How Are Nucleotides Produced: Detailed Insights

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Nucleotides are the unit monomers of nucleic acids.

In vivo i.e. inside a cell, there are exactly two methods for nucleotide synthesis- salvage and de novo. Salvage is to break down old nucleic acids whereas by the de novo method we synthesize new nucleotide molecules.

So how are nucleotides produced? The anabolic process of biochemically combining a phosphate group, a pentose sugar(ribose or deoxy-ribose) and a nitrogenous base is called de novo nucleotide synthesis. On the other hand, Nucleic acid destruction is a catabolic process, from which parts can be salvaged by the salvage pathway to produce new nucleotides as well.

How is a nucleotide formed in DNA?

Nucleotides are made up of trimeric monomers called nucleotides. Long chains of these nucleotides make up nucleic acid polymers like DNA and RNA.

 DNA nucleotide is made up of 3 main components- a pentose sugar(deoxyribose), a phosphate group and a nitrogenous base. A total of four different nitrogenous bases can be found in DNA including- Adenine, Guanine, Thymine and Cytosine.

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DNA nucleotide with Adenine nucleobase
Image: Wikipedia

All nucleotides are made up of three separate chemical subunits: a five-carbon sugar molecule, a nucleobase (together called a nucleoside), and one phosphate group. Depending on the sugar and nitrogenous base we can differ the nucleotide of the 2 different nucleic acids.

Where do nucleotides come from?

Nucleotides can be produced in vitro(outside a living organism) or in vivo(inside a living organism).

 Scientists often use groups like phosphoramidite in the laboratory to make nucleotides in vitro. Nucleotides can be produced from scratch(de novo mechanism) in the body(in vivo) or recycled through salvage mechanisms.

The salvage pathway is one in which a biological product is made from reaction intermediates produced while getting rid of a biomolecule. Nucleotide salvage, in which nucleotides (purine and pyrimidine) are produced from intermediates that are being catabolized or degraded.

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Nucleic acid composition
Image: Wikipedia

Bases and nucleosides that are generated during the breakdown of RNA and DNA are recovered via nucleotide salvage processes. This is significant in some organs since some tissues cannot be synthesised from scratch. The nucleotides can then be made from the recovered goods. Drug research is focusing on salvage routes, one of which is known as antifolates.

Instead of recycling or partial breakdown of byproducts, the de novo pathway refers to the production of new complex molecular compounds from simple molecules such as sugars or amino acids. Nucleotides, for example, are not required in the diet since they may be made from tiny precursor molecules like formate and aspartate. Methionine is an essential amino acid as the body cannot synthesize it from scratch. Hence the only way of input is via our diet.

Nucleotide synthesis pathway:

As discussed above nucleotide synthesis mainly occurs by 2 methods:

Here we will discuss the 2 processes in detail.

DE NOVO NUCLEOTIDE SYNTHESIS:

 De novo is a Latin word that translates to “from the beginning.” It may also mean “anew,” “from scratch,” or “from the outset.” The de novo pathway enzymes use 5-phosphoribosyl-1-pyrophosphate (PRPP) to produce new purine and pyrimidine nucleotides from “scratch bottom” by making use of simple biomolecules like amino acids and tetrahydrofolate.

 In comparison to the salvage process, this mechanism of nucleotide synthesis has a high energy need. Five of the 12 stages of de novo purine synthesis, for example, need ATP or GTP hydrolysis, although just one salvage cycle process does.

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UMP synthesis
Image: Wikipedia

Both of these biosynthetic pathways have something in common-  the presence of some proteins characterized as “housekeeping enzymes”. However seeing that they are quite essential to cellular regulations, they are thought to be present in minute quantities in all living cells. While the de novo route enzymes are assumed to be found in plastids, salvage cycle enzymes might be found in several compartments.

Free nitrogenous bases like Adenine (A), Guanine (G), Cytosine (C), Thymine (T), and Uracil (U) are not used in the de novo nucleotide pathway. Throughout the process, the purine ring is constructed either one atom or a few atoms at a time and connected to ribose. The pyrimidine ring is formed by attaching orotate to ribose phosphate and then converting it to common pyrimidine nucleotides.

SALVAGE PATHWAY:

Bases and nucleosides are recovered from RNA and DNA degradation or external sources and converted back to nucleotides via nucleotide salvage processes. This is significant in some organs since some tissues cannot be synthesized from scratch. The nucleotides can then be made from the recovered goods. Drug research is focusing on salvage routes, one of which is known as antifolates.

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IMP synthesis pathway
Image: Wikipedia

A variety of nucleases break down nucleic acids into their constituent nucleotides. Nucleosides are further broken down by several nucleobases and phosphatases. The component bases are released in the third stage of hydrolysis by nucleosidases and nucleoside phosphorylases.

The steps for purine and pyrimidine synthesis are slightly different. Here we will discuss some of them:

  • Pyrimidine salvage pathway

In the case of Uracil: Pyrimidine-nucleoside phosphorylase or simply uridine phosphorylase just substitutes free uracil on the anomeric-carbon-bonded phosphate of ribose 1-phosphate with uridine.

 Uridine kinase (also known as uridine–cytidine kinase) can then phosphorylate the nucleoside’s 5′-carbon to produce uridine monophosphate (UMP). UMP/CMP kinase converts UMP to uridine diphosphate, which nucleoside diphosphate kinase converts to uridine triphosphate.

In the case of Cytidine: Both nucleoside cytidine and deoxycytidine are usually salvaged by the enzyme cytidine deaminase, and converted to uridine and deoxyuridine respectively. Alternatively, they can be phosphorylated by uridine–cytidine kinase into cytidine monophosphate (CMP) or deoxycytidine monophosphate (DMP) by uridine–cytidine kinase (d-CMP).

 The enzyme UM Pkinase or CMP kinase converts dCMP to cytidine diphosphate or deoxycytidine diphosphate. This cytidine diphosphate is converted to cytidine triphosphate or deoxycytidine triphosphate by nucleoside diphosphate kinase enzyme.

In the case of thymine: Thymidine is recycled to produce dTMP by an enzyme called Thymidine kinase. Thymidine phosphorylase or pyrimidine-nucleoside phosphorylase, adds a 2-deoxy-alpha-D-ribose 1-phosphate group to thymine, creating the deoxynucleoside thymidine, which occurs when thymine binds to the 5’ C of deoxyribose. Thymidine kinase then phosphorylates this compound’s 5′-carbon to produce thymidine monophosphate (TMP). TMP may be phosphorylated by thymidylate kinase into thymidine diphosphate, which can then be phosphorylated by nucleoside diphosphate kinase into thymidine triphosphate.

  • Purine salvage pathway

In the case of guanine: A guanosine kinase recycles guanosine to produce GMP. A guanosine phosphorylase may convert it to guanine, which could then be turned to GMP by a guanine phosphoribosyltransferase.

In the case of Adenine: An adenosine kinase might use adenosine directly in the production of AMP, or an adenosine nucleosidase and an adenine phosphoribosyltransferase could use adenine.

 In the biosynthesis of IMP, adenine might be recycled through a series of processes mediated by four enzymes:

  1. an adenosine phosphorylase yielding adenosine,
  2. an adenosine deaminase producing inosine,
  3. an inosine phosphorylase that yields hypoxanthine    and
  4. an IMP synthesized by hypoxanthine phosphoribosyltransferase.

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