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Nucleotide Metabolism
Dr. Chalermchai Mitrpant
Outline
Content
Page
Function of nucleotide
Nucleotide synthesis
Purine synthesis and its regulation
Pyrimidine synthesis and its regulation
Synthesis of deoxyribonucleotide and its regulation
Purine Catabolism
Hyperuricemia and Gouty Arthritis
Pyrimidine catabolism
Medication involving in nucleotide metabolism
Biosynthesis of nucleotide coenzyme
References
1 2
3-6
3-4
5-6
6-7
6-9
7-9
9
10-11
11-12
13
Function of nucleotide
Nucleotide is an organic molecule consisting of a nucleoside linked with
a phosphate group and forming the basic constituent of deoxyribonucleic acid
(DNA) and ribonucleic acid (RNA). Nucleoside makes up of a five-carbon
sugar backbone (ribose or deoxyribose) and base. There are two types of
nucleotides i.e. purine and pyrimidine nucleotide, and nomenclature of bases is
elaborated in Figure 1. Purine nucleotide includes adenosine nucleotides and
guanosine nucleotides. Pyrimidine nucleotide includes cytidine nucleotides,
uridine nucleotides and thymidine nucleotides
Nucleotide is an important molecule possessing many functions in
mammalian cell as described below.
1. Monomeric unit of DNA or RNA
2. High energy molecule, ATP gives the energy through ATP hydrolysis
reaction to drive endergonic reaction in the cell (ATP coupling)
3. Some nucleotide such as cyclic AMP (cAMP) or cyclic GMP (cGMP)
functions a second messenger of some hormones
4. Nucleotide is a precursor for cofactor i.e. biopterin
5. Nucleotide is a component of some coenzymes
6. Nucleotide is an intermediate for biomolecule synthesis (viz. Glycogen,
phospholipid and tetrahydrofolate
7. Nucleotide plays important role in allosteric regulation of enzyme reaction
8. AMP/ADP/ATP cooperatively functions with adenylate cyclase enzyme
to control energy homeostasis in the cell
9. ADP is an important factor to stimulate platelet aggregation through
adenosine receptor P2Y12
Figure 1; Structure of nucleotide
(modified from Moran LA, Horton, RA, Scrimgeour, G, Perry, M. Nucleotide metabolism. 5th ed: Pearson publishing,
2011)
2 Nucleotide synthesis
There are two pathways for nucleotide synthesis i.e. de novo synthesis and
salvage pathway
1. De novo synthesis pathway is the pathway involves with different
enzymes to create nucleotide molecule.
2. Salvage pathway is the pathway that relies on recycling of degradative
product of DNA or RNA molecule.
Purine nucleotide synthesis and its regulation
1. De novo pathway; Synthesis of 5’ phosphoribosylamine 1’
pyrophosphate (PRPP) is one important regulatory step for de novo purine
synthesis. This enzyme is allosterically controlled by various molecules i.e.
inorganic phosphate (Pi), sulfate ion (SO4-), ADP and GDP as shown in Figure
ail2. Multistep enzymatic reactions shown in Figure 3 are required to create the
first nucleotide (inosine monophosphate (IMP)). IMP is subsequently converted
by a series of enzymes to be GMP and AMP in separate series of reactions.
The regulation of de novo purine synthesis is shown in Figure 4
Figure 2: Synthesis of PRPP
(modified from Moran LA, Horton, RA, Scrimgeour, G, Perry, M. Nucleotide metabolism. 5th ed: Pearson publishing,
2011)
Figure 3: De novo purine synthesis
(modified from Moran LA, Horton, RA, Scrimgeour, G, Perry, M. Nucleotide metabolism. 5th ed: Pearson publishing,
2011)
3 Figure 4: Regulation of de novo purine synthesis
(modified from Moran LA, Horton, RA, Scrimgeour, G, Perry, M. Nucleotide metabolism. 5th ed: Pearson publishing,
2011)
2. Purine salvage pathway; Two ribose sugar molecules can be used as
a substrate for purine salvage pathway i.e. PRPP and ribose-1-phosphate (R-1P) PRPP can combine to either hypoxanthine or guanine and become IMP and
GMP, respectively; this reaction is catalysed by Hypoxanthine Guanosine
phosphoribosyl transferase (HGPRT).
AMP can be salvaged from the
combination of PRPP and adenine by enzyme adenine phosphoribosyl
transferase (Figure 5). On the other hand, R-1-P can combine to either
adenine, hypoxanthine or guanine to synthesise nucleoside molecule by using
nucleoside phosphorylase (Figure 5). Deficiency in HGPRT leads to
neurological syndrome called Lesch-Nyhan Syndrome and deficiency in APRT
cause renal lithiasis and eventually renal failure.
Figure 5: Purine salvage pathway
(modified from Moran LA, Horton, RA, Scrimgeour, G, Perry, M. Nucleotide metabolism. 5th ed: Pearson publishing,
2011)
4 Pyrimidine synthesis and its regulation
1. De novo pyrimidine synthesis pathway; in mammalian, multisubunit
enzyme (CAD) is responsible for creating orotate, pyrimidine nucleus
substance. Orotate combines with PRPP by using phosphoribosyltransferase
to covalently link two molecules. OMP decarboxylase is the subsequent
enzyme converting OMP to UMP (Figure 6). Defect in the production of either
Orotate phosphoribosyltransferase or OMP decarboxylase is account for the
disease called Orotic aciduria. This group of patient will have a failure to thrive
and suffer from megaloblastic anemia and mental retardation.
Figure 6: De novo pyrimidine synthesis
(modified from Moran LA, Horton, RA, Scrimgeour, G, Perry, M. Nucleotide metabolism. 5th ed: Pearson publishing,
2011)
2.
Salvage pyrimidine synthesis pathway; Uracil can be recycled to
create UMP or uridine through catalysis of phosphoribosyltransferase or
nucleoside phosphorylase, respectively (Figure 7)
5 Figure 7: Salvage pyrimidine pathway
(modified from Moran LA, Horton, RA, Scrimgeour, G, Perry, M. Nucleotide metabolism. 5th ed: Pearson publishing,
2011)
Synthesis of deoxyribonucleotide and its regulation
Deoxyribonucleotide, a monomeric unit for DNA, cannot be directly synthesized
from de novo pathway. Ribonucleotide reductase (RNR) is an enzyme
responsible
for
converting
ribonucleoside
diphosphate
(NDP)
to
deoxyribonucleoside diphosphate (dNDP). Thioredoxin or glutaredoxin is
crucial coenzyme for this reaction.
Figure 8: Ribonucleotide reductase reaction and its allosteric regulation
(modified from 1. Logan DT. Closing the circle on ribonucleotide reductases. Nat Struct Mol Biol. 2011; 18(3): 251-3. 2.
Fairman JW, Wijerathna SR, Ahmad MF, Xu H, Nakano R, Jha S, et al. Structural basis for allosteric regulation of
human ribonucleotide reductase by nucleotide-induced oligomerization. Nat Struct Mol Biol. 2011; 18(3): 316-22. 3.
Moran LA, Horton, RA, Scrimgeour, G, Perry, M. Nucleotide metabolism. 5th ed: Pearson publishing; 2011)
6 Two main mechanisms utilised to regulate RNR activity are substrate specific
control and overall activity control. In substrate specific regulation, nucleosides
triphosphates are effector to increase specific dNDP production from
ribonucleoside diphosphate. On the other hand, ATP can stimulate the
production of dNDP while dATP suppress RNR activity (Figure 8). dTTP is the
only substrate for DNA which is not able to be synthesised from either salvage
pathway or RNR activity. Figure 9 shows the synthesis pathway of dTTP from
dCDP. This process requires tetrahydrofolate as a coenzyme to supply methyl
group (CH3) for dUMP.
Figure 9: Synthesis of dTTP
(modified from Moran LA, Horton, RA, Scrimgeour, G, Perry, M. Nucleotide metabolism. 5th ed: Pearson publishing,
2011)
Purine Catabolism
Nucleic acid can be disintegrated to be nucleotide, nucleoside or base as
shown in Figure 10. Purine base can be recycled to be nucleotide through
salvage pathway. Some other degraded products are further catabolised to
excrete in a form of uric acid. Adenosine deaminase (ADA) and purine
nucleoside phosphorylase (PNP) are two crucial enzymes for purine catabolism.
Loss of ADA or PNP activity leads to the excess of some deoxynucleotides i.e.
dATP or dGTP so that the immunity of affected child is substantially
compromised. Xanthine, Hypoxanthine, guanine are subjected to either
guanase or xanthine oxidase to produce uric acid.
Hyperuricemia and Gouty Arthritis
Uric is the final catabolite that is excreted by kidney in a form of urate salt.
Imbalance of uric acid production and uric excretion leads to increased uric acid
in the blood circulation, hyperuricemia, so that the urate crystal deposits in soft
tissue, joint and kidney, and can cause acute attack of joint inflammation, called
gouty arthritis. Hyperuricemia is caused by two main mechanisms i.e.
overproduction of uric acid and impaired excretion of urate crystal.
7 Figure 10: Purine catabolism pathway
(modified from Moran LA, Horton, RA, Scrimgeour, G, Perry, M. Nucleotide metabolism. 5th ed: Pearson publishing,
2011)
Three conditions can lead to overproduction of urate crystal
1. The excess of purine nucleotides from a defect in an enzyme in purine
salvage pathway. Deficiency in HGPRT is attributable to excess of
hypoxanthine and guanosine.
2. Tumour lysis syndrome is commonly found in patients with malignancy
who are on chemotherapy. Chemotherapy conjures up an acute tumour
cell death and the nucleic acid from lysed nuclei thereafter.
3. Deficiency in glucose-6-phosphatase causes increased glucose-6phosphate in which the excess of G-6-P stimulates pentose phosphate
pathway and the production of PRPP, an allosteric effector of purine de
novo synthesis.
Uric acid is filtered through glomeruli in the kidney and the excretion also
depends upon the balance between tubular secretion and tubular reabsorption
of urate salt at the proximal tubule. There is a polymorphism on ATP binding
cassette subfamily G member 2 (ABCG2), a urate transporter responsible for
tubular secretion (Figure 11). This non-synonymous mutation at amino acid
114 reduces renal urate excretion by 50%, and this polymorphism is
accountable for 10% of patient with gouty arthritis in Asia.
Treatment for acute gouty arthritis is Colchicine, an anti-inflammatory
molecule extracted from Colchicium autummale plant. Colchicine not only
reduces inflammation but also inhibit cell proliferation. The second medication
is allopurinol, a competitive inhibitor of xanthine oxidase. Reduced xanthine
oxidase activity leads to the reduction of uric acid with accumulation of
xanthine. Accumulated xanthine dissolves in the urine and is more amenable
for excretion compared to urate salt.
8 Figure 11: Uric excretion
(modified from Terkeltaub R. Update on gout: new therapeutic strategies and options. Nat Rev Rheumatol. 2010; 6(1): 30-8)
Pyrimidine catabolism
Uridine and thymidine can be catalysed by 5’ nucleotidase and nucleoside
phosphorylase, while cytidine or cytosine requires deaminase to convert them
to uridine or uracil (Figure 12). Uracil and thymine can be further degraded to
β-alanine (β-ALA) or β-aminoisobutyric acid (β-AIBA). Both are inessential
amino acid.
Figure 12: Catabolism of pyrimidine nucleotide
(modified from Moran LA, Horton, RA, Scrimgeour, G, Perry, M. Nucleotide metabolism. 5th ed: Pearson publishing,
2011)
9 Medication involving in nucleotide metabolism
1. Antimetabolite is an analogue of purine or pyrimidine in which this molecule
can interfere with nucleotide synthesis. This antimetabolite is typically used
to suppress tumour growth
a. 5-Fluorouracil; derivatives of this analogue can inhibit tumour
growth through 1) Inhibition of thymidylate synthase, essential
enzyme for dTMP synthesis, as shown in figure 9. 2) Promote
DNA damage through DNA repair mechanism. 3) Inhibit splicing
process (Figure 13).
b. 6-mercaptopurine is used to synthesise 6-MP monophosphate,
active analogue interfere with de novo purine synthesis pathway
(Figure 14).
Figure 13: Effect of 5-Fluorouracil on nucleotide metabolism
(modified from Moran LA, Horton, RA, Scrimgeour, G, Perry, M. Nucleotide metabolism. 5th ed: Pearson publishing,
2011)
Figure 14: Effect of 6-mercaptopurine on nucleotide metabolism
(modified from Moran LA, Horton, RA, Scrimgeour, G, Perry, M. Nucleotide metabolism. 5th ed: Pearson publishing,
2011)
10 2. Antifolate; Structure of this analogue is similar to the structure of folate so
that the antifolate agent can competitively impede the production of
tetrahydrofolate (THF). Reduction in THF interferes with thymidylate
synthase activity, and therefore inhibit DNA synthesis (Figure 13)
3. Glutamine antagonist; this analogue interferes the reaction requiring
gutamine, including conversion of IMP to GMP (Figure 3), conversion of
UTP to CTP (Figure 6).
4. Hydroxyurea; Anti-proliferative agent inhibit ribonucleotide reductase activity
so that DNA synthesis is impaired, especially in highly proliferative cell.
5. Antiviral medication
a. Acycloguanosine (Acyclovir); Acycloguanosine mono-, di-, triphosphate can only be produced by Herpes simplex virus specific
thymidine kinase. Acycloguanosine triphosphate is a substrate for
HSV DNA polymerase and the incorporation of this molecule
causes inibition of DNA synthesis process.
b. 3’-deoxyazidothymidine (AZT) can suppress proliferation of HIV
through HIV DNA polymerase.
Biosynthesis of nucleotide coenzyme
Nicotinamide adenine dinucleotide (NAD), Flavin adenine dinucleotide (FAD)
and Coenzyme A (CoA) are coenzymes essential for metabolism and
biosynthesis in mammalian cell.
1. Nicotinamide adenine dinucleotide (NAD) can be synthesised from
dietary nicotinic acid (Na), nicotinamide (Nm) or tryptophan (Trp) (Figure
15). Deficiency in Na or Typ causes Pellagra, the clinical syndrome
consisting of diarrhea, dermatitis and dementia.
Figure 15: Biosynthesis of Nicotinamide adenine dinucleotide
(modified from 1. Bogan KL, Brenner C. Nicotinic acid, nicotinamide, and nicotinamide riboside: a molecular evaluation of NAD+
precursor vitamins in human nutrition. Annu Rev Nutr. 2008; 28: 115-30. 2. Garten A, Petzold S, Korner A, Imai S, Kiess W.
Nampt: linking NAD biology, metabolism and cancer. Trends Endocrinol Metab. 2009; 20(3): 130-8)
11 2. Flavin adenine dinucleotide (FAD) is synthesised from riboflavin (Figure
16)
Figure 16: Biosynthesis of Flavin adenine dinucleotide
(modified from Berg JM, Tymoczko, J.L., Stryer, L., Gatto Jr. G J. Biochemistry. 7th ed. New York: W.H. Freeman and Company;
2012)
3. Coenzyme A is synthesised from pantothenate using multistep enzyme.
Cysteine is an amino acid required for the reaction (Figure 17).
Figure 17: Biosynthesis of Coenzyme A
(modified from Martinelli LK, Aldrich CC. Antimetabolite poisoning of cofactor biosynthesis. Chem Biol. 2012; 19(5): 543-4)
12 References
1.
Moran L, Horton R, Scrimgeour G, Perry M. Nucleotide Metabolism.
Principles of Biochemistry. 5th ed: Prentice Hall; 2011. p. 550-72.
2.
Berg JM, Tymoczko, J.L., Stryer, L., Gatto Jr. G J. Biochemistry. 7th ed.
Stryer L, editor. New York: W.H. Freeman and Company; 2012.
3.
Bogan KL, Brenner C. Nicotinic acid, nicotinamide, and nicotinamide
riboside: a molecular evaluation of NAD+ precursor vitamins in human nutrition.
Annu Rev Nutr. [Review]. 2008;28:115-30.
4.
Dorsam RT, Kunapuli SP. Central role of the P2Y12 receptor in platelet
activation. J Clin Invest. [Review]. 2004 Feb;113(3):340-5.
5.
Dzeja P, Terzic A. Adenylate kinase and AMP signaling networks:
Metabolic monitoring, signal communication and body energy sensing. Int J Mol
Sci. 2009 Apr;10(4):1729-72.
6.
Fairman JW, Wijerathna SR, Ahmad MF, Xu H, Nakano R, Jha S, et al.
Structural basis for allosteric regulation of human ribonucleotide reductase by
nucleotide-induced oligomerization. Nat Struct Mol Biol. [Research Support,
N.I.H., Extramural Research Support, Non-U.S. Gov't]. 2011 Mar;18(3):316-22.
7.
Garten A, Petzold S, Korner A, Imai S, Kiess W. Nampt: linking NAD
biology, metabolism and cancer. Trends Endocrinol Metab. [Research Support,
N.I.H., Extramural Research Support, Non-U.S. Gov't Review]. 2009
Apr;20(3):130-8.
8.
Logan DT. Closing the circle on ribonucleotide reductases. Nat Struct
Mol Biol. [Comment News]. 2011 Mar;18(3):251-3.
9.
Martinelli LK, Aldrich CC. Antimetabolite poisoning of cofactor
biosynthesis. Chem Biol. [Comment]. 2012 May 25;19(5):543-4.
10.
Miles EW, Rhee S, Davies DR. The molecular basis of substrate
channeling. J Biol Chem. [Review]. 1999 Apr 30;274(18):12193-6.
11.
Moran LA, Horton, R. A., Scrimgeour, G, Perry, M. Nucleotide
metabolism. 5th ed. Moran LA, editor: Pearson publishing; 2011.
12.
Papinazath T. THE EFFECTS OF PURINE NUCLEOSIDE
PHOSPHORYLASE (PNP) DEFICIENCY ON THYMOCYTE DEVELOPMENT.
Toronto: University of Toronto; 2010.
13.
Terkeltaub R. Update on gout: new therapeutic strategies and options.
Nat Rev Rheumatol. [Research Support, U.S. Gov't, Non-P.H.S. Review]. 2010
Jan;6(1):30-8.
13