Enzymatic synthesis of NAD+ with the specific incorporation of atomic labels

Kathleen A. Rising, Vern L. Schramm

Research output: Contribution to journalArticle

38 Citations (Scopus)

Abstract

An enzymatic synthesis is described for the production of NAD+ labeled with a radioactive or stable isotope at any desired position in the AMP or NMN+ portions of the molecule. In the first step, ten enzyme-catalyzed reactions are coupled for the synthesis of nicotinic acid adenine dinucleotide (NaAD+) from glucose, nicotinic acid, and ATP. NAD+ is formed from NaAD+ and glutamine in the second step. Oxidized nicotinamide adenine dinucleotide was synthesized with 3H, 14C, or 15N label specifically incorporated in the ribose or nicotinamide of the NMN+ portion of NAD+ as [H(N)1'-3H]NAD+, [H(N)2'-3H]NAD+, [H(N)4'-3H]NAD+, [H(N)5'-3H]NAD+, [C(N)1'-14C]NAD+, [C(N)5'-14C]NAD+, [N(N)1-15N, C(N)1'-14C]NAD+, and [N(N)1-15N, C(N)5'-14C]NAD+. Nuclear magnetic resonance spectroscopy of [H(N)2'-2H]NAD+ as well as enzymatic degradation were used to verify the position of labels. Appropriately labeled glucose, ribose 5-phosphate, or nicotinic acid were the starting materials and were converted to NAD+ using enzymes from the pentose pathway and the pathway for NAD+ de novo synthesis. Yields of purified NAD+ to 96% were obtained from starting glucose. The labeled NAD+ is catalytically competent and is chromatographically and spectrophotometrically indistinguishable from authentic NAD+. By using specifically labeled ATP as a precursor (Parkin, D.W.; Schramm, V.L. Biochemistry 1987, 26, 913-920), the method is readily adaptable for the synthesis of NAD+ with single or multiple atomic labels at various positions in the AMP portion of the molecule. NAD+ was synthesized from [8-14C]ATP to give [C(A)8-14C]NAD+ as an example. Together these methods provide a general scheme for the efficient synthesis of NAD+ of high purity with 3H, 14C, or other labels at any nonexchangeable position of the NMN+ or AMP portions of the NAD+ molecule.

Original languageEnglish (US)
Pages (from-to)6531-6536
Number of pages6
JournalJournal of the American Chemical Society
Volume116
Issue number15
DOIs
StatePublished - Jul 27 1994

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NAD
Labels
Adenosinetriphosphate
Glucose
Molecules
Acids
Enzymes
Biochemistry
Nuclear magnetic resonance spectroscopy
Isotopes
Phosphates
Adenosine Monophosphate
Degradation
Adenosine Triphosphate
Niacin
Pentoses
Ribose
Niacinamide

Keywords

  • biosynthesis
  • isotope
  • NAD synthetase
  • NADH
  • NMN
  • radiolabeled

ASJC Scopus subject areas

  • Chemistry(all)

Cite this

Enzymatic synthesis of NAD+ with the specific incorporation of atomic labels. / Rising, Kathleen A.; Schramm, Vern L.

In: Journal of the American Chemical Society, Vol. 116, No. 15, 27.07.1994, p. 6531-6536.

Research output: Contribution to journalArticle

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abstract = "An enzymatic synthesis is described for the production of NAD+ labeled with a radioactive or stable isotope at any desired position in the AMP or NMN+ portions of the molecule. In the first step, ten enzyme-catalyzed reactions are coupled for the synthesis of nicotinic acid adenine dinucleotide (NaAD+) from glucose, nicotinic acid, and ATP. NAD+ is formed from NaAD+ and glutamine in the second step. Oxidized nicotinamide adenine dinucleotide was synthesized with 3H, 14C, or 15N label specifically incorporated in the ribose or nicotinamide of the NMN+ portion of NAD+ as [H(N)1'-3H]NAD+, [H(N)2'-3H]NAD+, [H(N)4'-3H]NAD+, [H(N)5'-3H]NAD+, [C(N)1'-14C]NAD+, [C(N)5'-14C]NAD+, [N(N)1-15N, C(N)1'-14C]NAD+, and [N(N)1-15N, C(N)5'-14C]NAD+. Nuclear magnetic resonance spectroscopy of [H(N)2'-2H]NAD+ as well as enzymatic degradation were used to verify the position of labels. Appropriately labeled glucose, ribose 5-phosphate, or nicotinic acid were the starting materials and were converted to NAD+ using enzymes from the pentose pathway and the pathway for NAD+ de novo synthesis. Yields of purified NAD+ to 96{\%} were obtained from starting glucose. The labeled NAD+ is catalytically competent and is chromatographically and spectrophotometrically indistinguishable from authentic NAD+. By using specifically labeled ATP as a precursor (Parkin, D.W.; Schramm, V.L. Biochemistry 1987, 26, 913-920), the method is readily adaptable for the synthesis of NAD+ with single or multiple atomic labels at various positions in the AMP portion of the molecule. NAD+ was synthesized from [8-14C]ATP to give [C(A)8-14C]NAD+ as an example. Together these methods provide a general scheme for the efficient synthesis of NAD+ of high purity with 3H, 14C, or other labels at any nonexchangeable position of the NMN+ or AMP portions of the NAD+ molecule.",
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N2 - An enzymatic synthesis is described for the production of NAD+ labeled with a radioactive or stable isotope at any desired position in the AMP or NMN+ portions of the molecule. In the first step, ten enzyme-catalyzed reactions are coupled for the synthesis of nicotinic acid adenine dinucleotide (NaAD+) from glucose, nicotinic acid, and ATP. NAD+ is formed from NaAD+ and glutamine in the second step. Oxidized nicotinamide adenine dinucleotide was synthesized with 3H, 14C, or 15N label specifically incorporated in the ribose or nicotinamide of the NMN+ portion of NAD+ as [H(N)1'-3H]NAD+, [H(N)2'-3H]NAD+, [H(N)4'-3H]NAD+, [H(N)5'-3H]NAD+, [C(N)1'-14C]NAD+, [C(N)5'-14C]NAD+, [N(N)1-15N, C(N)1'-14C]NAD+, and [N(N)1-15N, C(N)5'-14C]NAD+. Nuclear magnetic resonance spectroscopy of [H(N)2'-2H]NAD+ as well as enzymatic degradation were used to verify the position of labels. Appropriately labeled glucose, ribose 5-phosphate, or nicotinic acid were the starting materials and were converted to NAD+ using enzymes from the pentose pathway and the pathway for NAD+ de novo synthesis. Yields of purified NAD+ to 96% were obtained from starting glucose. The labeled NAD+ is catalytically competent and is chromatographically and spectrophotometrically indistinguishable from authentic NAD+. By using specifically labeled ATP as a precursor (Parkin, D.W.; Schramm, V.L. Biochemistry 1987, 26, 913-920), the method is readily adaptable for the synthesis of NAD+ with single or multiple atomic labels at various positions in the AMP portion of the molecule. NAD+ was synthesized from [8-14C]ATP to give [C(A)8-14C]NAD+ as an example. Together these methods provide a general scheme for the efficient synthesis of NAD+ of high purity with 3H, 14C, or other labels at any nonexchangeable position of the NMN+ or AMP portions of the NAD+ molecule.

AB - An enzymatic synthesis is described for the production of NAD+ labeled with a radioactive or stable isotope at any desired position in the AMP or NMN+ portions of the molecule. In the first step, ten enzyme-catalyzed reactions are coupled for the synthesis of nicotinic acid adenine dinucleotide (NaAD+) from glucose, nicotinic acid, and ATP. NAD+ is formed from NaAD+ and glutamine in the second step. Oxidized nicotinamide adenine dinucleotide was synthesized with 3H, 14C, or 15N label specifically incorporated in the ribose or nicotinamide of the NMN+ portion of NAD+ as [H(N)1'-3H]NAD+, [H(N)2'-3H]NAD+, [H(N)4'-3H]NAD+, [H(N)5'-3H]NAD+, [C(N)1'-14C]NAD+, [C(N)5'-14C]NAD+, [N(N)1-15N, C(N)1'-14C]NAD+, and [N(N)1-15N, C(N)5'-14C]NAD+. Nuclear magnetic resonance spectroscopy of [H(N)2'-2H]NAD+ as well as enzymatic degradation were used to verify the position of labels. Appropriately labeled glucose, ribose 5-phosphate, or nicotinic acid were the starting materials and were converted to NAD+ using enzymes from the pentose pathway and the pathway for NAD+ de novo synthesis. Yields of purified NAD+ to 96% were obtained from starting glucose. The labeled NAD+ is catalytically competent and is chromatographically and spectrophotometrically indistinguishable from authentic NAD+. By using specifically labeled ATP as a precursor (Parkin, D.W.; Schramm, V.L. Biochemistry 1987, 26, 913-920), the method is readily adaptable for the synthesis of NAD+ with single or multiple atomic labels at various positions in the AMP portion of the molecule. NAD+ was synthesized from [8-14C]ATP to give [C(A)8-14C]NAD+ as an example. Together these methods provide a general scheme for the efficient synthesis of NAD+ of high purity with 3H, 14C, or other labels at any nonexchangeable position of the NMN+ or AMP portions of the NAD+ molecule.

KW - biosynthesis

KW - isotope

KW - NAD synthetase

KW - NADH

KW - NMN

KW - radiolabeled

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