Chemienzymatic synthesis of uridine nucleotides labeled with [15N] and [13C].
UTP, labeled with 15N and 13C (at all carbon atoms of the ribose moiety), was obtained enzymatically from [15N]uracil and [13C6]glucose. Eleven enzymes and suitable substrates reconstituted a metabolic pathway in which glucose was first transformed to 5-phosphoribosyl-1-pyrophosphate. The latter compound plus uracil yielded UMP in a second step by the reaction catalyzed by uracil phosphoribosyltransferase. UMP was subsequently phosphorylated to the corresponding di- and triphosphate. ATP, required for five phosphorylation reactions, was regenerated from creatine phosphate, whereas NADP+ necessary for the oxidation of glucose 6-phosphate and 6-phosphogluconate was recycled by glutamate dehydrogenase and excess of ammonia and alpha-oxoglutarate. Despite the number and complexity of the enzymatic steps, the synthesis of [15N, 13C]UTP is straightforward with an overall yield exceeding 60%. This method, extended and diversified to the synthesis of all natural ribonucleotides, is a more economical alternative for obtaining nucleic acids for structural analysis by heteronuclear NMR spectroscopy.
Gilles
A M
AM
Unité de Biochimie des Régulations Cellulaires, Institut Pasteur, Paris, France.
Cristea
I
I
Palibroda
N
N
Hilden
I
I
Jensen
K F
KF
Sarfati
R S
RS
Namane
A
A
Ughetto-Monfrin
J
J
Bârzu
O
O
D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism; D002247:Carbon Isotopes; D004798:Enzymes / Q000378:metabolism; D005656:Fungal Proteins / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D007553:Isotope Labeling / Q000379:methods*; D009682:Magnetic Resonance Spectroscopy; D009249:NADP / Q000378:metabolism; D009587:Nitrogen Isotopes; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000378:metabolism; D012274:Ribulosephosphates / Q000096:biosynthesis; D014498:Uracil / Q000378:metabolism; D014530:Uridine Diphosphate / Q000096:biosynthesis; D014542:Uridine Monophosphate / Q000096:biosynthesis; D014544:Uridine Triphosphate / Q000096:biosynthesis* / Q000737:chemistry
D001426:Bacterial Proteins; D002247:Carbon Isotopes; D004798:Enzymes; D005656:Fungal Proteins; D009587:Nitrogen Isotopes; D011994:Recombinant Proteins; D012274:Ribulosephosphates; C031524:ribulose 5-phosphate; D009249:NADP; D014498:Uracil; D014530:Uridine Diphosphate; D000255:Adenosine Triphosphate; D014542:Uridine Monophosphate; D005947:Glucose; D014544:Uridine Triphosphate