Pathway engineered enzymatic de novo purine nucleotide synthesis.
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.
Schultheisz
Heather L
HL
Department of Molecular Biology, The Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, MB33, La Jolla, California 92037, USA.
Szymczyna
Blair R
BR
Scott
Lincoln G
LG
Williamson
James R
JR
D000255:Adenosine Triphosphate / Q000138:chemical synthesis / Q000737:chemistry; D002247:Carbon Isotopes; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004798:Enzymes / Q000737:chemistry* / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006160:Guanosine Triphosphate / Q000138:chemical synthesis / Q000737:chemistry; D015394:Molecular Structure; D009587:Nitrogen Isotopes; D010957:Plasmids; D015202:Protein Engineering; D011685:Purine Nucleotides / Q000138:chemical synthesis* / Q000737:chemistry; D012313:RNA / Q000737:chemistry; D013379:Substrate Specificity
D002247:Carbon Isotopes; D004798:Enzymes; D009587:Nitrogen Isotopes; D011685:Purine Nucleotides; D012313:RNA; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate