseases illustrate the importance of maintaining a balanced supply of pyrimidine nucleotides for various cellular functions and processes.

Pyrimidine synthesis is essential for the production of DNA and RNA, which are the carriers of genetic information in all living organisms. Pyrimidine nucleotides also play important roles in various metabolic pathways, such as glycogen synthesis, phospholipid synthesis, and coenzyme formation . Moreover, pyrimidine derivatives have diverse biological activities that can be exploited for therapeutic purposes.

Some examples of pyrimidine derivatives with medicinal applications are:

• Fluorouracil: a fluorinated analog of uracil that inhibits thymidylate synthase, an enzyme required for DNA synthesis. It is used as an anticancer drug to treat various solid tumors.
• Azathioprine: a prodrug of 6-mercaptopurine, which inhibits purine and pyrimidine synthesis. It is used as an immunosuppressant drug to prevent organ rejection after transplantation and to treat autoimmune diseases.
• Acyclovir: a guanine analog that inhibits viral DNA polymerase. It is used as an antiviral drug to treat herpes simplex virus infections.
• Trimethoprim: a dihydrofolate reductase inhibitor that blocks the conversion of dihydrofolate to tetrahydrofolate, a cofactor required for purine and pyrimidine synthesis. It is used as an antibacterial drug to treat urinary tract infections and other infections caused by susceptible bacteria.

Pyrimidine synthesis is also involved in some genetic diseases that affect the metabolism or regulation of pyrimidine nucleotides. For example, orotic aciduria is a rare disorder caused by a deficiency of either orotate phosphoribosyltransferase or OMP decarboxylase, two enzymes involved in the de novo pyrimidine synthesis pathway. It is characterized by excessive excretion of orotic acid in urine, growth retardation, megaloblastic anemia, and immunodeficiency. Another example is Lesch-Nyhan syndrome, which is caused by a deficiency of hypoxanthine-guanine phosphoribosyltransferase, an enzyme involved in the purine salvage pathway. It is characterized by hyperuricemia, gout, nephrolithiasis, neurologic impairment, and self-mutilation.

In conclusion, pyrimidine synthesis is a vital process for the maintenance of cellular functions and genetic integrity. It also provides a source of potential drugs for various diseases. Therefore, understanding the molecular mechanisms and regulation of pyrimidine synthesis is important for both basic and applied research in biology and medicine.

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