Biosynthesis and degradation of nucleotides

1. Biosynthesis and degradation of nucleotides • Nucleotides are precursors of DNA and RNA, essential carriers of energy as ATP, GTP, NAD, FAD, CoA, etc., signaling mechanisms, and activated biosynthetic precursors. • Two pathways lead to nucleotide synthesis • de novo • salvage

2. de novo nucleotide synthesis • Appears identical among all organisms • Bases (guanine, etc.) are NOT intermediates in pathway • Purine rings not synthesized and attached to ribose, assembled on the ribose • Pyrimidine synthesized as orotate, attached to ribose phosphate and converted to nucleotides

3. Pyrimidines and purines share precursors • Phosphoribosyl pyrophosphate (PRPP) is a key intermediate for both (also involved in tryptophan and histidine synthesis also) • Amino acids are important precursors, glycine for purines, and aspartate for pyrimidines • Also, glutamine and aspartate serve as sources of amino groups in both purine and pyrimidine biosynthesis

4. PRPP serves as the foundation for purine nucleotide biosynthesis

5. Three atoms from glycine are added to the new amino group

6. This chain is extended by formate addition

7. An amine group is donated by glutamine

8. The FGAM ring is closed to form AIR

9. AIR is carboxylated to CAIR(unique because doesn’t use biotin)

10. A mutase rearranges the carboxylate

11. Aspartate donates an amino group in two steps to form AICAR

12. Another formate group is donated, carried by THF

13. Ring closure forms Inosinate (IMP)

14. Summary of purine atom origins

15. IMP is converted to purine nucleotides

16. Regulation of purine biosynthesis • Three major feedback loops • Primary regulation is AMP, GMP, and IMP inhibiting glutamine-PRPP amidotransferase (the first committed step) • IMP branchpoint to AMP and GMP is regulated independently by end products • Additional regulation is inhibition of PRPP synthesis (using ADP or GDP)

18. Pyrimidine biosynthesis from aspartate, PRPP, and carbamoyl phosphate • Base (as orotate) is made first then attached to ribose 5-phosphate • Orotate synthesis begins with aspartate reacting with carbamoyl phosphate to form a product which is cyclized to Dihydroorotate • Dihydroorotate is oxidized to orotate, which reacts with PRPP • This product can undergo subsequent reactions to form UMP, UTP, and CTP

20. Pyrimidine biosynthesis regulation • Mostly through the allosteric behavior of aspartate transcarbamoylase, which catalyzes the first step and is inhibited by CTP (inhibition can be prevented by ATP)

21. Nucleoside monophosphates are converted to nucleoside triphosphates • AMP  ADP (adenylate kinase) • ATP + NMP  ADP + NDP (nucleoside monophosphate kinases) • Nucleoside diphosphate kinase converts nucleoside diphosphates to triphosphates (generally ATP is phosphate donor)

22. From these pathways, you note that ribonucleotides are being generated • To get deoxyribonucleotides (precursors of DNA), the 2’ carbon atom must be reduced • Accomplished by an interesting enzyme ribonucleotide reductase • A pair of hydrogen atoms originating from NADPH are passed to ribonucleotide reductase by either glutaredoxin or thioredoxin to generate an activated enzyme intermediate

24. Ribonucleotide reductase catalytic mechanism includes free radicals

25. Regulation of ribonucleotide reductase • Both activity and substrate specificity is modulated by binding of effector molecules • At one binding site: ATP activates enzyme; dATP inactivates enzyme A second binding site monitors substrate binding

27. dTMP is generated from dUMP