Nucleotide Metabolism

Transcription

1 Nucleotide Metabolism Dr. Chalermchai Mitrpant Outline Content Page Function of nucleotide 2 Nucleotide synthesis 3-6 Purine synthesis and its regulation 3-4 Pyrimidine synthesis and its regulation 5-6 Synthesis of deoxyribonucleotide and its regulation 6-7 Purine Catabolism 6-9 Hyperuricemia and Gouty Arthritis 7-9 Pyrimidine catabolism 9 Medication involving in nucleotide metabolism Biosynthesis of nucleotide coenzyme References 13 1

2 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 2

3 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 (SO 4 - ), 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 Figure 3: De novo purine synthesis 3

4 Figure 4: Regulation of de novo purine synthesis 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-1- P) 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 4

5 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 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

6 Figure 7: Salvage pyrimidine pathway 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): 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): Moran LA, Horton, RA, Scrimgeour, G, Perry, M. Nucleotide metabolism. 5th ed: Pearson publishing; 6

7 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 (CH 3 ) for dump. Figure 9: Synthesis of dttp 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

8 Figure 10: Purine catabolism pathway 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-6- phosphate 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

9 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 9

10 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 Figure 14: Effect of 6-mercaptopurine on nucleotide metabolism 10

11 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: 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

12 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

13 References 1. Moran L, Horton R, Scrimgeour G, Perry M. Nucleotide Metabolism. Principles of Biochemistry. 5th ed: Prentice Hall; p Berg JM, Tymoczko, J.L., Stryer, L., Gatto Jr. G J. Biochemistry. 7 th ed. Stryer L, editor. New York: W.H. Freeman and Company; 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: Dorsam RT, Kunapuli SP. Central role of the P2Y12 receptor in platelet activation. J Clin Invest. [Review] Feb;113(3): Dzeja P, Terzic A. Adenylate kinase and AMP signaling networks: Metabolic monitoring, signal communication and body energy sensing. Int J Mol Sci Apr;10(4): 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] Mar;18(3): 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] Apr;20(3): Logan DT. Closing the circle on ribonucleotide reductases. Nat Struct Mol Biol. [Comment News] Mar;18(3): Martinelli LK, Aldrich CC. Antimetabolite poisoning of cofactor biosynthesis. Chem Biol. [Comment] May 25;19(5): Miles EW, Rhee S, Davies DR. The molecular basis of substrate channeling. J Biol Chem. [Review] Apr 30;274(18): Moran LA, Horton, R. A., Scrimgeour, G, Perry, M. Nucleotide metabolism. 5 th ed. Moran LA, editor: Pearson publishing; Papinazath T. THE EFFECTS OF PURINE NUCLEOSIDE PHOSPHORYLASE (PNP) DEFICIENCY ON THYMOCYTE DEVELOPMENT. Toronto: University of Toronto; Terkeltaub R. Update on gout: new therapeutic strategies and options. Nat Rev Rheumatol. [Research Support, U.S. Gov't, Non-P.H.S. Review] Jan;6(1):