Regulation of mammalian nucleotide metabolism and biosynthesis

Abstract

Nucleotides are required for a wide variety of biological processes and are constantly synthesized de novo in all cells. When cells proliferate, increased nucleotide synthesis is necessary for DNA replication and for RNA production to support protein synthesis at different stages of the cell cycle, during which these events are regulated at multiple levels. Therefore the synthesis of the precursor nucleotides is also strongly regulated at multiple levels. Nucleotide synthesis is an energy intensive process that uses multiple metabolic pathways across different cell compartments and several sources of carbon and nitrogen. The processes are regulated at the transcription level by a set of master transcription factors but also at the enzyme level by allosteric regulation and feedback inhibition. Here we review the cellular demands of nucleotide biosynthesis, their metabolic pathways and mechanisms of regulation during the cell cycle. The use of stable isotope tracers for delineating the biosynthetic routes of the multiple intersecting pathways and how these are quantitatively controlled under different conditions is also highlighted. Moreover, the importance of nucleotide synthesis for cell viability is discussed and how this may lead to potential new approaches to drug development in diseases such as cancer.

Figure 1.

De novo nucleotide biosynthesis: generation of activated ribose. 5-phosphoribose-1-pyrophosphate (PRPP) is the activated form of ribose used for nucleotide biosynthesis and is derived from ribose-5-phosphate from the pentose phosphate pathway (PPP).Ribose-5-phosphate is produced via both oxidative and non-oxidative branches of the PPP. The oxidative branch also generates two NADPH. The oxidative branch comprises the reactions catalyzed by G6PD, PGLS and PGD. The non-oxidative branch interconverts five carbon sugars with four and six carbon sugars using the transaldolase (TA) and transketolase (TK) reactions. HK: hexokinase; G6PD: glucose-6-phosphate dehydrogenase; PGLS: 6-phosphogluconolactonase; PGD: 6-phosphogluconate dehydrogenase; RPI: ribulose-5-phosphate isomerase; RPE: PGLS 3-epimerase; TK: transketolase; TA: transaldolase.

Figure 2.

Pyrimidine biosynthesis. CA: carbamoyl aspartate; DHO: dihydroorotate; OMP: orotate monophosphate. Enzyme names: (1) carbamoyl phosphate synthase II (CPSII); (2) aspartate transcarbamoylase (ATCase); (3) carbamoyl aspartate dehydratase = dihydroorotase [CAD encodes enzymes 1 + 2 + 3]; (4) dihydroorotate dehydrogenase; (5) orotate phosphoribosyltransferase; (6) orotidine-5-phosphate decarboxylase (OMP decarboxylase). The activities of 5 and 6 reside in a single bifunctional polypeptide encoded by the UMPS gene. Atom colors denotes origins: red from CO₂, green from aspartate and ultimately glucose or Gln, blue from Gln.

Figure 3.

Purine biosynthesis: synthesis of IMP. Various atoms of the purine ring originate from different sources, i.e. N3, N9 derive from the amido group of Gln (blue), N7, C5, C4 derive from Gly (green), C6 from CO₂ (black), N1 from the amino group of Asp (red) and C2, C8 from N¹⁰formyl-tetrahydrofolate. Enzyme names: (1) glutamine phosphoribosylpyrophosphate amidotransferase (PPAT); (2) glycinamide ribotide synthase (GART); (3) glycinamide ribotide transformylase (GART); (4) formylglycinamide synthase (PFAS); (5) aminoimidazole ribotide synthase (GART); (6) aminoimidazole ribotide carboxylase (PAICS); (7) succinylaminoimidazolecarboxamide ribotide synthase (PAICS); (8) adenylosuccinate lyase (ADSL); (9) aminoimidazole carboxamide ribotide transformylase (ATIC); (10) IMP cyclohydrolase (ATIC). IMP is the common precursor of AMP and GMP. The pathway from IMP to GMP and AMP are shown in Supplementary Figure S2.

Figure 4.

Glycine, serine and aspartate pathways. Synthesis of glycine and N⁵,N¹⁰-methylene tetrahydrofolate (N⁵,N¹⁰-CH₂-THF) from glucose via the One-Carbon pathway. N⁵,N¹⁰-CH₂-THF is further converted to N¹⁰-formyl-THF for incorporation into purine rings. Enzymes: 1: Hexokinase (HK); 2: 3-phosphoglycerate dehydrogenase (PHGDH); 3: phosphoserine aminotransferase (PSAT); 4: phosphoserine phosphatase (PSPH); 5: Serine Hydroxymethyltransferase (SHMT).

Figure 5.

Atom resolved tracing from glucose and glutamine into ribonucleotides. The ¹³C labels from ¹³C₆-Glc () are incorporated into the ribose unit (via PPP), uracil ring (via the Krebs cycle–pyrimidine synthesis path or PYR) of UMP or adenine ring (via the one-carbon or 1-C to purine synthesis path or PUR) of AMP (structures shown). The ¹³C () and ¹⁵N labels () from ¹³C₅,¹⁵N₂-Gln are expected to go into the uracil ring (via the anaplerotic glutaminolysis or GLS-Krebs cycle-PYR path) of UMP and the adenine ring (via the PUR path) of AMP. The color of the label for atomic positions in the UMP and AMP structures is matched with that of ¹³C or ¹⁵N label derived from the glucose or glutamine tracer, except for C4-C6 of UMP where glucose or Gln-derived ¹³C is not delineated. Three examples of labeled uracil ring delineate contribution of ¹³C from ¹³C₆-Glc or ¹³C₅,¹⁵N₂-Gln after one Krebs cycle turn without or with pyruvate carboxylation. The ¹³C labeling patterns of the Krebs cycle intermediates and Asp account for the ¹³C scrambling in succinate due to its symmetry and anaplerotic input (green arrows and ) from pyruvate carboxylation into the Krebs cycle after the first turn. Open circles: ¹²C; HK: hexokinase; G6PDH: glucose-6-phosphate dehydrogenase; PDH: pyruvate dehydrogenase; GLS: glutaminase; PCB: pyruvate carboxylase; OAA: oxaloacetate; αKG: α-ketoglutarate; exo: exocyclic.

Figure 6.

¹⁵N incorporation from [U-¹³C,¹⁵N]-glutamine into purines detected by HSQC. A549 cells were grown the presence of [U-¹³C,¹⁵N]-glutamine for 24 h. ¹H{¹⁵N} HSQC NMR spectra were recorded at 18.8 T using an INEPT delay optimized for two bond couplings in aromatic systems. The two ring ¹⁵N atoms derived from the amido N of Gln (blue) are indirectly detected by their coupled protons (red) as cross-peaks of N3 to H2 and N9 to H8 in the adenine ring of AXP. Reproduced with kind permission from Springer Science+Business Media from J. Biomolec NMR (Springer) J. Biomol NMR. 2011 April; 49(3–4): 267–280. doi:10.1007/s10858-011-9484-6 (figure 9).