Chem258 Xiayun Cheng

Pathway Engineered Enzymatic de Novo Purine Nucleotide S

ynthesis

Heather L. Schultheisz, Blair R. Szymczyna, Lincoln G. Scott, and J

ames R. Williamson

ACS Chem. Biol., 2008, 3 (8), 499-511

Outline

• Enzymatic synthesis

• Importance of making isotopically labeled nucleotides

• The chemistry of nucleotide biosynthesis

• Discussion of paper

• Conclusion

Enzymatic SynthesisOrganocatalysis?Mild, usually at ambient temperature and

atmospheric pressure Stereoselective and regioselective Capable of generating a wide variety of ch

iral compounds by using different classes of enzymes

Has been applied to many biomolecules and pharmaceuticals

Structures of Nucleotides

Phosphoester linkage Phosphoester linkage

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

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

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

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 stepPRPP is synthesized from ribose-5-phosphate from the pentose phosphate pathway

Pentose phosphate pathway

Purine Nucleotide Biosynthesis: de novo 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

Purine Nucleotide Biosynthesis: de novo pathway

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

Coenzymes for Oxidation/Reduction reaction

Nicotinamide adenine dinucleotide (NAD+)

Nicotinamide adenine dinucleotide phosphate

(NADP)

NADH is oxidized by the respiratory chain to generate ATPNADPH 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 serine13C-N10-formyl-THF: recyled from tetrahydrofolate, 13C of serine incorporated into 13C-N10-formyl-THFAspartate: recycled from fumarateGlutamine: recycled from α-ketoglutarate

Starting Materials

Black: stoichiometric isotopically labeled reagentsRed: phosphate and oxidizing equivalents as the driving forceBlue: recycled cofactors

List of Enzymes

U-15N-GTP13C-C-2,8-ATP

U-13C,15N-GTP U-13C-GTP

Products Synthesized

13C-C-2,8-ATP

β-13C-Serine

57%

23 enzymes

U-15N-GTP

15NH4Cl15N-glutamine24 enzymes

24%

U-13C,15N-GTP

13C-glucose15NH4Cl13C/15N-serineNaH13CO3

42%

27 enzymes

U-13C-GTP

13C-glucose15NH4Cl13C/15N-serineNaH13CO3

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