Chem258 Xiayun Cheng 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),

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

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

Structures of Nucleotides Phosphoester linkage

Why Need Isotopic Labeled Nucleotides? 13 C and 2 H labeled ribonucleotides have been used for NMR studies of RNA structures 13 C and 15 N 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

Synthesis of 13 C and 15 N labeled Nucleotides : Traditional Method Obtained from bacteria grown on a minimal medium 15 NH 4 Cl – sole nitrogen source 13 C-glucose – only carbon source Advantage: easy; good for large scale synthesis Weakness: Uniformly labeled; specific isotopic labeling patterns impossible

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

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

Pyrimidine Nucleotide Biosynthesis: de novo pathway Side chain of Gln

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

Purine Nucleotide Biosynthesis: de novo pathway

Synthesis of purine nucleotide ‘foundation’: Glutamine phosphoribosyl amidotransferase

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

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

Purine Nucleotide Biosynthesis: de novo pathway

AMP GMP

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

Nicotinamide adenine dinucleotide (NAD + ), = 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

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

Creatine phosphate Creatine

Glycine: from serine 13 C-N 10 -formyl-THF: recyled from tetrahydrofolate, 13 C of serine incorporated into 13C-N10-formyl-THF Aspartate: recycled from fumarate Glutamine: recycled from α-ketoglutarate

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

List of Enzymes

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

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

U- 15 N-GTP 15 NH 4 Cl 15 N-glutamine 24 enzymes 24%

U- 13 C, 15 N-GTP 13 C-glucose 15 NH 4 Cl 13 C/ 15 N-serine NaH 13 CO 3 42% 27 enzymes

U- 13 C-GTP 13 C-glucose 15 NH 4 Cl 13 C/ 15 N-serine NaH 13 CO 3 66% 26 enzymes

NMR Studies of Products

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