Lincoln Scott - Pathway engineered enzymatic de novo purine nucleotide synthesis.

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  ACS Chem Biol. 2008 Aug   15;3(8):499-511. doi: 10.1021/cb800066p.


  A general method for isotopic labeling of the purine base moiety   of nucleotides and RNA has been developed through biochemical   pathway engineering in vitro. A synthetic scheme was designed and   implemented utilizing recombinant enzymes from the pentose   phosphate and de novo purine synthesis pathways, with   regeneration of folate, aspartate, glutamine, ATP, and NADPH   cofactors, in a single-pot reaction. Syntheses proceeded quickly   and efficiently in comparison to chemical methods with isolated   yields up to 66% for 13C-, 15N-enriched ATP and GTP. The scheme   is robust and flexible, requiring only serine, NH4+, glucose, and   CO2 as stoichiometric precursors in labeled form. Using this   approach, U-13C- GTP, U-13C, 15N- GTP, 13C 2,8- ATP, and U-15N-   GTP were synthesized on a millimole scale, and the utility of the   isotope labeling is illustrated in NMR spectra of HIV-2   transactivation region RNA containing 13C 2,8-adenosine and 15N   1,3,7,9,2-guanosine. Pathway engineering in vitro permits complex   synthetic cascades to be effected, expanding the applicability of   enzymatic synthesis.

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