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Pathway engineered enzymatic de novo purine nucleotide synthesis

Academic Article
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Overview

authors

  • Schultheisz, Heather
  • Szymczyna, B. R.
  • Scott, L. G.
  • Williamson, James

publication date

  • August 2008

journal

  • ACS Chemical Biology  Journal

abstract

  • 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.

subject areas

  • Adenosine Triphosphate
  • Carbon Isotopes
  • Chromatography, High Pressure Liquid
  • Cloning, Molecular
  • Enzymes
  • Escherichia coli
  • Guanosine Triphosphate
  • Molecular Structure
  • Nitrogen Isotopes
  • Plasmids
  • Protein Engineering
  • Purine Nucleotides
  • RNA
  • Substrate Specificity
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Identity

PubMed Central ID

  • PMC2746247

International Standard Serial Number (ISSN)

  • 1554-8929

Digital Object Identifier (DOI)

  • 10.1021/cb800066p

PubMed ID

  • 18707057
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Additional Document Info

start page

  • 499

end page

  • 511

volume

  • 3

issue

  • 8

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