Biosynthesis of Purines and Pyrimidines | An important part of cellular metabolism

Biosynthesis of Purines

Image Credit : National Human Genome Research Institute (NHGRI) from Bethesda, MD, USA, Nanopore Sequencing (27820412495)CC BY 2.0

Biosynthesis of Purines and Pyrimidines | Biosynthesis of Nucleotides

Topics on Biosynthesis and Biotechnology

Contents

Nucleotide Biosynthesis

The pathways for the biosynthesis are classified into two different types: de novo pathway and salvage pathway. In the de novo pathways; nucleotide bases are synthesized from some simple compounds. The basic framework structure of the pyrimidine base is synthesized first and then gets attached to the ribose sugar. However, the purine base’s framework structure is synthesized in parts directly on a ribose sugar-based structure. 

5-phosphoribosyl-1-pyrophosphate (PRPP) + Amino acids + ATP + CO2 —> Nucleotide

In the salvage pathways, pre-formed bases are obtained, rearranged and rearranged on a ribose sugar unit. 

5-phosphoribosyl-1-pyrophosphate (PRPP) + Base —> Nucleotide

Both the salvage and de novo pathways operate to synthesize ribonucleotides. All the deoxyribonucleotides are produced from their corresponding ribonucleotides. The deoxyribose sugars are produced by the process of reduction of the ribose sugar present in a fully formed nucleotide. Moreover, methyl group that differentiate the thymine from the uracil (present in DNA and RNA respectively) is introduced in the last step of pathway. 

de novo synthesis of Pyrimidine ribonucleotide

In de novo synthesis for pyrimidines, origination of the basic structural framework ring is the first step. After this the ring gets attached to a ribose sugar to produce a pyrimidine nucleotide. 

Biosynthesis of Purines
(Biosynthesis of Pyrimidines): Basic Framework Structure of Pyrimidine Image credit : JyntoPyrimidine 2D numbers, marked as public domain, more details on Wikimedia

The ring of Pyrimidine is synthesized from aspartate and carbamoyl phosphate. Bicarbonate and ammonia are the precursors of carbamoyl phosphate. The synthesis of carbamoyl phosphate takes place by the utilization of bicarbonate and ammonia in a multistep process, with the utilization of two ATP molecules. This reaction is facilitated  cytosolic carbamoyl phosphate synthetase II

Carbamoyl phosphate behaves with aspartate to synthesize carbamoyl aspartate. This reaction is facilitated by aspartate transcarbamoylase. Carbamoylaspartate later undergo cyclization to produce dihydroorotate, it is then concerted into orotate by the process of oxidation.

5-phosphoribosyl-1-pyrophosphate (PRPP) + Base —> Nucleotide

Both the salvage and de novo pathways lead to the synthesis of ribonucleotides

Biosynthesis | Biosynthesis of Pyrimidines
Figure: Biosynthesis of Pyrimidines Image credit : BorisTM at English WikipediaNucleotides syn2, marked as public domain, more details on Wikimedia Commons

Orotate then attaches to the ribose, which is present in the form of PRPP. This is the activated form of ribose that is available for accepting nucleotide bases (PRPP is formed from the ribose-5-phosphate by following the pentose phosphate pathway after accepting pyrophosphate from ATP molecule). Orotate combines with PRPP to form a pyrimidine nucleotide orotidylate (OMP). The hydrolysis of pyrophosphate drives this reaction.

The enzyme named orotate phosphoribosyltransferase catalyzes the reaction requiring the production of orotidylate. This enzyme’s function is similar to the other phosphoribosyl transferases that add different groups to PRPP for the formation of other nucleotides. This orotidylate later decarboxylates to produce uridylate (UMP). UMP is an important pyrimidine nucleotide and a precursor to RNA. This reaction occurs in the presence of enzyme orotidylate decarboxylase. 

Synthesis of Cytidine (pyrimidine ribonucleotide)

Cytidine is synthesized from the uracil base of the UMP. Prior to the cytidine formation, UMP is transformed into UTP. Nucleoside monophosphates (NMP) are converted into nucleoside triphosphates (NTP) in the following reaction steps:

– Nucleoside monophosphates (UMP) are converted into nucleoside diphosphates (UDP) and then nucleoside triphosphates (UTP).

– The UTP formed can be converted into cytidine triphosphate (CTP) by displacing the carbonyl group with the amino group. 

Reduction of ribonucleotides to form deoxyribonucleotides

The precursors of Deoxyribonucleic acid (DNA) are deoxyribonucleotides; the reduction of ribonucleoside diphosphate forms these. This conversion is catalyzed by ribonucleotide reductase. The electrons are later transferred from NADPH to sulfhydryl groups or thiol groups present at the active site of the enzyme. This electron transfer is mediated by the help of proteins like thioredoxin and glutaredoxin. The dUMP is converted into dTMP by the addition of a methyl group. The methylene group and a hydride in this reaction is provided by N5, N10-methylenetetrahydrofolate. Later this N5, N10-methylenetetrahydrofolate transforms into dihydrofolate. Furthermore, this dihydrofolate undergoes reduction in the presence of NADPH to produce tetrahydrofolate. This reaction is facilitated by an enzyme known as dihydrofolate reductase.

Chemotherapeutic agents like methotrexate (amethopterin) and aminopterin inhibit the activity of dihydrofolate reductase. This folate analogue acted as a competitive inhibitor.

Purine ribonucleotide

The purine ring is assembled from a variety of precursors:

– Glutamine (N3 and N9)

– Glycine (C4, C5 and N7)

– Aspartate (N1)

– N10-formyltetra-hydrofolate (C2 and C8)

– CO2 (C6)

Biosynthesis of Purines
(Biosynthesis of Purines): Basic Framework Structure of Purine
Image credit :NEUROtikerPurin num2, marked as public domain, more details on Wikimedia Commons

de novo synthesis of purine (Biosynthesis of purines)

De novo synthesis of purine (Biosynthesis of purines) begins with simple substances such as bicarbonate and amino acids. The purine bases are assembled onto a ribose ring unlike pyrimidines,

Like pyrimidine biosynthesis, de novo purine biosynthesis, requires PRPP. However in the case of purines, PRPP gives the platform on which the nitrogenous bases are synthesized in multiple steps. In the first step, displacement of pyrophosphate takes place through ammonia instead of a preassembled base for producing 5-phosphoribosyl-1-amine. 

Glutamine PRPP amidotransferase catalyzes this reaction, which prevents wasteful hydrolysis of both the substrates. The enzyme amidotransferase considers the conformation of the active only for binding of PRPP and glutamine. This enzyme’s activity is inhibited by glutamine analogue azaserine, which as a result suppress the angiogenesis and malignancy.

Later, the addition of glycine, a series of formylation, amination and ring closure occurs. This series of reactions results into the formation of 5-aminoimidazole ribonucleotide. This 5-aminoimidazole ribonucleotide has the completed five-membered ring of the purine framework. The addition of carbon dioxide and a nitrogen atom from aspartate along with a formyl group takes part in ring closure or cyclization event. This ultimately forms inosinate (IMP) that is a purine ribonucleotide.

Biosynthesis of Purines
(Biosynthesis of Purines): de novo Biosynthesis of Purines
Image credit : BorisTM at English WikipediaNucleotides syn1, marked as public domain, more details on Wikimedia Commons

De novo purine biosynthesis proceeds as mentioned in the following steps:

  • The phosphorylation process activates the carboxylate group of a glycine. Glycine later couples with the amino group of 5-phosphoribosyl-1-amine. As a consequence a new amide bond comes into existence and the glycine (amino group) behaves as a nucleophile in the subsequent reaction steps.
  • The activated formate is then added to the amino group of glycine to produce formylglycinamide ribonucleotide. In few organisms, two different enzymes are involved in the catalysis of this step. One enzyme is involved in the transfers the formyl group while other enzyme initiates formate to form formyl phosphate. Formyl phosphate is then added to the amino group of glycine (source of formyl group is N10-formyltetrahydrofolate)
  • Amide group is then activated and converted into an amidine by the addition of ammonia (Source of ammonia in this step is glutamine).
  • The cyclization of Formylglycinamide ribonucleotide occurs to form a five-membered imidazole ring. This imidazole ring is characteristic of purines. This cyclization process is thermodynamically favourable and feasible.
  • The irreversibility of this reaction ensured by the consumption of one ATP molecule.
  • Bicarbonate undergoes phosphorylation and then reacts with exocyclic amino group. The product formed in the previous reaction then rearranges and transfers its carboxylate group to imidazole ring. Moreover, mammals do not need ATP for this step. Bicarbonate attaches to the exocyclic amino group, later it gets transferred to the imidazole ring.
  • The imidazole carboxylate group is further phosphorylated, and the aspartate’s amino group replaces the phosphate. This, a six-step reaction cascade links glycine, formate, ammonia, bicarbonate and aspartate to produce reaction intermediate that contains all but two of the atoms required for the formation of the purine ring.

Three more steps complete the ring synthesis. Fumarate, which is an intermediate in the Kreb’s cycle, is then removed, which in result facilitates the joining of nitrogen atom from aspartate to the imidazole ring. The amino group donated by the aspartate and the simultaneous removal of the fumarate stimulate the transformation of citrulline into arginine. Homologous enzymes are required to catalyze these steps into the two pathways. A formyl group is added to the nitrogen atom (the source of formyl group is N10-formyltetrahydrofolate) to form a terminal intermediate which triggers the cyclization process with the elimination of water molecules to form inosinate.

Formation of AMP and GMP

This IMP converts to either AMP or GMP is carried out in a two-step pathway completed at the expense of energy. (The synthesis of AMP requires GTP as their energy source, while GMP synthesis requires ATP). 

IMP —> XMP —> GMP

IMP is converted into XMP (xanthosine monophosphate) by tha e action of IMP dehydrogenase (utilize NAD as co-factor)

XMP isfurther converted into GMP (Guanosine monophosphate) by the action of XMP-glutamine amidotransferase.

IMP —> Adenylosuccinate —> AMP

IMP is converted into Adenylosuccinate by the action of enzyme Adenylosuccinate synthetase. Adenylosuccinate is further converted into AMP (Adenosine monophosphate) by the action of enzyme Adenylosuccinate Lyase.

Conversion of (NMP) nucleoside monophosphates to (NDP) nucleoside diphosphates and triphosphates (NTP). 

Nucleoside diphosphates (NDP) are synthesized from their corresponding nucleoside monophosphates (NMP) using a base-specific enzyme such as nucleoside monophosphate kinases. But, these kinases do not discriminate between ribose and deoxyriboses in the substrates. Generally, ATP is the main source of transferred phosphate since it is available in higher concentrations inside the cells as compared to the other nucleoside triphosphates.

For example, 

Adenylate kinase

AMP + ATP —> 2 ADP

Guanylate Kinase

GMP + ATP —> GDP + ADP

Nucleoside diphosphates (NDP) are converted into nucleoside triphosphates (NTP) by the action of nucleoside diphosphate kinase, this enzyme has broad specificity. Unlike the nucleoside monophosphate kinase (which has a narrow specificity). 

nucleoside diphosphate kinase helps in catalyzing both the following reactions:

GDP + ATP —> GTP + ATP

CDP + ATP —> CTP + ADP

Salvage pathways for biosynthesis of purines

Purines that are produced as a consequence of the degradation of nucleic acids inside the cell or that are obtained from the normal diet, but these purines can be again converted into (NTP) nucleoside triphosphates for reuse by the body. This process is known as salvage pathway for purines synthesis. This pathway involves two main enzymes: (APRT) adenine phosphoribosyltransferase and (HGPRT) hypoxanthine-guanine phosphoribosyltransferase. Both the enzymes utilize PRPP (which act as their prime source of ribose-5-phosphate).

APRT catalyze the reaction involving the formation of adenylate:

Adenine + PRPP —> Adenylate + PPi

HGPRT catalyzes the reaction involving the formation of inosinate (inosine monophosphate, IMP). It is a precursor molecule for synthesis of guanylate and adenylate.

Guanine + PRPP —> Guanylate + PPi

Hypoxanthine + PRPP —> inosinate + PPi

Similar salvage pathways exist for pyrimidines. Pyrimidine phosphoribosyl transferase will reconnect to uracil, but it does not connect cytosine, to PRPP.

Conclusions

Nucleotide biosynthesis which usually involves both biosynthesis of purines and pyrimidines takes place inside the cell as discussed in the article.

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FAQs

Q1. Purine vs pyrimidine (mark some differences between purines and pyrimidines)

Answer: Nitrogenous bases are broadly classified into two families; namely purines and pyrimidines. They are the building blocks or monomeric units of deoxyribonucleic acid (DNA) and Ribonucleic acid (RNA).

  • Purines are double ringed structures while pyrimidines contain a single ring. Both purines and pyrimidines are heterocyclic structures (Ring contains more than one type of constituent atoms).
  • Purines are of two basic types namely Adenine and Guanine. While, pyrimidines are of three basic types: Thymine, Cytosine and Uracil (it is present only in RNA in place of Thymine).
  • Purine degrades to form uric acid while pyrimidines break down to produce carbondi oxide, ammonia and beta amino acids.

Q2. Why Purine always base pair with pyrimidine

Answer: Due to the structural properties of the nitrogenous bases, purines and pyrimidines pairs with specificity. Adenine (A) always pairs with Thymine (T) whereas, Guanine (G) always pairs with Cytosine (C).

These combinations of nitrogenous bases have the tendency to form hydrogen bonds among them.

(A) Adenine forms two hydrogen bonds with (T) Thymine. Whereas, (G) Guanine forms three hydrogen bonds with (C) Cytosine.

About Dr. Abdullah Arsalan

I am Abdullah Arsalan , Completed my PhD in Biotechnology. I have 7 years of research experience. I have published 6 papers so far in the journals of international repute with an average impact factor of 4.5 and few more are in consideration. I have presented research papers in various national and international conferences. My subject area of interest is biotechnology and biochemistry with special emphasis on Protein chemistry, enzymology, immunology, biophysical techniques and molecular biology.

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