PowerPoint Presentation - Pathway Engineered Enzymatic De Novo

							            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


Chem258 Xiayun Cheng
                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   Phosphoester linkage
     Why Need Isotopic Labeled
          Nucleotides?
•     and 2H labeled ribonucleotides have
    13C

  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
 15NH Cl – sole nitrogen source
       4
 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
• Pyrimidines: assembled first and then
attached to ribose


                                              First
• Purines: directly assembled on already
  formed ribose ring

                                                First


• Deoxyribonucleotides are synthesized from
ribonucleotides by reduction at 3’
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
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:
Coenzymes for Oxidation/Reduction reaction




                        =


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




Nicotinamide adenine                       Nicotinamide adenine
dinucleotide                               dinucleotide phosphate
      (NAD+)                                       (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




Creatine phosphate
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
           Starting Materials




Black: stoichiometric isotopically labeled reagents
Red: phosphate and oxidizing equivalents as the driving force
Blue: recycled cofactors
List of Enzymes
    Products Synthesized




  13C-C-2,8-ATP   U-15N-GTP




U-13C,15N-GTP     U-13C-GTP
                57%


                      β-13C-Serine


13C-C-2,8-ATP          23 enzymes
            24%
                  15NH Cl
                       4
                  15N-glutamine

                  24 enzymes

U-15N-GTP
                      13C-glucose
                42%   15NH Cl
                           4
                      13C/15N-serine

                      NaH13CO3
                      27 enzymes
U-13C,15N-GTP
                  13C-glucose

            66%   15NH Cl
                       4
                  13C/15N-serine

                  NaH13CO3
U-13C-GTP         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