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Pathway Engineered Enzymatic de Novo Purine Nucleotide Synthesis

Pathway Engineered Enzymatic de Novo Purine Nucleotide Synthesis. Heather L. Schultheisz, Blair R. Szymczyna, Lincoln G. Scott, and James R. Williamson. ACS Chem. Biol. , 2008 , 3 (8), 499-511. Outline. Enzymatic synthesis Importance of making isotopically labeled nucleotides

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Pathway Engineered Enzymatic de Novo Purine Nucleotide Synthesis

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  1. Pathway Engineered Enzymatic de Novo Purine Nucleotide Synthesis Heather L. Schultheisz, Blair R. Szymczyna, Lincoln G. Scott, and James R. Williamson ACS Chem. Biol., 2008, 3 (8), 499-511

  2. Outline • Enzymatic synthesis • Importance of making isotopically labeled nucleotides • The chemistry of nucleotide biosynthesis • Discussion of paper • Conclusion

  3. Enzymatic Synthesis • Organocatalysis? • Mild, usually at ambient temperature and atmospheric pressure • Stereoselective and regioselective • Capable of generating a wide variety of chiral compounds by using different classes of enzymes • Has been applied to many biomolecules and pharmaceuticals

  4. Structures of Nucleotides Phosphoester linkage Phosphoester linkage

  5. Why Need Isotopic Labeled Nucleotides? • 13C and 2H labeled ribonucleotides have been used for NMR studies of RNA structures • 13C and 15N labeled nucleotides are used in NMR studies of RNA structure and dynamics • Reduce space crowding – a ‘spectral filter’ or to simplify the dipolar network for relaxation studies

  6. Synthesis of 13C and 15N labeled Nucleotides : Traditional Method • Obtained from bacteria grown on a minimal medium 15NH4Cl – sole nitrogen source 13C-glucose – only carbon source • Advantage: easy; good for large scale synthesis • Weakness: Uniformly labeled; specific isotopic labeling patterns impossible

  7. Basis for in vitro Enzymatic Synthesis of Nucleotides: Nucleotide Biosynthesis • de novo pathway Beginning from simple starting materials (eg. amino acids, bicarbonate) • Salvage pathway Bases generated by degradation of nucleic acids can be salvaged and recycled eg. Adenine + PRPP → AMP + PPi PRPP: 5-Phosphoribosyl-1-pyrophosphate

  8. First First Nucleotide Biosynthesis: de novo pathway • Pyrimidines: assembled first and then attached to ribose • Purines: directly assembled on already formed ribose ring • Deoxyribonucleotides are synthesized from ribonucleotides by reduction at 3’

  9. Side chain of Gln Pyrimidine Nucleotide Biosynthesis: de novo pathway

  10. Purine Nucleotide Biosynthesis: de novo pathway 5-Phosphoribosyl-1-pyrophosphate (PRPP) • PRPP provides the foundation on which the purine bases are constructed step by step • PRPP is synthesized from ribose-5-phosphate from the pentose phosphate pathway Pentose phosphate pathway

  11. Purine Nucleotide Biosynthesis: de novo pathway

  12. Purine Nucleotide Biosynthesis: de novo pathway Synthesis of purine nucleotide ‘foundation’: Glutamine phosphoribosyl amidotransferase

  13. Purine Nucleotide Biosynthesis: de novo pathway • Activation Mode • Catalyzed by enzymes with ATP grasp domains • Activation of carbonyl oxygen via phosphorylation, followed by displacement of phosphoryl group by amine or ammonia as nucleophile

  14. Purine Nucleotide Biosynthesis: de novo pathway Assembly of the purine ring: Activation of Gly

  15. Purine Nucleotide Biosynthesis: de novo pathway

  16. Purine Nucleotide Biosynthesis: de novo pathway

  17. Purine Nucleotide Biosynthesis: de novo pathway AMP GMP

  18. Purine Nucleotide Biosynthesis: de novo pathway AMP and GMP from IMP:

  19. Coenzymes for Oxidation/Reduction reaction = Nicotinamide adenine dinucleotide (NAD+),

  20. Coenzymes for Oxidation/Reduction reaction Nicotinamide adenine dinucleotide (NAD+) Nicotinamide adenine dinucleotide phosphate (NADP) • NADH is oxidized by the respiratory chain to generate ATP • NADPH serves as a reductant in biosynthetic processes

  21. Design of Enzymatic Synthesis • PRPP from pentose phosphate pathway • Using well established cofactor recycling schemes due to lack of some isotopically labeled starting materials

  22. Creatine Creatine phosphate

  23. Glycine: from serine 13C-N10-formyl-THF: recyled from tetrahydrofolate, 13C of serine incorporated into 13C-N10-formyl-THF Aspartate: recycled from fumarate Glutamine: recycled from α-ketoglutarate

  24. Starting Materials Black: stoichiometric isotopically labeled reagents Red: phosphate and oxidizing equivalents as the driving force Blue: recycled cofactors

  25. List of Enzymes

  26. Products Synthesized U-15N-GTP 13C-C-2,8-ATP U-13C,15N-GTP U-13C-GTP

  27. 13C-C-2,8-ATP 57% β-13C-Serine 23 enzymes

  28. U-15N-GTP 24% 15NH4Cl 15N-glutamine 24 enzymes

  29. U-13C,15N-GTP 13C-glucose 15NH4Cl 13C/15N-serine NaH13CO3 42% 27 enzymes

  30. U-13C-GTP 13C-glucose 15NH4Cl 13C/15N-serine NaH13CO3 66% 26 enzymes

  31. NMR Studies of Products

  32. Conclusions • Combined metabolic pathways in vitro; accurately controlled isotopic labeling ; one pot procedure • 4 types of isotopically labeled nucleotide synthesized on 1μM scale, yield up to 66% • Expensive starting materials; enzymes complicated to purify and easily lose activity • Future work: more specific labeling (eg.single carbon or nitrogen); combination of chemical synthesis with biosynthetic pathways

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