What are Nucleotides?

What are Nucleotides?

Nucleotides are organic molecules that serve as the building blocks of nucleic acids, such as DNA (deoxyribonucleic acid) and RNA (ribonucleic acid). They are composed of three primary components: a nitrogenous base, a five-carbon sugar, and one or more phosphate groups.

• Nitrogenous Base: The nitrogenous base found in nucleotides may be divided into two groups: purines and pyrimidines. Adenine (A) and guanine (G), two purines, both have a double-ring structure. Cytosine (C), thymine (T), and uracil (U), which have a single-ring structure, are examples of pyrimidines.
• Sugar: The sugar component of a nucleotide can be either ribose or deoxyribose. Ribose is a five-carbon sugar found in RNA nucleotides, while deoxyribose is a modified form of ribose lacking an oxygen atom and is found in DNA nucleotides.
• Phosphate Group: Nucleotides may contain one or more phosphate groups attached to the sugar molecule. The phosphate group(s) can be linked to the 5'-carbon position of the sugar (known as a 5'-phosphate) or to the 3'-carbon position (known as a 3'-phosphate).

The structure of a nucleotide (Sergeenko et al., 2020).

The nucleotides that makeup DNA and RNA's backbone establish phosphodiester linkages between the sugar and phosphate groups. The order of nitrogenous bases throughout the chain of nucleotides determines the genetic code and provides guidelines for protein synthesis.

Apart from their role in nucleic acids, nucleotides also serve various other functions in cellular metabolism. They act as carriers of chemical energy in the form of adenosine triphosphate (ATP), participate in signaling pathways, and serve as coenzymes in enzymatic reactions.

Biosynthesis of Nucleotides

The step-by-step building of nucleotides from simple precursor molecules is the de novo biosynthesis of nucleotides. Purine synthesis and pyrimidine synthesis are the two main mechanisms via which it happens.

Purine Synthesis Pathway

As part of the pentose phosphate route, ribose-5-phosphate is converted into phosphoribosyl pyrophosphate (PRPP), which initiates the purine synthesis process. A series of enzymatic processes transform PRPP into inosine monophosphate (IMP) after that. Adenosine monophosphate (AMP) and guanosine monophosphate (GMP) are two purine nucleotides that are produced from IMP. Amino acids, carbon dioxide, and a number of cofactors are needed for the production of purine nucleotides.

Pyrimidine Synthesis Pathway

The de novo synthesis of pyrimidine nucleotides like cytidine monophosphate (CMP), uridine monophosphate (UMP), and thymidine monophosphate (TMP) is a step in the pyrimidine production pathway. The process begins with the creation of carbamoyl phosphate, which is subsequently joined with aspartate to create carbamoyl aspartate. Carbamoyl aspartate undergoes a number of enzymatic processes to produce orotate, which is then transformed into UMP.

Salvage Pathways

In addition to de novo biosynthesis, cells can also acquire nucleotides through salvage pathways. Salvage pathways recycle nucleosides and free bases, salvaging them to regenerate nucleotides without the need for de novo synthesis. This process is energetically favorable and allows cells to efficiently utilize nucleotide precursors.

Purine Salvage Pathway

In the purine salvage pathway, nucleosides such as adenosine and guanosine are taken up by cells and converted into nucleotides through the action of specific enzymes. These salvage enzymes include adenosine kinase, guanosine kinase, and hypoxanthine-guanine phosphoribosyltransferase (HGPRT). The purine salvage pathway is particularly important in cells with high nucleotide turnover rates, such as rapidly dividing cells.

Pyrimidine Salvage Pathway

The pyrimidine salvage pathway involves the uptake of pyrimidine nucleosides, such as cytidine and uridine, followed by their conversion into nucleotides. Enzymes such as cytidine deaminase and uridine kinase catalyze these reactions. The pyrimidine salvage pathway allows cells to efficiently recycle and regenerate pyrimidine nucleotides.

Degradation of Nucleotides

Nucleotides can undergo degradation processes to maintain cellular nucleotide homeostasis and eliminate excess or damaged nucleotides. Degradation pathways differ for purine and pyrimidine nucleotides.

Purine Degradation

Purine nucleotides can be degraded into free bases, such as hypoxanthine, xanthine, and uric acid. This process occurs through a series of enzymatic reactions, including nucleotide phosphorylase, nucleotidase, and xanthine oxidase. The end product, uric acid, is excreted from the body in humans and some primates, while in other mammals, it is further degraded to more soluble compounds.

The purine nucleotide degradation pathway in the CNS (Eric et al., 2018).

Pyrimidine Degradation

Pyrimidine nucleotides undergo degradation through a series of enzymatic reactions. The degradation pathway differs for uracil and thymine. Uracil is converted to β-alanine and β-aminoisobutyric acid, while thymine is converted to β-aminoisobutyric acid and β-alanine. These degradation products can be further utilized in other metabolic pathways or excreted from the body.

Regulation of Nucleotide Metabolism

The biosynthesis, salvage, and degradation pathways of nucleotides are tightly regulated to maintain cellular nucleotide pools and meet the demands of DNA and RNA synthesis. Several key regulatory mechanisms exist to ensure balanced nucleotide metabolism.

Feedback Inhibition

Enzymes involved in nucleotide biosynthesis are subject to feedback inhibition by the end products of the pathway. For example, the final products of purine synthesis, AMP and GMP, can inhibit enzymes earlier in the pathway, ensuring that nucleotide synthesis is tightly regulated.

Allosteric Regulation

Allosteric regulation plays a significant role in controlling nucleotide metabolism. Enzymes involved in nucleotide biosynthesis and degradation can be allosterically regulated by various effectors, including nucleotides themselves and other metabolites. Allosteric regulation allows for fine-tuning of enzyme activity in response to cellular demands.

Hormonal Control

Hormones, such as insulin and glucagon, can influence nucleotide metabolism. For example, insulin promotes nucleotide biosynthesis by activating key enzymes in the pathway. Hormonal control ensures that nucleotide metabolism is coordinated with other metabolic processes and responds to physiological changes.

Genetic Regulation

Gene expression plays a crucial role in nucleotide metabolism. Transcription factors and regulatory elements control the expression of genes involved in nucleotide biosynthesis, salvage, and degradation. This genetic regulation allows for the adaptation of nucleotide metabolism in response to developmental, environmental, and cellular cues.

Analytical Techniques in Nucleotide Metabolomics Analysis

Creative Proteomics utilizes various cutting-edge analytical techniques to achieve accurate and comprehensive nucleotide metabolomics analysis. These techniques include:

Liquid Chromatography (LC):

• Reversed-Phase Chromatography: This is the most common LC mode used in nucleotide metabolomics analysis. It separates nucleotides based on their hydrophobicity, with more hydrophobic nucleotides eluting later. It utilizes a nonpolar stationary phase and a polar mobile phase.
• Ion-Exchange Chromatography: This mode separates nucleotides based on their charge. Positively charged nucleotides bind to a negatively charged stationary phase, while negatively charged nucleotides bind to a positively charged stationary phase. Elution is achieved by changing the ionic strength or pH of the mobile phase.
• Size-Exclusion Chromatography: Also known as gel filtration chromatography, this mode separates nucleotides based on their size. Larger nucleotides elute first, as they are excluded from the pores of the stationary phase, while smaller nucleotides penetrate the pores and elute later.

Mass Spectrometry (MS):

• Electrospray Ionization (ESI): ESI is widely used in nucleotide metabolomics analysis. It involves the ionization of nucleotides in solution by applying a high voltage, generating gas-phase ions. ESI can produce multiply charged ions, allowing for accurate mass measurements and improved sensitivity.
• Matrix-Assisted Laser Desorption/Ionization (MALDI): MALDI is an alternative ionization technique that involves embedding nucleotide samples in a matrix material and subjecting them to laser irradiation. This generates ions from the sample, which can then be detected and analyzed by the mass spectrometer.
• Tandem Mass Spectrometry (MS/MS): MS/MS is employed to obtain structural information about nucleotides and their fragments. It involves isolating a precursor ion of interest, fragmenting it, and analyzing the resulting fragments. This technique aids in the identification and confirmation of nucleotide structures.

Nuclear Magnetic Resonance (NMR) Spectroscopy:

• ¹H NMR Spectroscopy: ¹H NMR spectroscopy is commonly used in nucleotide metabolomics analysis. It detects the signals arising from the protons in nucleotides, providing valuable structural and quantitative information.
• ¹³C NMR Spectroscopy: ¹³C NMR spectroscopy is less commonly used but can provide additional structural information by detecting carbon-13 isotopes in nucleotides. It is particularly useful for studying carbon metabolism and metabolic flux analysis.

References

1. Sergeenko, Anna, Maria Yakunina, and Oleg Granichin. "Hamiltonian path problem solution using DNA computing." Cybernetics and Physics 9.1 (2020): 69-74.
2. Eric Nybo, S., and Jennifer T. Lamberts. "Integrated use of LC/MS/MS and LC/Q-TOF/MS targeted metabolomics with automated label-free microscopy for quantification of purine metabolites in cultured mammalian cells." bioRxiv (2018): 490300.

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