Nucleotides

Nucleotides are organic molecules consisting of a

nucleoside

and a

phosphate

. They serve as

monomer

ic units of the

nucleic acid

polymers

– deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), both of which are essential biomolecules within all life-forms on Earth. Nucleotides are obtained in the diet and are also synthesized from common nutrients by the liver.

Nucleotides are composed of three subunit molecules: a nucleobase, a

five-carbon sugar

(

ribose

or

deoxyribose

), and a phosphate group consisting of one to three

phosphate

s. The four nucleobases in DNA are

guanine

,

adenine

,

cytosine

and

thymine

; in RNA,

uracil

is used in place of thymine.

Nucleotides also play a central role in

metabolism

at a fundamental, cellular level. They provide chemical energy—in the form of the nucleoside triphosphates,

adenosine triphosphate

(ATP), guanosine triphosphate (GTP), cytidine triphosphate (CTP) and uridine triphosphate (UTP)—throughout the cell for the many cellular functions that demand energy, including:

amino acid

,

protein

and

cell membrane

synthesis, moving the cell and cell parts (both internally and intercellularly), cell division, etc.Alberts B, Johnson A, Lewis J, Raff M, Roberts K & Walter P (2002). ''Molecular Biology of the Cell'' (4th ed.). Garland Science. . pp. 120–121. In addition, nucleotides participate in cell signaling (cyclic guanosine monophosphate or cGMP and cyclic adenosine monophosphate or cAMP), and are incorporated into important cofactors of enzymatic reactions (e.g. coenzyme A,

FAD

,

FMN

, NAD, and

NADP⁺

).

In experimental

biochemistry

, nucleotides can be radiolabeled using radionuclides to yield radionucleotides.

Structure

A nucleotide is composed of three distinctive chemical sub-units: a five-carbon sugar molecule, a nucleobase—the two of which together are called a

nucleoside

—and one

phosphate group

. With all three joined, a nucleotide is also termed a "nucleoside ''mono''phosphate", "nucleoside ''di''phosphate" or "nucleoside ''tri''phosphate", depending on how many phosphates make up the phosphate group.

In

nucleic acid

s, nucleotides contain either a

purine

or a

pyrimidine

base—i.e., the nucleobase molecule, also known as a nitrogenous base—and are termed ''ribo''nucleotides if the sugar is ribose, or ''deoxyribo''nucleotides if the sugar is deoxyribose. Individual phosphate molecules repetitively connect the

sugar-ring

molecules in two adjacent nucleotide monomers, thereby connecting the nucleotide monomers of a nucleic acid end-to-end into a long chain. These chain-joins of sugar and phosphate molecules create a 'backbone' strand for a single- or double helix. In any one strand, the chemical orientation ( directionality) of the chain-joins runs from the 5'-end to the 3'-end (''read'': 5 prime-end to 3 prime-end)—referring to the five carbon sites on sugar molecules in adjacent nucleotides. In a double helix, the two strands are oriented in opposite directions, which permits base pairing and complementarity between the base-pairs, all which is essential for

replicating

or transcribing the encoded information found in DNA.

Nucleic acids then are polymeric macromolecules assembled from nucleotides, the

monomer-units of nucleic acids

. The purine bases

adenine

and

guanine

and pyrimidine base

cytosine

occur in both DNA and RNA, while the pyrimidine bases

thymine

(in DNA) and

uracil

(in RNA) occur in just one. Adenine forms a base pair with thymine with two hydrogen bonds, while guanine pairs with cytosine with three hydrogen bonds.

In addition to being building blocks for the construction of nucleic acid polymers, singular nucleotides play roles in cellular energy storage and provision, cellular signaling, as a source of phosphate groups used to modulate the activity of proteins and other signaling molecules, and as enzymatic cofactors, often carrying out

redox

reactions. Signaling cyclic nucleotides are formed by binding the phosphate group twice to the same sugar molecule, bridging the 5'- and 3'-

hydroxyl group

s of the sugar. Some signaling nucleotides differ from the standard single-phosphate group configuration, in having multiple phosphate groups attached to different positions on the sugar. Nucleotide cofactors include a wider range of chemical groups attached to the sugar via the glycosidic bond, including

nicotinamide

and

flavin

, and in the latter case, the ribose sugar is linear rather than forming the ring seen in other nucleotides.

Synthesis

Nucleotides can be synthesized by a variety of means both

in vitro

and in vivo.

In vitro, protecting groups may be used during laboratory production of nucleotides. A purified

nucleoside

is protected to create a

phosphoramidite

, which can then be used to obtain analogues not found in nature and/or to synthesize an oligonucleotide.

In vivo, nucleotides can be synthesized de novo or recycled through salvage pathways. The components used in de novo nucleotide synthesis are derived from biosynthetic precursors of carbohydrate and

amino acid

metabolism, and from ammonia and carbon dioxide. The liver is the major organ of de novo synthesis of all four nucleotides. De novo synthesis of pyrimidines and purines follows two different pathways. Pyrimidines are synthesized first from aspartate and carbamoyl-phosphate in the cytoplasm to the common precursor ring structure orotic acid, onto which a phosphorylated ribosyl unit is covalently linked. Purines, however, are first synthesized from the sugar template onto which the ring synthesis occurs. For reference, the syntheses of the

purine

and

pyrimidine

nucleotides are carried out by several enzymes in the cytoplasm of the cell, not within a specific organelle. Nucleotides undergo breakdown such that useful parts can be reused in synthesis reactions to create new nucleotides.

Pyrimidine ribonucleotide synthesis

The synthesis of the pyrimidines CTP and UTP occurs in the cytoplasm and starts with the formation of carbamoyl phosphate from

glutamine

and CO₂. Next, aspartate carbamoyltransferase catalyzes a condensation reaction between aspartate and carbamoyl phosphate to form carbamoyl aspartic acid, which is cyclized into 4,5-dihydroorotic acid by dihydroorotase. The latter is converted to orotate by dihydroorotate oxidase. The net reaction is:

:(''S'')-Dihydroorotate + O₂ → Orotate + H₂O₂

Orotate is covalently linked with a phosphorylated ribosyl unit. The covalent linkage between the ribose and pyrimidine occurs at position C₁ of the

ribose

unit, which contains a pyrophosphate, and N₁ of the pyrimidine ring. Orotate phosphoribosyltransferase (PRPP transferase) catalyzes the net reaction yielding orotidine monophosphate (OMP):

:Orotate +

5-Phospho-α-D-ribose 1-diphosphate (PRPP)

→ Orotidine 5'-phosphate + Pyrophosphate

Orotidine 5'-monophosphate is decarboxylated by orotidine-5'-phosphate decarboxylase to form uridine monophosphate (UMP). PRPP transferase catalyzes both the ribosylation and decarboxylation reactions, forming UMP from orotic acid in the presence of PRPP. It is from UMP that other pyrimidine nucleotides are derived. UMP is phosphorylated by two kinases to uridine triphosphate (UTP) via two sequential reactions with ATP. First, the diphosphate from UDP is produced, which in turn is phosphorylated to UTP. Both steps are fueled by ATP hydrolysis:

:ATP + UMP → ADP + UDP

:UDP + ATP → UTP + ADP

CTP is subsequently formed by the amination of UTP by the catalytic activity of CTP synthetase. Glutamine is the NH₃ donor and the reaction is fueled by ATP hydrolysis, too:

:UTP + Glutamine + ATP + H₂O → CTP + ADP + P_i

Cytidine monophosphate (CMP) is derived from cytidine triphosphate (CTP) with subsequent loss of two phosphates.

Purine ribonucleotide synthesis

The atoms that are used to build the purine nucleotides come from a variety of sources:

The de novo synthesis of purine nucleotides by which these precursors are incorporated into the purine ring proceeds by a 10-step pathway to the branch-point intermediate

IMP

, the nucleotide of the base

hypoxanthine

. AMP and GMP are subsequently synthesized from this intermediate via separate, two-step pathways. Thus, purine moieties are initially formed as part of the ribonucleotides rather than as free bases.

Six enzymes take part in IMP synthesis. Three of them are multifunctional:
* GART (reactions 2, 3, and 5)
* PAICS (reactions 6, and 7)
* ATIC (reactions 9, and 10)

The pathway starts with the formation of PRPP. PRPS1 is the

enzyme

that activates R5P, which is formed primarily by the

pentose phosphate pathway

, to PRPP by reacting it with

ATP

. The reaction is unusual in that a pyrophosphoryl group is directly transferred from ATP to C₁ of R5P and that the product has the α configuration about C1. This reaction is also shared with the pathways for the synthesis of

Trp

,

His

, and the pyrimidine nucleotides. Being on a major metabolic crossroad and requiring much energy, this reaction is highly regulated.

In the first reaction unique to purine nucleotide biosynthesis, PPAT catalyzes the displacement of PRPP's pyrophosphate group (PP_i) by an amide nitrogen donated from either

glutamine

(N),

glycine

(N&C), aspartate (N),

folic acid

(C₁), or CO₂. This is the committed step in purine synthesis. The reaction occurs with the inversion of configuration about ribose C₁, thereby forming β- 5-phosphorybosylamine (5-PRA) and establishing the anomeric form of the future nucleotide.

Next, a glycine is incorporated fueled by ATP hydrolysis, and the carboxyl group forms an amine bond to the NH₂ previously introduced. A one-carbon unit from folic acid coenzyme N₁₀-formyl-THF is then added to the amino group of the substituted glycine followed by the closure of the imidazole ring. Next, a second NH₂ group is transferred from glutamine to the first carbon of the glycine unit. A carboxylation of the second carbon of the glycin unit is concomitantly added. This new carbon is modified by the addition of a third NH₂ unit, this time transferred from an aspartate residue. Finally, a second one-carbon unit from formyl-THF is added to the nitrogen group and the ring is covalently closed to form the common purine precursor inosine monophosphate (IMP).

Inosine monophosphate is converted to adenosine monophosphate in two steps. First, GTP hydrolysis fuels the addition of aspartate to IMP by adenylosuccinate synthase, substituting the carbonyl oxygen for a nitrogen and forming the intermediate adenylosuccinate. Fumarate is then cleaved off forming adenosine monophosphate. This step is catalyzed by adenylosuccinate lyase.

Inosine monophosphate is converted to guanosine monophosphate by the oxidation of IMP forming xanthylate, followed by the insertion of an amino group at C₂. NAD⁺ is the electron acceptor in the oxidation reaction. The amide group transfer from glutamine is fueled by ATP hydrolysis.

Pyrimidine and purine degradation

In humans, pyrimidine rings (C, T, U) can be degraded completely to CO₂ and NH₃ (urea excretion). That having been said, purine rings (G, A) cannot. Instead, they are degraded to the metabolically inert

uric acid

which is then excreted from the body. Uric acid is formed when GMP is split into the base guanine and ribose. Guanine is deaminated to xanthine which in turn is oxidized to uric acid. This last reaction is irreversible. Similarly, uric acid can be formed when AMP is deaminated to IMP from which the ribose unit is removed to form hypoxanthine. Hypoxanthine is oxidized to xanthine and finally to uric acid. Instead of uric acid secretion, guanine and IMP can be used for recycling purposes and nucleic acid synthesis in the presence of PRPP and aspartate (NH₃ donor).

Prebiotic synthesis of nucleotides

Theories about how

life

arose require knowledge of chemical pathways that permit formation of life’s key building blocks under plausible prebiotic conditions. The RNA world hypothesis holds that in the primordial soup there existed free-floating ribonucleotides, the fundamental molecules that combine in series to form

RNA

. Complex molecules like RNA must have arisen form small molecules whose reactivity was governed by physico-chemical processes. RNA is composed of

purine

and

pyrimidine

nucleotides, both of which are necessary for reliable information transfer, and thus Darwinian

evolution

. Becker et al. showed how pyrimidine

nucleoside

s can be synthesized from small molecules and

ribose

, driven solely by wet-dry cycles.Becker S, Feldmann J, Wiedemann S, Okamura H, Schneider C, Iwan K, Crisp A, Rossa M, Amatov T, Carell T. Unified prebiotically plausible synthesis of pyrimidine and purine RNA ribonucleotides. Science. 2019 Oct 4;366(6461):76-82. doi: 10.1126/science.aax2747. PMID: 31604305 Purine nucleosides can be synthesized by a similar pathway. 5’-mono- and di-phosphates also form selectively from phosphate-containing minerals, allowing concurrent formation of polyribonucleotides with both the purine and pyrimidine bases. Thus a reaction network towards the purine and pyrimidine RNA building blocks can be established starting from simple atmospheric or volcanic molecules.

Unnatural base pair (UBP)

An unnatural base pair (UBP) is a designed subunit (or nucleobase) of

DNA

which is created in a laboratory and does not occur in nature. Examples include

d5SICS

and

dNaM

. These artificial nucleotides bearing hydrophobic nucleobases, feature two fused aromatic rings that form a (d5SICS–dNaM) complex or base pair in DNA. ''E. coli'' have been induced to replicate a plasmid containing UBPs through multiple generations. This is the first known example of a living organism passing along an expanded genetic code to subsequent generations.

Medical applications of synthetic nucleotides

Several nucleotide derivatives have been used as antivirals against

hepatitis

and

HIV

. Tenofovir disoproxil,

Tenofovir alafenamide

and

Sofosbuvir

are examples of NRTI used against hepatitis. Whereas certain drugs like

Mericitabine

,

Lamivudine

,

Entecavir

and

Telbivudine

for example are nucleosides, but they are metabolized into their bioactive nucleotide forms through phosphorylation.

Length unit

Nucleotide (abbreviated "nt") is a common unit of length for single-stranded nucleic acids, similar to how base pair is a unit of length for double-stranded nucleic acids.

Abbreviation codes for degenerate bases

The IUPAC has designated the symbols for nucleotides. Apart from the five (A, G, C, T/U) bases, often degenerate bases are used especially for designing PCR primers. These nucleotide codes are listed here. Some primer sequences may also include the character "I", which codes for the non-standard nucleotide

inosine

. Inosine occurs in tRNAs and will pair with adenine, cytosine, or thymine. This character does not appear in the following table, however, because it does not represent a degeneracy. While inosine can serve a similar function as the degeneracy "D", it is an actual nucleotide, rather than a representation of a mix of nucleotides that covers each possible pairing needed.

See also

*

Biology

*

Chromosome

* Gene
* Genetics
*
*
*

References

Further reading

*
*Freisinger, E., & Sigel, R. K. (2007)
From nucleotides to ribozymes—a comparison of their metal ion binding properties
''Coordination Chemistry Reviews'', ''251''(13-14), 1834-1851.

External links

Abbreviations and Symbols for Nucleic Acids, Polynucleotides and their Constituents
(IUPAC)

Provisional Recommendations 2004
(IUPAC)

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Nucleotides, *
DNA
Molecular biology