22.4: Biosynthesis and Degradation of Nucleotides

Search Fundamentals of Biochemistry

 

 

Learning Goals (ChatGPT o3-mini)

1. Describe the Biological Importance of Nucleotides

   • Explain how nucleotides serve dual roles as building blocks for nucleic acids and as key carriers of biological energy (e.g., ATP, GTP) and signaling molecules.
   • Discuss the diverse metabolic fates of amino acids in relation to nucleotide synthesis and degradation.

2. Identify the Precursors and Sources of Atoms in Nucleotide Synthesis

   • Summarize the contributions of amino acids—such as glutamine, aspartate, glycine, and serine—to the synthesis of purine and pyrimidine rings.
   • Interpret Figure 1 to explain the origin of atoms in the nucleotide bases.

3. Compare and Contrast Purine and Pyrimidine Biosynthesis Pathways

   • Outline the key steps and enzymes involved in the de novo purine biosynthesis pathway (leading to IMP) including the role of multifunctional enzymes (e.g., TGART, PAICS, ATIC) and the importance of PRPP.
   • Explain how the salvage pathway recycles purine bases and how enzymes like APRT and HGPRT contribute to nucleotide homeostasis.
   • Describe the de novo pyrimidine biosynthesis pathway, highlighting how the nucleobase is synthesized directly as a ribonucleotide and the role of key substrates such as aspartate, carbamoyl phosphate, and 5,10-meTHF.

4. Understand the Regulation of Nucleotide Biosynthesis

   • Discuss the rate-limiting steps in both purine (e.g., PPAT) and pyrimidine (e.g., CAD complex) synthesis and how these pathways are regulated by substrate availability, feedback inhibition, and enzyme phosphorylation.
   • Explain the concept of the purinosome as a dynamic multienzyme complex that facilitates de novo purine synthesis and how its assembly is coordinated with mitochondrial metabolism.

5. Outline Nucleotide Interconversion and Deoxynucleotide Formation

   • Describe the enzymatic conversion of ribonucleotides to deoxynucleotides, emphasizing the role of ribonucleotide reductase (RNR) and its free-radical mechanism.
   • Recognize the importance of deoxynucleotide synthesis in DNA replication and repair, and how RNR activity is regulated through both allosteric effectors and transcriptional control.

6. Summarize the Pathways for Nucleotide Degradation

   • Detail the catabolic routes for purine degradation, including the conversion of nucleotides to uric acid.
   • Summarize the degradation pathway for pyrimidines and how these products are further processed and excreted.
   • Relate nucleotide turnover and salvage pathways to overall cellular metabolism and energy balance.

7. Integrate Nucleotide Metabolism with Cellular Function and Disease

   • Explain how disruptions in nucleotide synthesis, salvage, or degradation can affect energy production, signal transduction, and gene regulation.
   • Discuss the role of nucleotide metabolism in rapidly proliferating cells (e.g., cancer cells) and how targeting these pathways can be a therapeutic strategy.

These learning goals aim to equip you with a comprehensive understanding of the metabolic pathways of nucleotides, highlighting their synthesis from amino acids, regulatory mechanisms, and their critical roles in cellular physiology and pathology.

Introduction

We conclude our exploration of metabolic pathways with the biosynthesis and breakdown of nucleotides, the monomers that comprise nucleic acids. We cannot forget the important role of ATP as the universal carrier of biological free energy, as well as the nucleotides involved in signal transduction, such as GTP in heterotrimeric G proteins and small G proteins, and ATP as a substrate in protein phosphorylation by kinases. As with the other later sections on metabolism, we won't focus much on detailed reaction mechanisms or enzyme structures, with one exception: the enzyme that converts nucleotides to deoxynucleotides.

Nucleotide synthesis is often included in chapters on amino acid metabolism as almost every atom in the purine and pyrimidine ring derives from them, as shown in Figure \(\PageIndex{1}\).

Figure \(\PageIndex{1}\): Source of atoms in nucleotide bases. Lieu, E.L., Nguyen, T., Rhyne, S., et al. Amino acids in cancer. Exp Mol Med 52, 15–30 (2020). https://doi.org/10.1038/s12276-020-0375-3. Creative Commons Attribution 4.0 International License, http://creativecommons.org/licenses/by/4.0/.

For purines, glutamine and aspartate provide the nitrogen for the nucleotide rings. They also provide the NH₃s for the ring substituents (glutamine for adenine and aspartate for guanine). Glycine and formate provide the carbon atoms for the rings. The one-carbon molecule formate, which derives from glycine, is also added to purine rings. Glycine provides a one-carbon unit indirectly through the main carrier of activated one-carbon units, 5,10-meTHF, which is converted to formate through 10-formyl THF.

Pyrimidines are much smaller, and their synthetic pathway reflects that. Instead of being synthesized as nucleobases, as in the case of purines, they are formed as ribonucleotides by being linked to phosphoribosyl pyrophosphate (PRPP). Glutamine and aspartate again provide the ring C and N atoms. The one-carbon unit derives from serine-to-glycine conversion. A methyl group from the activated 1C donor, 5,10-meTHF, is added to dUMP to make dTMP.

Purine Synthesis

The material below derives from De Vitto, H.; Arachchige, D.B.; Richardson, B.C.; French, J.B. The Intersection of Purine and Mitochondrial Metabolism in Cancer. Cells 2021, 10, 2603. https://doi.org/10.3390/cells10102603. Creative Commons Attribution License

Mammals have two pathways for purine synthesis: a de novo pathway and a salvage pathway, which allows for the recycling of nucleotide bases. The salvage pathway is typically sufficient, as purine bases are derived from nucleic acid breakdown. The resulting free bases (adenine, guanine, and hypoxanthine are connected to phosphoribosyl pyrophosphate (PRPP) to form nucleoside monophosphates (NMP) using either adenine phosphoribosyltransferase (APRT) to form AMP or hypoxanthine-guanine phosphoribosyltransferase (HGRT) to form IMP and GMP. PRPP is a substrate in both the salvage and de novo pathways. The overall de novo and salvage pathways for purine synthesis are described in detail in Figure \(\PageIndex{2}\).

Figure \(\PageIndex{2}\): Purine metabolic pathways. De Vitto, H.; Arachchige, D.B.; Richardson, B.C.; French, J.B. The Intersection of Purine and Mitochondrial Metabolism in Cancer. Cells 2021, 10, 2603. https://doi.org/10.3390/cells10102603. Creative Commons Attribution License

The conserved de novo biosynthesis pathway for generating IMP consists of 10 chemical steps, catalyzed by six gene products in humans. These include the trifunctional enzyme TGART, composed of GAR synthetase (GARS), GAR transformylase (GARTfase), and AIR synthetase (AIRS) domains; the bifunctional enzymes PAICS, composed of CAIR synthetase/AIR carboxylase (CAIRS) and SAICAR synthetase (SAICARS), and ATIC, composed of AICAR transformylase (AICART) and IMP cyclohydrolase (IMPCH); and three monofunctional enzymes, phosphoribosyl amidotransferase (PPAT), formyl glycin amidine ribonucleotide synthetase (FGAMS), and adenylosuccinate lyase (ADSL). Downstream IMP is converted to (1) GMP through stepwise reactions of IMP dehydrogenase (IMPDH) followed by GMP synthetase (GMPS) and (2) AMP via adenylosuccinate synthetase (ADSS) followed by ADSL. The salvage pathway requires PRPP to generate IMP and GMP through one-step reactions mediated by hypoxanthine phosphoribosyltransferase (HPRT), utilizing hypoxanthine and guanine bases. AMP is generated by adenine phosphoribosyltransferase (APRT), which utilizes the adenine base and PRPP as substrates. Mitochondria supply precursors for purine de novo biosynthesis, including glycine, N10-formyl THF, and aspartic acid, through their one-carbon cycle (1C cycle) and tricarboxylic acid cycle (TCA).

The de novo pathway kicks in when there is high demand for purines. Six enzymes are required for the 10-step pathway. Three of these are multifunctional enzymes catalyzing multiple steps in the pathway, comprising the two bifunctional enzymes phosphoribosylaminoimidazole carboxylase (PAICS) and AICAR transformylase/inosine monophosphate cyclohydrolase (ATIC), and the trifunctional enzyme glycinamide ribonucleotide transformylase (TGART). When active, the pathway is limited by both substrate availability and the reaction rate of its initial step, the conversion of PRPP to phosphoribosylamine (PRA) by phosphoribosylpyrophosphate amidotransferase (PPAT). The final product of the de novo biosynthesis pathway, IMP, is a substrate for the production of both AMP and GMP. Six ATPs are used to make one IMP from PRPP. None are required in the salvage pathway.

PPAT is also called Glutamine phosphoribosylpyrophosphate amidotransferase or amidophosphoribosyltransferase.  It catalyzes the rate-limiting step and is tightly regulated. PPAT possesses two nucleotide-binding sites near the active site, allowing for feedback control by downstream purine nucleotides via allosteric inhibition.

Figure \(\PageIndex{3}\) shows an interactive iCn3D model of the Glutamine Phosphoribosylpyrophosphate Amidotransferase from Arabidopsis thaliana (6LBP)

Figure \(\PageIndex{3}\): Glutamine Phosphoribosylpyrophosphate Amidotransferase from Arabidopsis thaliana (6LBP). (Copyright; author via source). Click the image for a popup or use this external link:  https://structure.ncbi.nlm.nih.gov/i...uWnf1bTq1odk88

The plant enzyme is a homotetramer, with each subunit having a Fe₄S₄ center (spacefill).  The active site residues in each subunit (DDS 432-444 and DS 369-370) are shown as colored sticks and labeled.

Regulation of IMP production also occurs through enzyme phosphorylation. For example, Thr 397 on PPAT is phosphorylated by protein kinase B (PKB). PRPP concentrations also affect the rate. Another regulation of the flux through the de novo pathway involves the condensation of enzymes into the purinosome, which contains PPAT, TGART, formylglycinamidine ribonucleotide synthetase (FGAMS), PAICS, adenylosuccinate lyase (ADSL), and ATIC. The purinosome also interacts with the mitochondria, allowing for high local concentrations of ATP.

A cartoon view of the purinosome is shown in Figure \(\PageIndex{4}\).

Figure \(\PageIndex{4}\): Cartoon showing the purinosome. Baresova et al (2018) PLoS ONE 13(7): e0201432. https://doi.org/10.1371/journal.pone.0201432. Creative Commons Attribution License,

Figure \(\PageIndex{5}\) shows another view of de novo IMP synthesis in which the origin of each atom in the purine ring is shown in color.

Figure \(\PageIndex{5}\): De novo IMP synthesis showing the origin of atoms in IMP.

Figure \(\PageIndex{6}\) shows an expanded view of the conversion of IMP to GTP and ATP.

Figure \(\PageIndex{6}\): Conversion of IMP to GTP and ATP

Pyrimidine Synthesis

As mentioned in the introduction, pyrimidines have a much simpler biosynthetic pathway. Instead of being synthesized as nucleobases as in the case of purines, they are made as ribonucleotides as they are linked to phosphoribosyl pyrophosphate (PRPP). Glutamine and aspartate again provide the ring C and N atoms. The one-carbon unit derives from serine-to-glycine conversion. A methyl group from the activated 1C donor, 5,10-meTHF, is added to dUMP to make dTMP.

The first step in the pathway for pyrimidine synthesis is the condensation of aspartate and carbamoyl phosphate. We have seen the synthesis of carbamoyl phosphate in the urea cycle by the enzyme carbamoylphosphate synthase I (CPSI) in Chapter 18.3 but present the reaction again in Figure \(\PageIndex{7}\).

Figure \(\PageIndex{7}\): Synthesis of carbamoyl phosphate

A different cytosolic version of the enzyme, CPS II, is used to synthesize both arginine and pyrimidine nucleotides. It uses glutamine as a donor of NH₃.

The pathway for the synthesis of UTP and CTP is shown in Figure \(\PageIndex{7}\). It does not explicitly show the synthesis of carbamoylphosphate, which is an integral part of the pathway and a rate-limiting step in pyrimidine synthesis.

Figure \(\PageIndex{8}\): De novo synthesis of UDP, UTP, and CTP

UDP and CDP can be converted to dCDP and dUDP, and then on to dCPT and dUTP, and to dTMP as shown in Figure \(\PageIndex{9}\).

Figure \(\PageIndex{9}\): Synthesis of dTMP

Some of the material below derives from Li et al. Int. J. Mol. Sci. 2021, 22(19), 10253; https://doi.org/10.3390/ijms221910253. Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/)

In purine synthesis, we observed multifunctional enzymes that catalyze multiple steps, as well as the assembly of these enzymes into the purinosome within the de novo pathway. Analogously, three different enzyme activities that catalyze the first three combined rate-limiting steps of pyrimidine synthesis, Carbamoyl-phosphate synthetase, Aspartate transcarbamoylase, and Dihydroorotase, are found in a single, multifunctional protein referred to as CAD. Its structure is a hexamer of a 243K monomer. It has four domains that include

• glutamine amidotransferase (GATase) which "moves" HCO₃⁻, glutamine, and ATP to the CPSIIase domain
• carbamoylphosphate synthetase II (CPSIIase): This has two parts, CPSaseA, and CPSase B. They combine functionally with GATase to form a glutamine-dependent carbamoylphosphate synthase (CPSase)
• aspartate transcarbamylase (ATCase) acts as a homotrimer
• dihydroorotase (DHOase) catalyzes the reversible cyclization reaction.

The CAD protein is a fusion protein that encodes these four enzymatic activities of the pyrimidine pathway. Figure \(\PageIndex{10}\) shows the domain structure of the CAD protein.

Figure \(\PageIndex{10}\): Domain structure of CAD

The red represents glutamine amidotransferase and the blue the carbamoyl phosphate synthase ATP binding domain. More specifically, the following amino acid stretches comprise the different domains: GATase (2-365), CPSase A (395-933), CPSase B (934-1455), DHOase (1456-1788), and ATCase (1918-2225)

Figure \(\PageIndex{11}\) shows an interactive iCn3D model of the AlphaFold predicted model of the CAD protein (P27708)

Figure \(\PageIndex{11}\): AlphaFold predicted model of the CAD protein (P27708). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...5V2XUmfsz1qjZ6

The GATase domain is magenta, the CPSase domains are orange, the DHOase domain is yellow, and the ATCase domain is cyan. The structure of the disordered loops could not be modeled.

Ribonucleotide reductases (RNRs)

Ruskoski and. Boal. J. Biol. Chem. (2021) 297(4) 101137. DOI:https://doi.org/10.1016/j.jbc.2021.101137. CC-BY license (http://creativecommons.org/licenses/by/4.0/).

Ribonucleotide reductases (RNRs), also called ribonucleoside-diphosphate reductase, catalyze the oxidation of the C2'-OH on the ribose ring to C2'-H through a free radical mechanism for the oxidation of all NDPs, including ADP, GDP, CDP, and UDP (which converts to dUDP and in a different reaction to dTDP). The reaction is as follows:

[thioredoxin]-dithiol + ribonucleoside 5'-diphosphate ↔ [thioredoxin]-disulfide + 2'-deoxyribonucleoside 5'-diphosphate + H₂O

To refresh your memory, thioredoxin is a small protein (12 kDa) that is part of a complex with thioredoxin reductase and thioredoxin-interacting protein. It has two key sulfhydryls at the active site, which act as reducing agents as they are converted to a disulfide, as shown in Figure \(\PageIndex{12}\).

Figure \(\PageIndex{12}\): Reduced thioredoxin and its oxidized form

This single enzyme is so critical to cellular life that we will examine it more closely.

There are several classes of these enzymes, including Ia-Ie, II, and III. The class I enzymes generally use a di-transition metal complex as a cofactor, whereas class II enzymes use adenosylcobalamin. We will focus on class I enzymes, which have two subunits, designated as α and β, or M1 and M2, respectively. The NDP binds to an α/M1 subunit active site, which is only developed in the dimer. The β/M2 subunit is often referred to as the radical-generating subunit, as it contains the transition metal complex that generates a free tyrosyl radical cation, which is critical to the reaction.

Almost all class I ribonucleotide reductases (RNRs) use transition metal ions located in the β/M2 subunit in the catalytic cycle for the dehydroxylation of the 2' OH on the ribose ring of the nucleotide. The metal ion complex a β/M2 subunit tyrosine to a tyrosine free radical cation, which oxidizes an active site cysteine in the α/M1 subunit to form a thiol radical cation (Cys•⁺), called a thiyl radical. This abstracts a H• from the 3'C on the ribose of the substrate, forming a 3'C radical cation. This facilitates a dehydration reaction, which leads to the dehydroxylation of the 2' OH, regenerating the thiyl radical. Reducing equivalents to restore the catalytic function of the enzyme come from the oxidation of a thioredoxin disulfide bound in the other subunit of the protein or a formate.

Given the importance of these enzymes, they must be highly regulated. There are two regulatory sites:

• a specificity site: determines nucleotide (NDP) specificity
• an activity site: regulates catalytic activity

The specificity and activity sites are in the α/M1 subunit, where allosteric regulators dNTPs and ATP bind to different sites. When ATP is bound, the enzyme uses CDP and UDP as substrates. When dGTP is bound, ADP is the preferred substrate. Finally, when dTTP is bound, GDP is the preferred substrate. The enzyme is inhibited by dATP binding to the actual active site.

Figure \(\PageIndex{13}\)s shows an abbreviated mechanism and cartoon showing the activities of the two subunits.

Figure \(\PageIndex{13}\): Abbreviated mechanism and a cartoon showing the activities of the two subunits. in class I RNR. A, universal mechanism for nucleotide reduction in RNRs. B, diagram of the steps involved in radical translocation in class I RNRs. Ruskoski and. Boal. J. Biol. Chem. (2021) 297(4) 101137. DOI:https://doi.org/10.1016/j.jbc.2021.101137. CC-BY license (http://creativecommons.org/licenses/by/4.0/)

Radical formation starts at tyrosine 122 in the metal center site in the β/M2 subunit. Electron transfer then occurs across the two subunits from a very distant active site Cys 439 in the α subunit, which enables the formation of the thiyl radical cation (Cys•⁺).

The structure of an E. coli Type IA enzyme with bound ligands has been determined after considerable effort that involved trapping a long-lived intermediate. It required the replacement of Tyr 122 in both β chains with 2,3,5-trifluorotyrosine, which allowed the structure to be determined by cryo-EM. Tyr 122 in the β chain initiates the electron transfer process by becoming the Tyr 122^.+ radical cation.

A detailed mechanism showing both electron transfer to Y122^.+ and accompanying proton transfer is shown in Figure \(\PageIndex{14}\).

Figure \(\PageIndex{14}\): Pathway of electron and proton transfers for the formation of the thiyl radical cation (Cys•⁺). after Kang et al. Science (2020). DOI: 10.1126/science.aba6794)

The path for electron flow from the sulfur of C439 in the β subunit to regenerate Y122 is shown. That electron transfer occurs over a very long distance of 35 Å as it hops from the original sulfur donor to the acceptor.

Figure \(\PageIndex{15}\) shows an interactive iCn3D model of the holocomplex of E. coli class Ia ribonucleotide reductase with GDP and TTP (6W4X).

Figure \(\PageIndex{15}\): Holocomplex of E. coli class Ia ribonucleotide reductase with GDP and TTP (6W4X). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...71ubUDRovXURG8

The structure is a tetramer of two α chains (different shades of gray) and two β chains (different shades of cyan). The GDP and TTP in the α subunit, as well as the Fe cluster in the β subunit, are shown in CPK spacefill and labeled. The key side chains in the α and β chains that participate in electron transfer over the 35 Å distance are shown in CPK-colored sticks and labeled. The Fe²⁺ ions are in the form of a μ-oxo-diron complex (2 Fe ions coordinated by one oxide).

Figure \(\PageIndex{16}\) shows specificity and catalytic sites of the biologically active tetramer form for Class 1 RNR as well as the transition metal cofactors (Fe, Mn, or both) in various Class 1 RNRs.

Figure \(\PageIndex{16}\): Quaternary structure of the active holoenzyme complex in class I RNR (PDB accession code 6W4X). Insets show the location of the active site in the catalytic α subunit (middle top) and the metallo- or radical cofactor (middle bottom and far right) in the β subunit.

The tyrosyl free radical forms on binding of dioxygen to the transition metal ion center electron and subsequent loss of an electron from tyrosine, as shown in Figure \(\PageIndex{17}\):

Figure \(\PageIndex{17}\): Cofactor assembly mechanisms for class I RNRs. Manganese-dependent enzymes are highlighted in purple. Superoxide-dependent RNRs are highlighted in hot pink. Subclasses that require an NrdI activase are indicated with a yellow box. Metal-centered Cys oxidants are shown in green, and Tyr-derived radical Cys oxidants are shown in blue.

The mechanisms of cofactor actions are not fully understood. O₂ initially adds to the Fe²⁺/Fe²⁺ cluster, forming a peroxo-Fe³⁺/Fe³⁺ intermediate.

Structural features of the active site for different class I RNRs along with redox-active transition metal ions are shown in Figure \(\PageIndex{18}\).

Figure \(\PageIndex{18}\): Comparison of metal-binding sites in class I RNRs

The PDB codes for each structure are as follows: A (3N3A), B (4M1I), C (6CWP), and D (6EBO). Water molecules are shown as red spheres.

Now, let's examine the regulation of Class IA RNRs in more detail. Let's consider perhaps the most important allosteric regulators, which bind 15 Å from the active site and affect RNR enzymatic activity:

• dATP: inhibits RNRs when it binds to the α subunit
• ATP: reverses the inhibition by dATP

dATP/ATP also affects RNR enzyme specificity as they tilt the preference of RNR towards pyrimidine substrates, where TTP and dGTP promote purine substrate binding. These same rules apply to human and E. coli RNRs, with the locations of the active sites also being the same.

The allosteric regulators appear to affect the quaternary structure of the enzyme.

In E. Coli, the binding of dADP converts the structure of the enzyme from an active α₂β₂ form to an inhibited α₄β₄ ring structure. When dATP is bound, a "cone" domain in α forms interactions with the β subunit, leading to the formation of a dimer of the α₂β₂ tetramer. ATP reverses this effect by displacing dATP and pushing the equilibrium towards the active α₂β₂ form. This is shown in Panel A of Figure \(\PageIndex{19}\).

 

Figure \(\PageIndex{19}\): Comparison of mechanisms of allosteric regulation of activity for E. coli and human RNRs. Brignole et al. 3.3-Å resolution cryo-EM structure of human ribonucleotide reductase with substrate and allosteric regulators bound eLife 7:e31502. https://doi.org/10.7554/eLife.31502. Attribution 4.0 International (CC BY 4.0).

Panel B above illustrates the regulation of the human enzyme, which appears to be quite different. In the absence of either dATP or ATP, RNRs exist just as α₂ dimers. On binding either dATP or ATP, the α₂ dimer is converted to an α₆ (a trimer of dimers) structure. Both are inactive in the absence of β subunits (which provide the metal cofactor site)

• When β₂ is added to the dATP-bound α₆ hexamer, the hexamer becomes stabilized, but the resulting complex is inhibited.
• When β2 is added to the ATP-bound α6 hexamer, the hexamer becomes destabilized and breaks down into smaller, active structures.

Hence, the ratio of cellular dATP/ATP changes the aggregation state of the RNR and hence its activity in both E. Coli and humans.

Figure \(\PageIndex{20}\) shows an interactive iCn3D model of the Human ribonucleotide reductase large subunit (alpha) with dATP and CDP (6AUI).

Figure \(\PageIndex{20}\): Human ribonucleotide reductase large subunit (alpha) with dATP and CDP (6AUI). (Copyright; author via source). Click the image for a popup or use this external link:https://structure.ncbi.nlm.nih.gov/i...w1sFwwYSAD4nG6

Each of the α subunits in the hexamer is shown in a different color. dATPs (allosteric inhibitors) are shown in spacefill, red. CDPs (substrates) are shown in spacefill yellow.

The hole in the middle of the structure prevents β₂ from binding in a catalytically productive manner, rendering the structure inactive.

A specific loop, loop 2, assists in the determination of RNRs' specificity. It is between the base of dATP and the base of the substrate CDP. The backbone of the loop interacts with the adenine base and orients Gln 288 in the active site to interact with the cytosine of the substrate CDP. In both systems, backbone atoms of this loop ‘read out’ the adenine base and position Gln288 into the active site to recognize the cytosine of CDP. Figure \(\PageIndex{20}\) gives further details on the origin of substrate specificity.

Figure \(\PageIndex{21}\): Determinants of substrate specificity are conserved from E. coli to humans.

Panel (A) shows residues of human α (blue) interacting with CDP (carbons in orange) in the active site and dATP (carbons in yellow) in the specificity site. Density for CDP in the orange mesh and for dATP in the yellow mesh.

Panel (B) zooms in on dATP in the specificity site. Water molecules and oxygen atoms are in red, nitrogen in blue, magnesium in green, and phosphate in gold.

Panel (C) zooms in on CDP in the active site.

Panel (D) shows an overlay of human α from the α₆ EM structure in blue with E. coli α from the α₄β₄-CDP-dATP cocrystal structure in gray (PDB: 5CNS) shows a nearly identical loop 2 conformation positioning Gln288 and Arg293 (Gln294 and Arg298 in E. coli).

Panel (E) shows an overlay of human α from the α₆ EM structure in blue with the crystal structure of human α with N- and C-termini truncated (residues 77–742) cocrystallized with dATP in tan (PDB: 2WGH) shows similar positioning of dATP but an altered conformation of loop 2 in the absence of bound CDP. The CDP shown is from the α₆ EM structure.

Panel (F) shows an overlay of human α from the α₆ EM structure in blue with equivalent residues of yeast α structure with CDP and AMPPNP in brown (PDB: 2CVU) and shows a conformation of loop 2 that is distinct from that seen in structures of E. coli and human α

Transcriptional regulation of RNRs

Given the importance of this key enzyme, it should come as no surprise that its levels are regulated at the transcription level. As the activity of RNR is determined by its polymeric quaternary state, the transcriptional activation of the genes for RNRs is controlled in bacteria by quaternary states of the RNR-specific transcriptional repressor NrdR. The transcription factors bind to a specific DNA sequence called an NrdR box, which precedes the transcription start site for RNR, where RNA polymerase binds.

The NrdR protein acts to repress the synthesis of RNR genes. DATP/ATP ratios control its aggregation state. When dATP is high, the NrdR binds to DNA and represses the synthesis of the RNR gene. In contrast, when ATP is high, the protein does not bind to DNA, and hence it can not repress transcription. The aggregation state of NrdR controls the association /dissociation of the repressor. When abundant, NrdR exists as a 12-mer complex with two molecules of ATP bound per monomer. This ATP-bound 12-mer can't bind DNA, so transcription of the gene for RNR is not repressed. As dATP increases, one ATP is displaced, so each monomer has one dATP and one ATP bound. This causes the NrdR to covet to an 8-mer. A 4-mer (tetrameric) version of this protein binds to the NrdR box sequence at the start of the RNR gene, repressing its synthesis.

Figure \(\PageIndex{22}\) illustrates the dodecameric, octameric, and tetrameric structures of NrdR, along with their corresponding functions.

Figure \(\PageIndex{22}\): Mechanism of NrdR function involving dodecameric, octameric, and tetrameric structures. Rozman Grinberg, I., Martínez-Carranza, M., Bimai, O. et al. A nucleotide-sensing oligomerization mechanism that controls NrdR-dependent transcription of ribonucleotide reductases. Nat Commun 13, 2700 (2022). https://doi.org/10.1038/s41467-022-30328-1. Creative Commons Attribution 4.0 International License. http://creativecommons.org/licenses/by/4.0/.

Panel (a) shows a surface representation of the cryo-EM maps for the dodecameric, octameric, and DNA-bound tetrameric NrdR structures.

Panel (b) shows a cartoon representation of the ATP-loaded NrdR tetramer (left) and the dATP/ATP-loaded tetramer (right). Chains A, B, C, and D are colored beige, green, pink, and blue, respectively.

Panel (c) shows the interface between the ATP cones in chain A (beige) and chain B (green) in the ATP-loaded dodecamer.

Panel (d) shows the dATP/ATP-loaded tetramer.

Panels c, and d were made from the same perspective, based on an alignment of the ATP-cones in chains A and B in both structures.

Figure \(\PageIndex{23}\) shows an interactive iCn3D model of the Streptomyces coelicolor dATP/ATP-loaded NrdR in complex with its cognate DNA (7P3F).

Figure \(\PageIndex{23}\): Streptomyces coelicolor dATP/ATP-loaded NrdR in complex with its cognate DNA (7P3F). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...2u3HMRPmJHe9X9

The monomers of the NrdR tetramer are shown in different colors. The yellow spheres represent dATP, and the orange ones represent ATP.

Pyrimidine Salvage Pathway

There is also a salvage pathway as shown in Figure \(\PageIndex{24}\).

Figure \(\PageIndex{24}\): Pyrimidine salvage pathway (after Wang et al. Frontiers in Oncology, 11 (2021). https://www.frontiersin.org/article/...nc.2021.684961. DOI=10.3389/fonc.2021.684961

Cells at rest utilize the salvage pathway's reactants, which are derived from the degradation of nucleic acids.

Nucleotide Degradation

Purines

The pathway for purine degradation is shown in Figure \(\PageIndex{25}\).

Figure \(\PageIndex{25}\): Purine degradation

Pyrimidine Degradation

The pathway for pyrimidine degradation is shown in Figure \(\PageIndex{26}\).

Figure \(\PageIndex{26}\): Pyrimidine degradation

Summary

This chapter concludes our exploration of metabolic pathways by focusing on the biosynthesis, interconversion, and degradation of nucleotides—the essential building blocks of nucleic acids—and their critical roles in energy metabolism and signal transduction.

1. Importance and Roles of Nucleotides

• Fundamental Functions:
  Nucleotides not only serve as the monomeric units of DNA and RNA but also function as universal energy carriers (e.g., ATP, GTP) and key signaling molecules. Their metabolism is intricately connected with the overall energy status and regulatory mechanisms of the cell.

• Integration with Amino Acid Metabolism:
  Many atoms in the purine and pyrimidine rings are derived from amino acids (e.g., glutamine, aspartate, glycine, serine). This link emphasizes the interconnectedness of amino acid and nucleotide metabolism.

2. Purine Biosynthesis

• De Novo Pathway:
  The purine de novo synthesis pathway builds the purine ring on a ribose-phosphate backbone (derived from PRPP) in a series of 10 steps catalyzed by multifunctional enzymes (such as the trifunctional TGART, bifunctional PAICS and ATIC, along with several monofunctional enzymes). Key nitrogen atoms come from glutamine and aspartate, while glycine and one-carbon units (from 5,10-methylenetetrahydrofolate) contribute essential carbon atoms. The end product, inosine monophosphate (IMP), serves as a branch point for the formation of both AMP and GMP.

• Salvage Pathway:
  To conserve energy, cells recycle free purine bases from nucleic acid turnover. Enzymes like adenine phosphoribosyltransferase (APRT) and hypoxanthine-guanine phosphoribosyltransferase (HGPRT) use PRPP to convert these bases back into nucleoside monophosphates.

3. Pyrimidine Biosynthesis

• Direct Assembly as Ribonucleotides:
  Unlike purines, pyrimidine rings are synthesized as part of the ribonucleotide. The pathway starts with the formation of carbamoyl phosphate (using CPS II, which uses glutamine as the nitrogen donor) and aspartate, leading to the production of UMP. Subsequent phosphorylation steps yield UTP and CTP, and the methylation of deoxy-UMP (using 5,10-meTHF) produces dTMP.

4. Deoxynucleotide Formation

• Ribonucleotide Reductase (RNR):
  RNR catalyzes the reduction of ribonucleotides to deoxyribonucleotides—a reaction critical for DNA synthesis and repair. This enzyme employs a radical mechanism and is tightly regulated through allosteric effectors (ATP, dATP, dGTP, dTTP) that modulate both substrate specificity and overall enzymatic activity. The regulation of RNR ensures balanced deoxynucleotide pools and prevents DNA damage.

5. Nucleotide Degradation

• Purine and Pyrimidine Catabolism:
  Nucleotide degradation involves a series of reactions that break down purine and pyrimidine bases into waste products (such as uric acid for purines) that are excreted. These pathways not only remove excess nucleotides but also recycle components for reuse in biosynthetic pathways.

6. Regulation and Integration with Cellular Metabolism

• Feedback and Allosteric Regulation:
  The de novo synthesis of purines is controlled by feedback inhibition (e.g., PPAT is allosterically inhibited by downstream nucleotides), and both purine and pyrimidine pathways are responsive to substrate availability and energy status. In addition, formation of multi-enzyme complexes like the purinosome optimizes metabolic flux by co-localizing pathway enzymes with mitochondrial sources of ATP and one-carbon units.

• Interplay with Energy Metabolism:
  Nucleotide metabolism is linked to the central energy-generating pathways, including glycolysis and the citric acid cycle. For example, intermediates from the TCA cycle (e.g., oxaloacetate and α-ketoglutarate) are precursors for amino acids that feed into nucleotide biosynthesis.

• Clinical Relevance:
  Disruptions in nucleotide metabolism can lead to imbalances in energy production and signal transduction, contributing to various diseases (such as cancer, where rapidly proliferating cells require robust nucleotide synthesis). Understanding these pathways also lays the groundwork for therapeutic strategies targeting nucleotide metabolism in disease.

This chapter integrates the biochemistry of nucleotide synthesis, interconversion, and degradation with broader cellular functions. It emphasizes how nucleotide metabolism is interwoven with amino acid metabolism, energy production, and regulatory networks that maintain cellular homeostasis and support growth, proliferation, and response to environmental cues.