Enzymatic synthesis of guanine nucleotides labeled with 15N at the 2-amino group of the purine ring.
GMP and dGMP labeled with 15N at the 2-amino group of the purine ring was obtained enzymatically from NH4Cl (> 99 at.% 15N) and from IMP or dIMP, respectively, by several reactions involving IMP-dehydrogenase, GMP-synthetase, adenylate kinase, and creatine kinase. The first three enzymes were obtained by overexpression in Escherichia coli of the corresponding genes. The isotope content of the primary amino group of guanine determined by mass spectrometry after acid hydrolysis of nucleotides was found higher than 98 at.% 15N. The proton NMR spectrum of [15N]GMP in solution in the absence of nitrogen decoupling showed a doublet with a coupling constant of 92 Hz. When nitrogen decoupling was used during the acquisition time, the doublet was replaced by a single peak at 6.47 ppm, indicating that the corresponding proton is bound to 15N.
Bouhss
A
A
Unité de Biochimie des Régulations Cellulaires, Institut Pasteur, Paris, France.
Sakamoto
H
H
Palibroda
N
N
Chiriac
M
M
Sarfati
R
R
Smith
J M
JM
Craescu
C T
CT
Bârzu
O
O
D000263:Adenylate Kinase; D019731:Carbon-Nitrogen Ligases; D003001:Cloning, Molecular; D003402:Creatine Kinase; D003848:Deoxyguanine Nucleotides / Q000138:chemical synthesis; D004926:Escherichia coli / Q000254:growth & development; D006150:Guanine Nucleotides / Q000096:biosynthesis / Q000138:chemical synthesis*; D006157:Guanosine Monophosphate / Q000138:chemical synthesis; D007168:IMP Dehydrogenase; D007202:Indicators and Reagents; D007291:Inosine Monophosphate; D007553:Isotope Labeling / Q000379:methods; D007700:Kinetics; D008025:Ligases; D013058:Mass Spectrometry / Q000379:methods; D009587:Nitrogen Isotopes; D009943:Organophosphorus Compounds; D011994:Recombinant Proteins
D003848:Deoxyguanine Nucleotides; D006150:Guanine Nucleotides; D007202:Indicators and Reagents; D009587:Nitrogen Isotopes; D009943:Organophosphorus Compounds; D011994:Recombinant Proteins; D007291:Inosine Monophosphate; C023477:diisopropyl methylphosphonate; C007257:2'-deoxyguanosine 5'-phosphate; D006157:Guanosine Monophosphate; D007168:IMP Dehydrogenase; D003402:Creatine Kinase; D000263:Adenylate Kinase; D008025:Ligases; D019731:Carbon-Nitrogen Ligases; C020828:GMP synthase (glutamine-hydrolyzing)