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22.4: Biosynthesis and Degradation of Nucleotides

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    15181
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    Purine  synthesis

    This section is derived from 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/.

    The carbon and nitrogen atoms in the bases of both purines and pyrimidine nucleotides come from amino acids as shown in Figure 2 below.

    Amino acids in cancerFig2.svg

    Figure \(\PageIndex{2}\):  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/.

    Purine biosynthesis requires formate, bicarbonate, and three amino acids: aspartate, glycine, and glutamine. While glutamine and aspartate act as the nitrogen source for both nucleobases (N1 from aspartate and N3 and N9 from glutamine) and the amino group of purines (glutamine for adenine and aspartate for guanine), glycine can contribute to purine biosynthesis in two ways: by direct incorporation into the purine backbone (C4, C5, and N7) or by producing one-carbon units for biochemical reactions involved in purine biosynthesis (C2 and C8) (Fig. 2c)5. The critical carrier of one-carbon units in the latter process is 5,10-meTHF. 5,10-meTHF is further converted to formate (10-formyl THF), contributing C2 and C8 carbons to the nucleobase4,7,61.

    Aspartate, glycine, and glutamine provide nitrogen, and glycine and one-carbon units from the folate cycle (as a form of formate) provide carbon for purines. Glycine is formate’s indirect precursor through one-carbon metabolism, providing formate for biochemical reactions in purine biosynthesis.

    Pyrimidine biosynthesis is simpler than that of purine. In contrast to purines that are synthesized as ribonucleotides rather than as nucleobases, pyrimidines are synthesized first as nucleobases and then conjugated to phosphoribosyl pyrophosphate (PRPP) to yield the corresponding ribonucleotide. The pyrimidine ring is derived from glutamine, aspartate, and bicarbonate. For pyrimidine synthesis, aspartate acts as both carbon and nitrogen donors (N1, C4, C5, and C6), whereas glutamine contributes to N3 of the nucleobase and amino group of cytosine (Fig. 2c)4. The one-carbon unit derived from serine to glycine conversion is required for thymidylate synthesis. 5,10-meTHF serves as a one-carbon donor to transfer a methyl group to deoxyuridine monophosphate (dUMP) and produce deoxythymidine monophosphate (dTMP), a reaction catalyzed by thymidylate synthase (TS)7,8.  Aspartate and glutamine are the main amino acids involved in pyrimidine synthesis. Carbon (C) is in yellow, and nitrogen (N) is in green. Aspartate and glutamine are the main amino acids involved in pyrimidine synthesis. Carbon (C) is in yellow, and nitrogen (N) is in green.

     


    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

     

    Nucleotides, beyond forming the fundamental building blocks of the genetic code and its expression, serve a variety of active biochemical roles in the biology of the cell. Consisting of either a single-ring pyrimidine or a fused-ring purine carbon–nitrogen nucleobase and a ribose/deoxyribose-phosphate moiety, nucleotides and their derivatives act as energy carriers to drive enzymatic reactions, mediate signaling within and between cells, and play numerous vital roles in the regulation of metabolism beyond their own homeostasis [1,2]. Due to the central nature of nucleotides in cellular function, it is critical that cells maintain an uninterrupted supply of both pyrimidines and purines. In mammals, this is achieved for purines by two main pathways, salvage from existing bases and de novo biosynthesis, with significant activity occurring in the liver [3]. Under normal physiological conditions, most of the purine pool is generated by the salvage pathway with the nucleic acid breakdown process, leading to the release of free purine nucleobases in the form of adenine, guanine, and the hypoxanthine base of inosine monophosphate (IMP) [4]. These free bases are attached to phosphoribosyl pyrophosphate (PRPP) to form purine nucleoside monophosphates by either adenine phosphoribosyltransferase (APRT), which mediates adenosine monophosphate (AMP) formation, or hypoxanthine-guanine phosphoribosyltransferase (HGRT), which acts on hypoxanthine to form IMP and guanine to form guanosine monophosphate (GMP) (Figure 1) [5]. As a critical precursor of de novo biosynthesis, PRPP plays an important role in maintaining both de novo biosynthetic and salvage pathways, with the distribution and balance of the nucleotide pool in each cell type being vital for regular cellular activities [6].

    Under high cellular purine demands exceeding the capacity of salvage, the nucleotide requirement is met by upregulation of the de novo biosynthetic pathway [4]. This is a highly conserved pathway that produces AMP and GMP using metabolic precursors including PRPP, several amino acids (glutamine, aspartic acid, and glycine), small molecule cofactors N10-formyl Tertrahydrofolate (THF) and Nicotinamide Adenine Dinucleotide (NAD+), and existing adenosine triphosphate (ATP) and guanosine triphosphate (GTP). In humans, de novo biosynthesis requires a sequence of ten distinct reactions catalyzed by six enzymes. 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) [7,8]. When active, the pathway is limited both by substrate availability and by the reaction rate of its initial step, the conversion of PRPP to phosphoribosylamine (PRA) by phosphoribosylpyrophosphate amidotransferase (PPAT) [9]. The final product of the de novo biosynthesis pathway, IMP, is the precursor for both AMP and GMP, which are formed via two further enzymatic reactions; in total, the energy from hydrolysis of six ATP molecules to adenosine diphosphate (ADP) is required to synthesize one molecule of IMP from PRPP, whereas nucleotide salvage is not dependent on stored phosphate bond energy.

    As the presumed primary rate-limiting step of purine biosynthesis, PPAT activity is tightly regulated. PPAT possesses two nucleotide-binding sites near the active site, allowing for feedback control by downstream purine nucleotides via allosteric inhibition [10,11]. Furthermore, there is a growing body of evidence that signaling pathway enzymes such as protein kinase B (PKB) and ribosomal protein S6 kinase (S6K) influence IMP production directly through the phosphorylation of purine biosynthetic enzymes [12]. One such modification is the Thr397 phosphorylation of PPAT by PKB, detected in purine supplemented conditions only and affecting downstream inosine monophosphate (IMP) production [13]. Similarly, as the limiting metabolic input, regulation of PRPP levels affects the rate of purine synthesis. Analysis of PRPP in different growth stages in HTC116 colon cancer cells demonstrated that rates of purine synthesis via both salvage and the de novo pathways increased by 5 and 3.3 fold, respectively, from the end of the G1 phase to the beginning of the S phase, with the de novo increase attributed to an increase in intracellular phosphate stimulating PRPP synthetase activity [14]. More broadly, high-throughput global proteomic studies have revealed 174 post-translational modifications within the six enzymes across the purine de novo biosynthetic pathway [13].

    In an additional mechanism of pathway regulation, still incompletely understood, purine de novo synthesis enzymes cluster into large complexes termed purinosomes, presumably to increase pathway efficiency and isolate reactive intermediates. A purinosome core of PPATTGART, and formylglycinamidine ribonucleotide synthetase (FGAMS) interacts with PAICS, adenylosuccinate lyase (ADSL), and ATIC, which also transiently interact with each other, suggesting the possibility of association-dependent regulation of the pathway as a whole [15]. Furthermore, super-resolution microscopy studies demonstrate that these purinosomes colocalize with mitochondria, potentially positioning them in areas of high ATP and metabolite concentrations to promote forward flux through the pathway [16].

     

    PurineSynthesisMetFig1.svg

    Figure 1. 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 schematic representation shows the de novo and salvage pathways and their crosstalk with mitochondria. The conserved de novo biosynthesis pathway to generate IMP consists of 10 chemical steps catalyzed by 6 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), formylglycinamidine 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) utilizing 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).


    PLOS

    10 reaction steps catalysed by six enzymes, four of which are multifunctional. Once synthesized, they are efficiently recycled by the enzymes of the salvage pathway and eventually removed from cells in the form of uric acid or allantoin 

    Because de novo purine synthesis (DNPS) produces unstable and/or toxic intermediates [1], the enzymes would need proximity to ensure this vital metabolic function. Knowledge of the composition and regulation of this multienzyme structure, the purinosome, would have important implications regarding human diseases and the treatment of cancer, inflammation and infections. The existence of purinosome has been therefore addressed by various biochemical, molecular and structural approaches

     

    purinosomecartoonPLOS.png

    Baresova et al (2018) PLoS ONE 13(7): e0201432. https://doi.org/10.1371/journal.pone.0201432.  Creative Commons Attribution License,

    The initial substrate in DNPS is phosphoribosyl pyrophosphate (PRPP). Six enzymes are involved in DNPS and the purinosome multienzyme complex: phosphoribosyl pyrophosphate amidotransferase (PPAT), the trifunctional enzyme GART (glycinamide ribonucleotide synthetase/glycinamide ribonucleotide transformylase/aminoimidazole ribonucleotide synthetase), phosphoribosylformylglycinamidine synthetase (PFAS), the bifunctional enzyme PAICS (phosphoribosylaminoimidazole carboxylase/phosphoribosylaminoimidazolesuccinocarboxamide synthetase), adenylosuccinate lyase (ADSL), and the bifunctional enzyme ATIC (5-aminoimidazole-4-carboxamide ribonucleotide transformylase/inosine monophosphate cyclohydrolase). The final product is inosine monophosphate (IMP). IMP is converted into adenosine monophosphate (AMP) and guanosine monophosphate (GMP) and is also degraded to uric acid via the degradation pathway. The hypoxanthine intermediate can be recycled by the enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT) into IMP or GMP.

     

     

     

    IMP synthesis

    IMP_PURINE1_Synthesis.05212.svg

     

     

    Source of Atoms in IMP Rings

    IMP_PURINE1_SourceAtomsIMP052122.svg

     

     

    no w continue

     

     

    IMP_PURINE1_toGTP_ATP52122.svg

     

    Pyrmidine Synthesis

    1st ref

    Pyrimidine biosynthesis is simpler than that of purine. In contrast to purines that are synthesized as ribonucleotides rather than as nucleobases, pyrimidines are synthesized first as nucleobases and then conjugated to phosphoribosyl pyrophosphate (PRPP) to yield the corresponding ribonucleotide. The pyrimidine ring is derived from glutamine, aspartate, and bicarbonate. For pyrimidine synthesis, aspartate acts as both carbon and nitrogen donors (N1, C4, C5, and C6), whereas glutamine contributes to N3 of the nucleobase and amino group of cytosine (Fig. 2c)4. The one-carbon unit derived from serine to glycine conversion is required for thymidylate synthesis. 5,10-meTHF serves as a one-carbon donor to transfer a methyl group to deoxyuridine monophosphate (dUMP) and produce deoxythymidine monophosphate (dTMP), a reaction catalyzed by thymidylate synthase (TS)7,8.  Aspartate and glutamine are the main amino acids involved in pyrimidine synthesis. Carbon (C) is in yellow, and nitrogen (N) is in green. Aspartate and glutamine are the main amino acids involved in pyrimidine synthesis. Carbon (C) is in yellow, and nitrogen (N) is in green.


    Li et al. Int. J. Mol. Sci. 202122(19), 10253; https://doi.org/10.3390/ijms221910253.  Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/)

    CAD (Carbamoyl-phosphate synthetase 2, Aspartate transcarbamoylase, and Dihydroorotase) is a multifunctional protein that participates in the initial three speed-limiting steps of pyrimidine nucleotide synthesis.

    It was also found that carbamyl phosphate synthetases (CPSases) CPS-1 and CPS-2 provide CAP pools for arginine and pyrimidine synthesis, respectively [4,5,6]. In animals, aspartate transcarbamoylase (ATC) and dihydroorotase (DHO) were subsequently co-purified with CPS-2. These three enzymes form a single multi-enzymatic protein named CAD to participate in the de novo pyrimidine pathway in mammals

    CAD is a multifunctional protein that takes part in the initial three speed-limiting steps of pyrimidine nucleotide synthesis. Moreno-Morcillo et al. have shown that CAD is a hexamer of a 243 kDa polypeptide chain [15]. Human CAD involves the concerted action of four domains: glutamine amidotransferase (GATase), carbamylphosphatesynthetase II (CPSIIase), aspartate transcarbamylase (ATCase), and dihydroorotase (DHOase) (Figure 1A). CPSIIase consists of two highly homologous fragments, which are designated as CPSase A and CPSase B. Specifically, GATase and CPSase (CPSase A and CPSase B) jointly form the glutamine-dependent CPSase. GATase transfer HCO3, glutamine, and ATP to form carbamoylphosphate (CP) at the site of CPSIIase domain. CP formation is the first rate-limiting step for the nucleotide synthesis [16,17]. We will discuss the details of CPSIIase and its regulations in the latter text. ATCase, consisted of a catalytic homotrimer, catalyzes carbamoylphosphate (CP) and aspartate (Asp) into carbamoyl aspartate (CA-asp) [18]. The DHOase domain catalyzes the reversible cyclization of CA-asp to dihydroorotate (DHO), the first cyclic compound of de novo pyrimidine nucleotide synthesis pathway [15,19,20]. Dihydroorotate dehydrogenase (DHODH) subsequently reduces DHO to orotate in mitochondria [21]. Finally, uridine monophosphate synthase (UMPS) converts orotic acid to produce the end product uridine monophosphate (UMP)

    uniprot:  CAD protein is a 'fusion' protein encoding four enzymatic activities of the pyrimidine pathway (GATase, CPSase, ATCase and DHOase).

    This protein is involved in step 12 and 3 of the subpathway that synthesizes (S)-dihydroorotate from bicarbonate.1 Publication This subpathway is part of the pathway UMP biosynthesis via de novo pathway, which is itself part of Pyrimidine metabolism.

    CAD Protein Domain PFam.png

    red:  glutamine amidotransferase

    blue 1: carbamoyl phosphate synthase ATP bindiong

    back to paper

    • GATase (2-365), 
    • CPSilase A (395-933)
    • CPSilase B (934-1455)
    • DHOase (1456-1788)
    • ATCase (1918-2225)

    put in iCn3E

     

    AlphaFold predicted model of the CAD protein (P27708)

     

    Figure \(\PageIndex{5}\) below shows an interactive iCn3D model of the AlphaFold predicted model of the CAD protein (P27708)

    AlphaFold predicted model of the CAD protein (P27708).png

    NIH_NCBI_iCn3D_Banner.svg Figure \(\PageIndex{5}\): 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 CPSilase domains orange, the DHOase domain yellow and the ATCase domain cyan. 

     

     

    PyrmidineSyn_AsptoUTP_CTP052122.svg

     

    Now deoxythymidine-5-monophosphate synthesis

    PyrmidineSyn_CTPtoThyminePPP052122.svg

     

     

     

    Below from:

    https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5410357/

    “INPUTS” OF ONE-CARBON METABOLISM

    As mentioned above, one-carbon metabolism acts as an integrator of the cell nutrient status by redistributing carbon groups from certain aminoacids, usually serine and glycine, (called “inputs”) to generate various compounds (“outputs”) that serve as building blocks for cell biosynthesis and also maintain the redox and methylation states of cells []. Serine can be obtained exogenously (i.e. imported from outside of the cell) as well as endogenously by de novo synthesis (see details below and in Figure ​Figure1).1). Glycine can be also transported through the plasma membrane []. Alternatively, it can be generated from serine through an enzymatic conversion in either cytoplasm or mitochondria. Furthermore, glycine can also be synthesized from threonine as was shown for mouse embryonic stem cells [].

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    Figure 1

    Schematic representation of the compartmentalization and enzymatic reactions of one-carbon metabolism

    One-carbon metabolism acts as a gauge of the cell nutrient status by redistributing carbon groups from serine and glycine, called “inputs”, to generate various compounds, called “outputs” (shown in black boxes) that serve as building blocks for cell biosynthesis. Also, they maintain the redox and methylation states of cells. Serine and Glycine can be imported through the membrane (shown as green layer) into the cells or it can be synthesized from the intermediate of glycolysis – 3-PG. Metabolic cycles are denoted as circles. Critical enzymes are shown in red. Carriers of one-carbon groups are shown in yellow. For example, 5,10-methyleneTHF provides one-carbons for thymidylate synthesis, catalyzed by the enzyme called Tymidylate Synthase. The positions of one-carbons used for the synthesis of purines (C2, C4, C5, and C8 carbons of purine rings) are indicated. Folate cycle is tightly connected with Methionine cycle. Folate cycle operates both in the cytoplasm and in mitochondria (magenta colored circle) and are linked through Tetra Hydro Folate (THF).

    In theory, both serine and glycine can be potential donors of 1C-groups for one-carbon metabolism. However, the actual relationship between serine and glycine metabolism is far more complex. The integrated scheme summarizing the crosstalk of serine and glycine metabolic pathways is presented in Figure ​Figure11.

    Serine

    There are evidences that cancer cells usually demonstrate increased serine and glycine biosynthesis and uptake [, , , ]. De novo serine synthesis consists of three steps and involves the conversion of 3-phosphoglycerate (3-PG, an intermediate of glycolysis) to 3-phosphopyruvate (3-PP) by the Phosphoglycerate Dehydrogenase (PHGDH) (Figure ​(Figure1).1). The next step involves conversion of 3-PP to 3-phosphoserine (3-PS) which is mediated by the Phosphoserine Aminotransferase (PSAT1) using glutamate for this transamination. As the final step, the phosphate ester is hydrolyzed by the Phosphoserine Phosphatase (PSPH), resulting in production of serine. Apparently, different cancer cells promote expression of the corresponding enzymes to increase the biosynthesis of serine [, , ].

    It has been shown that cancer cells utilize up to 10% of glycolytic intermediate 3-PG for serine biosynthesis []. PHGDH is amplified in a number of cancers, including 6% of breast cancers and 40% of melanomas []. Moreover, experiments using siRNA demonstrated that attenuation of PHGDH expression was associated with slow cell growth of non-malignant cells. On the contrary, ectopic expression of PHGDH in the non-cancerous MCF10A breast epithelial cell line disrupted acinar morphogenesis and induced other phenotypic alterations that may predispose cells to transformation [].

    There are also evidences of de-regulated expression in cancer of two other enzymes of serine biosynthesis – PSAT1 [, , ] and PSPH [, ].

    Besides serine, which represents a critically important “input” of one-carbon metabolism and nucleotide biosynthesis, there is another important metabolite generated at the transamination step of serine biosynthesis - α-ketoglutarate (αKG) []. αKG is the entry point through which glutamine supplies carbon to the tricarboxylic acid (TCA) cycle during cell growth, enabling the production of a number of essential biosynthetic precursors [].

    Taken these results together, it is evident that serine is a hub of one-carbon metabolism and therefore its overexpression is an important feature of different malignancies.

    Glycine

    Whereas the importance of serine for the enhanced proliferation of cancer cells is generally accepted, the impact of glycine on this process is the topic for intense debates.

    Jain with colleagues [] applied a mass-spectrometry approach to measure the consumption and release of 219 metabolites from the media across the NCI-60 panel of cancer cell lines and combined these data with the pre-existing atlas of gene expression. The integrated analysis identified glycine consumption as well as expression of the mitochondrial glycine biosynthetic pathway (SHMT2, MTHFD2 and MTHFD1L) to be strongly correlated with the rates of proliferation across all cancer cell lines.

    However, other works have shown that cancer cells fail to consume glycine when serine is abundant [, ]. For instance, Labuschange (2014) [] showed that cancer cells preferentially consumed serine rather than glycine, and the high level of serine uptake paralleled with glycine efflux. Moreover, according to their results, the excess of glycine even inhibited cell growth. Biochemically, high levels of glycine inhibited the metabolic transformation of the former into purines, required for DNA replication, by driving instead the intracellular glycine-to-serine conversion. Glycine was converted to serine at the expense of 5,10-methylenTHF, thus depleting its intracellular pool and hence slowing the cell growth. Based on these data, it has been proposed that cancer cells release the excess of glycine thereby limiting its intracellular concentration to facilitate serine uptake and serine-to-glycine conversion []. Notably, it is the serine-to-glycine conversion process that yields the 5,10-methyleneTHF metabolite required for the maintenance of maximal levels of nucleotide synthesis and proliferation. Serine is converted to glycine by two isoforms of Serine hydroxymethyltransferase (SHMT1 and SHMT2) that correspond to the cytosolic and mitochondrial forms, respectively. The excess amount of glycine, the product of this reaction, can reverse this reaction and therefore should be removed from the cell []. On the related note is the fact that two different cancer in vivo models also demonstrated that excess of dietary glycine inhibited the development of tumors [, ]. Collectively, glycine biosynthesis is deemed as the central process which sustains one-carbon metabolism and rapid proliferation.

    As mentioned above, glycine metabolism is intimately linked with purine biosynthesis and defines the sensitivity to mycophenolate, tiazofurin, alanosine and other inhibitors of purine biosynthesis []. In general, glycine can contribute to purine biosynthesis in two ways: by direct incorporation into the purine ring or indirectly, by providing one-carbon units for biochemical reactions involved in the purine ring biosynthesis. The latter can be derived by either synthesis of glycine from serine (the SHMT-catalyzed reaction), or alternatively, by glycine degradation (oxidization to CO2 by a highly evolutionary conserved glycine cleavage system - GCS) (Figure ​(Figure1).1). It is important to note that the incorporation of glycine into purine nucleotides does not involve oxidation by GCS but is rather mediated by SHMT-derived glycine []. These data suggest that SHMT-catalyzed glycine synthesis together with direct incorporation of glycine into the purine rings link glycine production with purine biosynthesis.

    As mentioned above, the glycine degradation is exerted by GCS. This system consists of four mitochondrial proteins: the T-protein (GCST or AMT (aminomethyltransferase)), P-protein (GLDC (glycine dehydrogenase)), L-protein (GCSL or DLD (dihydrolipoyl dehydrogenase)), and H-protein (GCSH). It converts glycine to CO2 in the following reaction: Glycine + THF + NAD+ = 5,10-methylene-THF + CO2 + NH3 + NADH2 []. Importantly, the products of this enzymatic reaction, 5,10-methyleneTHF and NADH, are required for the nucleotide biosynthesis.

    Apparently, GCS is critical for efficient elimination of the excess of glycine. Components of GCS, especially GLDC, are frequently overexpressed in different malignancies and this is linked with cancer progression. Tedeschi and colleagues [] showed that about 28% of lung-, 19% of breast-, 9% of prostate-, 30% of colorectal-, 23% of brain- and 21% of ovarian cancers exhibit a significant up-regulation of the 1C-metabolism gene signature, including GLDC. The data from other groups support the notion on overexpression of GCS components in cancer [, ].

    Zhang and colleagues [] have established GLDC as instrumental for the growth and tumorigenesis of tumor-initiating cells derived from the primary NSCLC. Overexpression of GLDC promotes cellular transformation and induces dramatic changes in glycolysis and serine/glycine metabolism, leading to changes in pyrimidine metabolism and cancer cell proliferation. Furthermore, its aberrant regulation often correlates with poor survival of lung cancer patients.

    Another study [] has shown that GCS may cooperate with SHMT2 to ensure survival and progression of tumors. For example, GLDC-mediated cleavage of the excessive amount of glycine supported the growth of glioma cells with active SHMT2 under ischemic conditions. On the contrary, when the activity of GLDC was inhibited, cells with high levels of SHMT2 were selectively killed. This is explained by the fact that the excess of glycine generated by SHMT2 was subsequently converted into two toxic molecules, aminoacetone and methylglyoxal, which normally are metabolized by GLDC [].

    Taken these data together, one can reckon that despite the fact that glycine biosynthesis is the hub of one-carbon metabolism, the excess of glycine itself can be detrimental to the tumor cell proliferation and needs to be strictly controlled by either its export or by GCS-mediated clearance.

    FOLATE AND METHIONINE CYCLES

    The “core” part of the one-carbon metabolism comprises the Folate and Methionine cycles, which are linked together. These two cycles integrate cell nutrient status using 1C-groups from glycine and serine as “inputs” to generate different “outputs” such as nucleotides, glutathione, SAM, and other metabolites, which are required for DNA and RNA biosynthesis, as well as for the maintenance of the redox and epigenetic cell states.

    Folate cycle

    Folates are referred to the family of B9 vitamins []. They are naturally present in different sources of food or can be synthesized chemically (e.g. folic acid) as dietary supplements. Folates function as carriers that distribute one-carbon groups from “inputs” to “outputs” (Figure ​(Figure11 and ​and2).2). Once transported to the cell, the vitamin undergoes covalent modification by polyglutamination. It is further substituted by the one-carbon moiety in the N5 and/or N10 position at different oxidation levels: formate (10-formylTHF), formaldehyde (5,10-methyleneTHF), or methanol (5-methylTHF) [].

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    Figure 2

    Folate cycle, its “outputs” and the energy balance

    Critical enzymes of the Folate cycle are shown. TetraHydroFolate (THF) is a carrier that distributes one-carbon groups (1C-group) from serine to different “outputs” – thymidylates, purines, SAM, GSH, etc (shown in black boxes). After accepting the 1C-group, THF undergoes modifications that alter its oxidation states: 10-formylTHF, 5,10-methyleneTHF, 5-methylTHF (shown in different background colors). Donated carbon and nitrogen atoms corresponding to their numbers in the pyrimidine and purine rings are shown in brackets. Red asterisks indicate the enzymes that are currently being explored as drug targets. Enzymes marked with orange asterisks are considered as potential drug targets. Folate cycle can provide cells with additional source of energy. Two molecules of NADPH are synthesized in cytoplasm in reactions catalyzed by DHFR (conversion of DHF to THF) and MTHFD1 (conversion of 5,10-methylenTHF to 5,10-methenylTHF), as well as in mitochondria by MTHFD2L (conversion of 5,10-methylenTHF to 5,10-methenyl THF). One molecules of NADPH is used by MTHFR which links Folate cycle to the Methionine cycle. Also, ATP can be synthesized during MTHFD1- (cytoplasm) or MTHFD1L-mediated (mitochondria) conversion of 10-formylTHF to THF.

    As mentioned above, there are only two direct sources of 1C-groups in one-carbon metabolism – serine and glycine. Thus, the central reaction of the Folate cycle is conversion of serine to glycine by SHMT1 and SHMT2 enzymes. By transferring the 1C-group from serine and THF, this reaction generates 5,10-methyleneTHF – the first donor of one-carbon group in the folate cycle. Another source of 5,10-methyleneTHF comes from the enzymatic cleavage of glycine by an enzyme called glycine decarboxylase (GLDC), which resides in mitochondria.

    In turn, 5,10-methyleneTHF can be used in three ways (Figure ​(Figure2).2). First, it can serve as 1C-donor for the initial step of thymidylate biosynthesis, a reaction catalyzed by thymidylate synthase (TS). In this reaction 5,10-methyleneTHF provides one-carbon group for the pyrimidine biosynthesis and is oxidized into dihydrofolate (DHF). In the next reaction dihydrofolate reductase (DHFR) reduces DHF to THF enclosing this metabolic loop.

    Second, 5,10-methyleneTHF can be used by a cytosolic enzyme Methylenetetrahydrofolate reductase 1 (MTHFD1), or mitochondrial tandem enzymes Methylenetetrahydrofolate reductases MTHFD2L/MTHFD2, to generate 10-formylTHF. 10-formylTHF is a 1C-donor for the two reactions of purine biosynthesis catalyzed by Trifunctional enzyme Phosphoribosylglycinamide Formyltransferase/ Synthetase/ Phosphoribosylaminoimidazole Synthetase (GART) and Bifunctional 5-Aminoimidazole-4-Carboxamide Ribonucleotide Formyltransferase/IMP Cyclohydrolase (ATIC), both of which in turn generate THF.

    Third, 5,10-methyleneTHF is used by Methylentetrahydrofolatereductase (MTHFR) to generate methylTHF. The latter donates a methyl group to homocycteine resulting in the formation of methionine and THF. By this way the Folate cycle is coupled with Methionine cycle. Finally, THF is converted into 5,10-methyleneTHF by SHMT1 and SHMT2 thus enclosing the Folate cycle.

    Methionine cycle

    Another arm of the 1C-metabolic process is the methionine cycle (Figure ​(Figure3).3). It starts with methionine synthesis from homocysteine and methylTHF catalyzed by methionine synthase (MS). Subsequently, methionine adenyltransferase (MAT) synthesizes SAM, the main donor of methyl groups in the cell. After demethylation, SAM is converted to S-adenosylhomocysteine (SAH). Finally, S-adenosyl homocysteine hydrolase (SAHH) mediates de-adenylation of SAHH resulting in homocysteine and full turn of the cycle.

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    Figure 3

    Folate cycle is coupled with Methionine cycle

    During Folate cycle MTHFR reduces 5,10-methyleneTHF to 5-methylTHF. Subsequently, 5-methylTHF donates its carbon group to convert homo-cysteine (hcystein) to methionine by methionine synthase (MS), hence initiating Methionine cycle. In turn, methionine is used by methionine adenosyltransferase (MAT) to generate S-adenosylmethionine (SAM) – the principal donor of methyl groups for DNA and proteins methylation. Thus, SAM is used by different methyltransferases, resulting in S-adenosylhomocysteine after its demethylation. Finally, S-adenosylhomocysteine hydrolase (SAHH) mediates deadenylation of S-adenosylhomocysteine to hcysteine, enclosing the methionine cycle. Homocysteine can be used by cystathionine synthase (CBS), which converts it to cystathionine. In turn, cystathionine is a substrate for cystathionine gamma-lyase (CTH), which uses it for synthesis of cysteine. Cysteine is required for the synthesis of proteins as well as for generation of taurine and glutathione, the latter is one of the critical molecules for redox homeostasis.

    “OUTPUTS” OF ONE-CARBON METABOLISM

    The Folate and Methionine cycles mediate redistribution of 1C-groups results in biosynthesis of a number of important compounds including nucleotides, several aminoacids, and GSH and SAM molecules that are critical for the maintenance of cell redox status and epigenetic homeostasis (Figure ​(Figure1).1). All of these compounds are necessary for the rapid proliferation of cancer cells.

    The biosynthesis of nucleotides

    One of the main outputs of one-carbon metabolism is biosynthesis of nucleotides. This is one of the molecular processes that constrains quickly proliferating cancer cells rate-limiting as it provides building blocks for DNA synthesis, purines and pyrimidines. The biosynthesis of both purine and pyrimidine (thymidylate) nucleotides requires cofactors generated through 1C-metabolism pathways.

    Pyrimidines

    The pyrimidine ring is composed of three fragments: C4 to C6 and N1 atoms are provided by aspartate, whereas C2 is derived from HCO3, while N3 – from glutamine. The central precursor for generating pyrimidines is uridine-monophosphate (UMP). At first, the pyrimidine ring is constructed followed by conjugation with phospho-ribosyl-pyrophosphate (PRPP) (for details see Figure ​Figure4A).4A). The synthesis of UMP does not require the 1C-cofactors. UMP through UDP can be than converted to either dUMP (for subsequent dTTP synthesis) or other nucleotides UTP, CTP and dCTP. The conversion of dUMP to dTMP is mediated by thymidylate synthase (TS) and requires 5,10-methylenTHF as 1C-donor. This reaction is very important for nucleotide biosynthesis and TS is a target for several approved drugs in cancer therapy (see below).

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    Figure 4

    Major pathways of the nucleotide biosynthesis

    A. Shown are the reactions of the biosynthesis of pyrimidines: The participating enzymes are shown in red. The input compounds, intermediates and the resulting products are indicated (future positions of atoms in the nucleotide are indicated in brackets). Reactions are numbered in sequential order. Thus, reactions 1, 2 and 3 are catalyzed by tri-functional enzyme Carbamoyl Dehydrogenase (CAD); 4 – Dihydroorotate Dehydrogenase (DHODH);5 and 6 – bifunctional Uridine Monophosphate Synthetase (UMPS); 7,8,9 - Nucleoside Diphosphate Kinase (NME); 10 - Thymidylate synthase (TS), 11 - CTP Synthase (CTPS). Red asterisks indicate the enzymes that are currently being explored as drug targets. Enzymes marked with orange asterisks are considered as potential drug targets. B. The biosynthesis of purines. Abbreviations are the same as in part A.

    Purines

    The purine ring is composed of different components (see Figure ​Figure4B):4B): glycine, which is the most used precursor (donates C4, C5 and N7 atoms), HCO3 - the donor of C6, glutamine (the donor of N3 and N9 atoms), aspartate (the donor for N1), and 1C-cofactor - N10-formylTHF which is a donor for C2 and C8. The central intermediate for purines is inosine-monophosphate (IMP). In contrast to pyrimidines, the purine synthesis starts with PRPP and subsequent step-by-step construction of the purine ring (for details see Figure ​Figure4).4). The IMP generation from PRPP needs five enzymes, three of which are multifunctional (GART, PAICS, ATIC). Reactions, catalyzed by GART and ATIC (the adding atoms C2 and C8 for purine ring) require N10-formylTHF produced by MTHFD1 and MTHFD2 during the folate cycle.

    IMP can be converted in the two-steps reactions either into AMP or GMP. The gateway to guanine nucleotides is controlled by IMPDH, making it an “enzyme of consequence” for virtually every organism. This reaction is the first rate-limiting step in guanine nucleotide biosynthesis (Figure ​(Figure4B4B).

    Thus, the biosynthesis of nucleotides is critical for rapidly proliferating cancer cells to ensure the timely DNA replication and therefore represents a promising target for anticancer therapy (see below).

    Summary

    One-carbon metabolism and nucleotide biosynthesis stand in one line with such cancer-related metabolic alterations as increased glycolysis, pentose-phosphate pathway and an acquired ability of de novo synthesis of fatty acids. According to the growing body of evidences, these metabolic features are common to different types of tumors and are considered now as one of the “hallmarks of cancer” [, , ]. They provide metabolic plasticity to cancer cells which has an impact on different features such as gene expression [], epigenetic control [] and drug resistance []. One carbon metabolism provides “building blocks” (nucleotides, certain aminoacids) as well as contributes to epigenetic (SAM for DNA and protein methylation) and redox (glutathione) homeostasis for rapidly proliferating cancer cells.

    The high importance of one-carbon metabolism for cancer cells is reflected in more than 60 years application of its inhibitor (MTX) for a cancer treatment. But, one can notice, that despite the a relatively big number of enzyme operating in one-carbon metabolism and nucleotide biosynthesis, only a few of them are currently used as anticancer targets. They are only: DHFR, TS and RNR. Recently, IMPDH inhibitors became to be used and it will be interesting to see in next years if they are efficient to treat tumors.

    Thus, an important question is whether other enzymes of one-carbon metabolism and nucleotide biosynthesis can be efficiently targeted by anticancer therapies without excessive toxicity to normal cells? For instance, as discussed above, there are number of potent GART inhibitors, but none of them has successfully passed through clinical trials due to their high toxicity. A related question is whether it is possible in general to target GART without cytotoxicity to normal cells?

    Nucleotide Degradation

    Purines

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    Pyrimidine Degradation

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    22.4: Biosynthesis and Degradation of Nucleotides is shared under a not declared license and was authored, remixed, and/or curated by Henry Jakubowski.

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