Nucleotide- Definition, Characteristics, Biosynthesis, Functions

Nucleotides are the basic building blocks of nucleic acids, such as DNA and RNA. Nucleic acids are polymers made of long chains of nucleotides. A nucleotide consists of three components: a sugar molecule, a phosphate group and a nitrogen-containing base .

The sugar molecule in a nucleotide can be either ribose in RNA or deoxyribose in DNA. The main difference between these two sugars is that ribose has an -OH group at the 2` position, while deoxyribose lacks it. The sugar molecules are present in their β-furanose form, which is a closed five-membered ring structure.

The nitrogen-containing base in a nucleotide can be one of four types: adenine (A), cytosine (C), guanine (G) or thymine (T) in DNA, or uracil (U) instead of thymine in RNA. These bases are derived from two parent compounds: purines and pyrimidines. Purines have a double-ring structure and include adenine and guanine. Pyrimidines have a single-ring structure and include cytosine, thymine and uracil.

The nitrogen-containing base is linked covalently to the sugar molecule by an N-glycosidic bond, which connects the N-1 position of pyrimidines or the N-9 position of purines to the C-1 position of the sugar. The phosphate group is attached to the 5` position of the sugar by an ester bond. A nucleotide can have one, two or three phosphate groups, forming a nucleoside monophosphate (NMP), diphosphate (NDP) or triphosphate (NTP), respectively.

Nucleotides are joined together by phosphodiester bonds, which link the 5` phosphate group of one nucleotide to the 3` hydroxyl group of another nucleotide. This forms a backbone of alternating sugar and phosphate units, with the bases projecting outwards. The sequence of bases along the nucleic acid chain determines the genetic information encoded by the molecule.

Nucleotides can also form hydrogen bonds with each other based on their complementary base pairing. In DNA, adenine pairs with thymine by two hydrogen bonds, and guanine pairs with cytosine by three hydrogen bonds. In RNA, uracil replaces thymine and pairs with adenine by two hydrogen bonds. These base pairs form the rungs of the double helix structure of DNA or the secondary structures of RNA.

Nucleotides have various functions in living cells. They are involved in energy storage and transfer (e.g., ATP), enzyme cofactors (e.g., NAD+), signaling molecules (e.g., cAMP), and regulation of gene expression (e.g., ppGpp). Nucleotides are synthesized by two pathways: de novo synthesis, which builds nucleotides from simple molecules, and salvage pathway, which recycles nucleotides from degraded nucleic acids. The biosynthesis of nucleotides is regulated by feedback inhibition and allosteric mechanisms.

Nucleotides are organic molecules composed of three subunit molecules: a nucleobase, a five-carbon sugar (ribose or deoxyribose), and a phosphate group consisting of one to three phosphates. The nucleobases are derived from two-parent compounds – purines and pyrimidines. The four nucleobases in DNA are guanine, adenine, cytosine and thymine; in RNA, uracil is used in place of thymine.

The sugar component may either be ribose or deoxyribose. Ribose is the sugar component of the nucleotides that make up RNA. The deoxyribose sugar is the sugar component of DNA. The main difference is seen at the second position of the pentose structure, in the case of 2` – deoxyribose there is an absence of an alcohol group/ oxy group/ -OH group at the second position, hence the name. In the case of D – ribose pentose the –OH group is present at the second position. In both types, the pentoses are present in their β-furanose form which is a close five-membered ring structure.

The nitrogenous base is linked covalently to pentose sugar by an N- glycosidic bond (N-1 in the case of pyrimidines and N-9 in the case of purines is linked to the C-1 carbon atom of the pentoses.). The consecutive nucleotides in DNA and RNA are linked by phospho-diester linkages, the 5phosphate group of one nucleotide is linked with the 3 hydroxyl group of another nucleotide and the complementary nucleotides are linked by hydrogen bonds. Adenosine and Thymine are bonded by two hydrogen bonds and Guanine and Cytosine are bonded by three hydrogen bonds. The backbone of the DNA is composed of the chain of nucleotides linked to each other.

Nucleotides can be classified into two types based on their function: structural nucleotides and non-structural nucleotides. Structural nucleotides are those that form part of the nucleic acid polymers, such as DNA and RNA. Non-structural nucleotides are those that have other roles in metabolism, signaling, and enzymatic reactions. Some examples of non-structural nucleotides are:

• Adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytidine triphosphate (CTP) and uridine triphosphate (UTP) – these nucleotides provide chemical energy for various cellular functions and act as phosphoryl group donors.
• Cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) – these nucleotides act as second messengers in response to hormones and other signals.
• Nicotinamide adenine dinucleotide (NAD), flavin adenine dinucleotide (FAD), coenzyme A (CoA) – these nucleotides act as cofactors for many enzymes involved in oxidation-reduction reactions, acetyl group transfer, etc..

Nucleotides are the building blocks of DNA and RNA, and they also have important roles in energy metabolism, signal transduction, and enzyme cofactors. Nucleotides can be synthesized by two pathways: de novo synthesis and salvage pathway.

De novo synthesis of nucleotides

In this pathway, nucleotides are synthesized from simple precursors such as amino acids, sugars, and carbon dioxide. The pathway for purine and pyrimidine nucleotides is different and requires different enzymes and substrates.

Purine nucleotides

Purine nucleotides are composed of a purine base (adenine or guanine) attached to a ribose sugar and one or more phosphate groups. The purine ring is built on the ribose sugar by adding atoms from various sources such as glutamine, glycine, aspartate, and formate. The first step is the formation of phosphoribosyl pyrophosphate (PRPP) from ribose-5-phosphate and ATP. PRPP then reacts with glutamine to form phosphoribosylamine (PRA), which is the first committed step in purine synthesis. PRA then undergoes a series of reactions involving 10 different enzymes to form inosine monophosphate (IMP), which is the common precursor for adenine and guanine nucleotides. IMP can be converted to AMP by adding an amino group from aspartate and a formyl group from N10-formyltetrahydrofolate (N10-THF), or to GMP by adding an amino group from glutamine and an oxygen atom from water. Both reactions require ATP as an energy source. AMP and GMP can then be phosphorylated to ADP and GDP by nucleoside monophosphate kinases, and further to ATP and GTP by nucleoside diphosphate kinases.

Pyrimidine nucleotides

Pyrimidine nucleotides are composed of a pyrimidine base (cytosine, uracil, or thymine) attached to a ribose sugar and one or more phosphate groups. The pyrimidine ring is synthesized first and then attached to the ribose sugar. The first step is the formation of carbamoyl phosphate from glutamine, carbon dioxide, and ATP by the enzyme carbamoyl phosphate synthetase II (CPS II). Carbamoyl phosphate then reacts with aspartate to form N-carbamoylaspartate, which is cyclized to dihydroorotate by the enzyme dihydroorotase. Dihydroorotate is then oxidized to orotate by the enzyme dihydroorotate dehydrogenase, which requires NAD+ as a cofactor. Orotate then reacts with PRPP to form orotidine-5`-monophosphate (OMP), which is decarboxylated to uridine-5`-monophosphate (UMP) by the enzyme orotidine-5`-phosphate decarboxylase. UMP can be converted to CMP by adding an amino group from glutamine, or to TMP by adding a methyl group from N5,N10-methylene tetrahydrofolate (N5,N10-mTHF). Both reactions require ATP as an energy source. UMP, CMP, and TMP can then be phosphorylated to UDP, CDP, and TDP by nucleoside monophosphate kinases, and further to UTP, CTP, and TTP by nucleoside diphosphate kinases.

Salvage pathway of nucleotides

In this pathway, nucleotides are recycled from free bases or nucleosides that are released from DNA or RNA degradation or dietary intake. The salvage pathway is more economical than the de novo pathway, as it requires less energy and fewer precursors.

Purine nucleotides

Free purine bases (adenine, guanine, or hypoxanthine) can be salvaged by reacting with PRPP to form purine nucleotides. This reaction is catalyzed by two enzymes: adenine phosphoribosyltransferase (APRT) for adenine, and hypoxanthine-guanine phosphoribosyltransferase (HGPRT) for guanine and hypoxanthine. Free purine nucleosides (adenosine or guanosine) can also be salvaged by phosphorylation to form purine nucleotides. This reaction is catalyzed by two enzymes: adenosine kinase for adenosine, and guanosine kinase for guanosine.

Pyrimidine nucleotides

Free pyrimidine bases (cytosine, uracil, or thymine) can be salvaged by reacting with PRPP to form pyrimidine nucleotides. This reaction is catalyzed by two enzymes: cytosine phosphoribosyltransferase (CPRT) for cytosine, and uracil phosphoribosyltransferase (UPRT) for uracil. Thymine cannot be salvaged by this pathway, as there is no enzyme that can transfer a ribose sugar to thymine. Free pyrimidine nucleosides (cytidine, uridine, or deoxythymidine) can also be salvaged by phosphorylation to form pyrimidine nucleotides. This reaction is catalyzed by three enzymes: cytidine kinase for cytidine, uridine kinase for uridine, and thymidine kinase for deoxythymidine.

De novo synthesis of nucleotides is the biochemical pathway in which nucleotides are synthesized from simple precursor molecules, such as sugars, amino acids and carbon dioxide. De novo synthesis of nucleotides can be divided into two pathways: purine synthesis and pyrimidine synthesis.

Purine synthesis

Purine synthesis involves the formation of a purine ring, which is a six-membered ring fused to a five-membered ring, attached to a ribose-5-phosphate sugar. The purine nucleotides are adenosine monophosphate (AMP) and guanosine monophosphate (GMP), which contain the bases adenine and guanine, respectively.

The purine synthesis starts with the formation of 5-phosphoribosyl-1-pyrophosphate (PRPP) from ribose-5-phosphate and ATP. PRPP is then converted to phosphoribosylamine (PRA) by the enzyme glutamine phosphoribosylpyrophosphate amidotransferase (GPAT), which uses glutamine as a nitrogen donor. PRA is then modified by several enzymes, using glycine, formate, aspartate and carbon dioxide as sources of carbon and nitrogen atoms, to form inosine monophosphate (IMP), which is the common precursor of AMP and GMP.

IMP can be converted to AMP by the enzyme adenylosuccinate synthetase, which uses aspartate and GTP as substrates, and then to adenylosuccinate lyase, which releases fumarate. Alternatively, IMP can be converted to GMP by the enzyme IMP dehydrogenase, which uses NAD+ and water as substrates, and then to GMP synthetase, which uses glutamine and ATP as substrates.

The following is a summary of the steps involved in purine synthesis:

Ribose-5-phosphate + ATP → PRPP + AMP (PRPP synthetase)
PRPP + Glutamine → PRA + Glutamate + PPi (GPAT)
PRA + Glycine + ATP → GAR + ADP + Pi (GAR synthase)
GAR + N10-formyl-THF → FGAR + THF (GAR transformylase)
FGAR + ATP → FGAM + ADP + Pi (FGAM synthase)
FGAM + N10-formyl-THF → AIR + THF (AIR synthetase)
AIR + CO2 → CAIR (AIR carboxylase)
CAIR + Aspartate + ATP → SAICAR + ADP + Pi (SAICAR synthetase)
SAICAR → AICAR + Fumarate (SAICAR lyase)
AICAR + N10-formyl-THF → FAICAR + THF (AICAR transformylase)
FAICAR → IMP + H2O (IMP cyclohydrolase)
IMP + Aspartate + GTP → Adenylosuccinate + GDP + Pi (Adenylosuccinate synthetase)
Adenylosuccinate → AMP + Fumarate (Adenylosuccinate lyase)
IMP + NAD+ + H2O → XMP + NADH + H+ (IMP dehydrogenase)
XMP + Glutamine + ATP → GMP + Glutamate + ADP + Pi (GMP synthetase)

Pyrimidine synthesis

Pyrimidine synthesis involves the formation of a pyrimidine ring, which is a six-membered ring with two nitrogen atoms, attached to a ribose-5-phosphate sugar. The pyrimidine nucleotides are uridine monophosphate (UMP) and cytidine monophosphate (CMP), which contain the bases uracil and cytosine, respectively.

The pyrimidine synthesis starts with the formation of carbamoyl phosphate from bicarbonate and glutamine by the enzyme carbamoyl phosphate synthetase II (CPS II), which is located in the cytosol. Carbamoyl phosphate then reacts with aspartate to form N-carbamoylaspartate by the enzyme aspartate transcarbamoylase (ATCase). N-carbamoylaspartate then undergoes a series of reactions, catalyzed by dihydroorotase, dihydroorotate dehydrogenase, orotate phosphoribosyltransferase and orotidine-5`-monophosphate decarboxylase, to form UMP.

UMP can be converted to CMP by the enzyme CTP synthetase, which uses glutamine and ATP as substrates. UMP and CMP can also be phosphorylated to form UDP and CDP, respectively, by the enzyme nucleoside monophosphate kinase, which uses ATP as a substrate. UDP and CDP can then be converted to dUDP and dCDP, respectively, by the enzyme ribonucleotide reductase, which uses NADPH and thioredoxin as substrates. dUDP and dCDP can then be phosphorylated to form dUTP and dCTP, respectively, by the enzyme nucleoside diphosphate kinase, which uses ATP as a substrate. dUTP can then be hydrolyzed to form dUMP and PPi by the enzyme dUTPase. dUMP can then be methylated to form dTMP by the enzyme thymidylate synthase, which uses N5,N10-methylene-THF as a methyl donor.

The following is a summary of the steps involved in pyrimidine synthesis:

Bicarbonate + Glutamine + 2 ATP → Carbamoyl phosphate + Glutamate + 2 ADP + Pi (CPS II)
Carbamoyl phosphate + Aspartate → N-carbamoylaspartate + Pi (ATCase)
N-carbamoylaspartate → L-dihydroorotate + H2O (Dihydroorotase)
L-dihydroorotate + NAD+ → Orotate + NADH + H+ (Dihydroorotate dehydrogenase)
Orotate + PRPP → OMP + PPi (Orotate phosphoribosyltransferase)
OMP → UMP + CO2 (OMP decarboxylase)
UMP + ATP → UDP + ADP (Nucleoside monophosphate kinase)
UDP + ATP → UTP + ADP (Nucleoside diphosphate kinase)
UTP + Glutamine + ATP → CTP + Glutamate + ADP + Pi (CTP synthetase)
CTP + ATP → CDP + ADP (Nucleoside monophosphate kinase)
CDP + ATP → CTP + ADP (Nucleoside diphosphate kinase)
UDP + NADPH + Thioredoxin → dUDP + NADP+ + Thioredoxin disulfide (Ribonucleotide reductase)
dUDP + ATP → dUTP + ADP (Nucleoside diphosphate kinase)
dUTP → dUMP + PPi (dUTPase)
dUMP + N5,N10-methylene-THF → dTMP + THF (Thymidylate synthase)
dTMP + ATP → dTDP + ADP (Nucleoside monophosphate kinase)
dTDP + ATP → dTTP + ADP (Nucleoside diphosphate kinase)
CDP + NADPH + Thioredoxin → dCDP + NADP+ + Thioredoxin disulfide (Ribonucleotide reductase)
dCDP + ATP → dCTP + ADP (Nucleoside diphosphate kinase)

The salvage pathway of nucleotides is a process that recycles the free bases and nucleosides that are formed during the degradation of RNA and DNA. This pathway is important for some tissues that cannot synthesize nucleotides from scratch (de novo synthesis). The salvaged bases and nucleosides can then be converted back into nucleotides by using activated ribose-5-phosphate (phosphoribosyl pyrophosphate, PRPP) as a donor of the sugar and phosphate groups.

The salvage pathway of nucleotides involves different enzymes that catalyze the transfer of PRPP to the free bases or nucleosides. There are two types of enzymes that can transfer PRPP to free purine bases: adenine phosphoribosyltransferase (APRT) and hypoxanthine-guanine phosphoribosyltransferase (HGPRT). APRT can transfer PRPP to adenine, forming adenosine monophosphate (AMP). HGPRT can transfer PRPP to hypoxanthine or guanine, forming inosine monophosphate (IMP) or guanosine monophosphate (GMP), respectively. These nucleotides can then be phosphorylated to form the corresponding triphosphates (ATP, GTP).

The salvage pathway of pyrimidine nucleotides involves different enzymes that can either transfer PRPP to free pyrimidine bases or phosphorylate pyrimidine nucleosides. For example, uracil phosphoribosyltransferase (UPRT) can transfer PRPP to uracil, forming uridine monophosphate (UMP). Uridine kinase (UK) can phosphorylate uridine or cytidine to form UMP or cytidine monophosphate (CMP), respectively. Thymidine kinase (TK) can phosphorylate thymidine or deoxyuridine to form thymidine monophosphate (TMP) or deoxyuridine monophosphate (dUMP), respectively. These nucleotides can then be converted to their corresponding diphosphates and triphosphates by other kinases.

The salvage pathway of nucleotides is regulated by feedback inhibition and allosteric modulation of some enzymes. For example, PRPP synthetase, which catalyzes the formation of PRPP from ribose-5-phosphate and ATP, is inhibited by ADP and GDP. PRPP amidotransferase, which catalyzes the first step of purine de novo synthesis, is inhibited by AMP and GMP. HGPRT is inhibited by IMP and GMP. UPRT is inhibited by UMP. UK is inhibited by UTP and CTP.

The salvage pathway of nucleotides is essential for maintaining a balanced supply of nucleotides for various cellular functions, such as DNA and RNA synthesis, energy metabolism, signal transduction, and coenzyme formation. Defects in some enzymes of this pathway can cause genetic disorders, such as Lesch-Nyhan syndrome, which is caused by a deficiency of HGPRT and leads to excessive production and accumulation of uric acid.

Some possible sentences to conclude the point 5 are:

• Therefore, the salvage pathway of nucleotides is an important alternative to de novo synthesis that allows the reuse of nitrogenous bases and nucleosides from various sources.
• In summary, the salvage pathway of nucleotides is a way of recycling the components of RNA and DNA degradation or exogenous intake into functional nucleotides by using PRPP as a common precursor.
• Thus, the salvage pathway of nucleotides is a vital process that conserves energy and resources by reutilizing the free bases and nucleosides that are generated from different pathways or obtained from the environment.

Nucleotide biosynthesis is a highly regulated process that uses multiple metabolic pathways across different cell compartments and several sources of carbon and nitrogen. The processes are regulated at the transcription level by a set of master transcription factors but also at the enzyme level by allosteric regulation and feedback inhibition.

The regulation of nucleotide biosynthesis is performed by feedback control or repression, where the end products of the pathway inhibit the activity or expression of the first enzyme. For example, CTP inhibits aspartate transcarbamoylase (ATCase), the first enzyme of pyrimidine biosynthesis, while AMP and GMP inhibit phosphoribosylpyrophosphate amidotransferase (PRPP amidotransferase), the first enzyme of purine biosynthesis.

The regulation of nucleotide biosynthesis is also performed by feed-forward activation, where the intermediates or precursors of the pathway stimulate the activity or expression of a later enzyme. For example, PRPP activates ATCase, while ATP and GTP activate PRPP amidotransferase.

The regulation of nucleotide biosynthesis is also performed by cross-regulation, where the products of one pathway affect the activity or expression of enzymes in another pathway. For example, ATP activates carbamoyl phosphate synthetase II (CPS II), the second enzyme of pyrimidine biosynthesis, while UTP inhibits it. Similarly, GTP activates adenylosuccinate synthetase (ASS), an enzyme involved in AMP synthesis from IMP, while ATP activates IMP dehydrogenase (IMPDH), an enzyme involved in GMP synthesis from IMP.

Nucleotide biosynthesis can also be inhibited by various molecules that interfere with the enzymes or substrates of the pathway. For example, nucleotide analogs, such as 5-fluorouracil and 6-mercaptopurine, can compete with natural nucleotides for incorporation into DNA or RNA, leading to faulty replication or transcription. Precursor/substrate analogs, such as azaserine and acivicin, can inhibit glutamine-dependent enzymes involved in purine and pyrimidine synthesis. Inhibitors of folic acid pathway, such as methotrexate and trimethoprim, can block the formation of tetrahydrofolate (THF) derivatives that are essential for one-carbon transfer reactions in nucleotide synthesis.

Nucleotide biosynthesis is also influenced by bacterial pathogenesis, as some bacteria can exploit the host`s nucleotide pool or synthesize their own nucleotides to survive and cause infection. Regulators of nucleotide biosynthesis are emerging as important for control of the expression of virulence factors. For example, IMPDH regulates the expression of type III secretion system in Pseudomonas aeruginosa and Salmonella enterica. De novo nucleotide biosynthesis is targeted by purpose-specific molecules to facilitate bacterial survival during interspecies competition. For example, colibactin is a genotoxin produced by Escherichia coli that inhibits purine synthesis in competing bacteria.

In summary, nucleotide biosynthesis is a complex and dynamic process that is tightly regulated by various mechanisms and factors to maintain cellular homeostasis and respond to environmental changes.

Nucleotides are organic molecules that serve as the basic structural units for DNA and RNA. They also play a central role in metabolism, cell signaling, and enzyme regulation. Some of the major functions of nucleotides are:

• Energy storage and transfer: Nucleotides provide chemical energy in the form of nucleoside triphosphates, such as ATP, GTP, CTP, and UTP, for many cellular functions that demand energy. ATP is considered the currency of cell and plays a crucial role in various pathways and acts as a phosphoryl group donor. Hydrolysis of ATP yields a large amount of energy which can be utilized by the cell for different functions.
• Cell signaling: Nucleotides participate in cell signaling as second messengers, such as cAMP and cGMP, which are produced in response to hormones and other chemical signals. cAMP and cGMP regulate many cellular processes, such as gene expression, cell growth, differentiation, apoptosis, and ion channel opening.
• Enzyme regulation: Nucleotides act as coenzymes or cofactors, which are required to catalyze many biochemical reactions by enzymes. Examples of nucleotide-derived coenzymes are coenzyme A, NAD+, NADP+, FAD, FMN, etc. These coenzymes participate in various metabolic pathways, such as glycolysis, Krebs cycle, oxidative phosphorylation, fatty acid synthesis and oxidation, etc.
• Nucleic acid synthesis: Nucleotides are the building blocks of DNA and RNA, which store and transmit genetic information. DNA and RNA are composed of four types of nucleotides each: adenine (A), guanine (G), cytosine (C), and thymine (T) for DNA; and adenine (A), guanine (G), cytosine (C), and uracil (U) for RNA. The nucleotides are linked by phosphodiester bonds to form polynucleotide chains. The sequence of nucleotides determines the genetic code that specifies the amino acid sequence of proteins.

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