nicotinamide-hypoxanthine-dinucleotide and nicotinamide-guanine-dinucleotide

Nicotinamide adenine dinucleotide (NAD) is an important coenzyme that regulates various metabolic pathways, including glycolysis, β-oxidation, and oxidative phosphorylation. Additionally, NAD serves as a substrate for poly(ADP-ribose) polymerase (PARP), sirtuin, and NAD glycohydrolase, and it regulates DNA repair, gene expression, energy metabolism, and stress responses. Many studies have demonstrated that NAD metabolism is deeply involved in aging and aging-related diseases. Previously, we demonstrated that nicotinamide guanine dinucleotide (NGD) and nicotinamide hypoxanthine dinucleotide (NHD), which are analogs of NAD, are significantly increased in Nmnat3-overexpressing mice. However, there is insufficient knowledge about NGD and NHD in vivo. In the present study, we aimed to investigate the metabolism and biochemical properties of these NAD analogs. We demonstrated that endogenous NGD and NHD were found in various murine tissues, and their synthesis and degradation partially rely on Nmnat3 and CD38. We have also shown that NGD and NHD serve as coenzymes for alcohol dehydrogenase (ADH) in vitro, although their affinity is much lower than that of NAD. On the other hand, NGD and NHD cannot be used as substrates for SIRT1, SIRT3, and PARP1. These results reveal the basic metabolism of NGD and NHD and also highlight their biological function as coenzymes.

Topics: Aging; Animals; Guanine Nucleotides; Guanosine Triphosphate; Inosine Triphosphate; Mice; NAD; Poly(ADP-ribose) Polymerases; Sirtuins

Cyclic ADP ribose (cADPR) is a Ca(2+)-mobilizing intracellular second messenger synthesized from NAD by ADP-ribosyl cyclases (ADPR cyclases). In animals, cADPR targets the ryanodine receptor present in the sarcoplasmic/endoplasmic reticulum to promote Ca(2+) release from intracellular stores to increase the concentration of cytosolic free Ca(2+) in Arabidopsis (Arabidopsis thaliana), and cADPR has been proposed to play a central role in signal transduction pathways evoked by the drought and stress hormone, abscisic acid, and the circadian clock. Despite evidence for the action of cADPR in Arabidopsis, no predicted proteins with significant similarity to the known ADPR cyclases have been reported in any plant genome database, suggesting either that there is a unique route for cADPR synthesis or that a homolog of ADPR cyclase with low similarity might exist in plants. We sought to determine whether the low levels of ADPR cyclase activity reported in Arabidopsis are indicative of a bona fide activity that can be associated with the regulation of Ca(2+) signaling. We adapted two different fluorescence-based assays to measure ADPR cyclase activity in Arabidopsis and found that this activity has the characteristics of a nucleotide cyclase that is activated by nitric oxide to increase cADPR and mobilize Ca(2.)

Topics: ADP-ribosyl Cyclase; Arabidopsis; Arabidopsis Proteins; Calcium; Cytosol; Guanine Nucleotides; NAD; Niacinamide; Nitric Oxide; Signal Transduction

Readily synthesized nicotinamide adenine dinucleotide (NAD(+)) analogues have been used to investigate aspects of the cyclization of NAD(+) to cyclic adenosine 5'-O-diphosphate ribose (cADPR) catalyzed by the enzyme adenosine 5'-O-diphosphate (ADP) ribosyl cyclase and to produce the first potent inhibitors of this enzyme. In all cases, inhibition of Aplysia californica cyclase by various substrate analogues was found to be competitive while inhibition by nicotinamide exhibited mixed-behavior characteristics. Nicotinamide hypoxanthine dinucleotide (NHD(+)), nicotinamide guanine dinucleotide (NGD(+)), C1'-m-benzamide adenine dinucleotide (Bp(2)A), and C1'-m-benzamide nicotinamide dinucleotide (Bp(2)N) were found to be nanomolar potency inhibitors with inhibition constants of 70, 143, 189, and 201 nM, respectively. However, NHD(+) and NGD(+) are also known substrates and are slowly converted to cyclic products, thus preventing their further use as inhibitors. The symmetrical bis-nucleotides, bis-adenine dinucleotide (Ap(2)A), bis-hypoxanthine dinucleotide (Hp(2)H), and bis-nicotinamide dinucleotide (Np(2)N), exhibited micromolar competitive inhibition, with Ap(2)A displaying the greatest affinity for the enzyme. 2',3'-Di-O-acetyl nicotinamide adenine dinucleotide (AcONAD(+)) was not a substrate for the A. californica cyclase but also displayed some inhibition at a micromolar level. Finally, inhibition of the cyclase by adenosine 5'-O-diphosphate ribose (ADPR) and inosine 5'-O-diphosphate ribose (IDPR) was observed at millimolar concentration. The nicotinamide aromatic ring appears to be the optimal motif required for enzymatic recognition, while modifications of the 2'- and 3'-hydroxyls of the nicotinamide ribose seem to hamper binding to the enzyme. Stabilizing enzyme/inhibitor interactions and the inability of the enzyme to release unprocessed material are both considered to explain nanomolar inhibition. Recognition of inhibitors by other ADP ribosyl cyclases has also been investigated, and this study now provides the first potent nonhydrolyzable sea urchin ADP ribosyl cyclase and cADPR hydrolase inhibitor Bp(2)A, with inhibition observed at the micromolar and nanomolar level, respectively. The benzamide derivatives did not inhibit CD38 cyclase or hydrolase activity when NGD(+) was used as substrate. These results emphasize the difference between CD38 and other enzymes in which the cADPR cyclase activity predominates.

Topics: ADP-ribosyl Cyclase; ADP-ribosyl Cyclase 1; Animals; Antigens, CD; Antigens, Differentiation; Aplysia; Binding Sites; Enzyme Inhibitors; Guanine Nucleotides; Kinetics; Multienzyme Complexes; NAD; NAD+ Nucleosidase; Ovum; Sea Urchins; Substrate Specificity

Poly(ADP-ribosyl) transferase (pADPRT) catalyzes the transfer of the ADP-ribose moiety from NAD+ onto proteins as well as onto protein-bound ADP-ribose. As a result, protein-bound polymers of ADP-ribose are formed. pADPRT itself contains several acceptor sites for ADP-ribose polymers and may attach polymers to itself (automodification). In this study the influence of substitutions in the purine base of NAD+ on the polymerization reaction was investigated. The adenine moiety of NAD+ was replaced by either guanine, hypoxanthine or 1,N6-ethenoadenine. These analogs served as substrates for polymer synthesis as judged from the extent of automodification of the enzyme and the sizes of the polymers formed. Time course experiments revealed that 1,N6-etheno NAD+ (epsilon-NAD+) and nicotinamide hypoxanthine dinucleotide (NHD+) were rather poor substrates as compared to NAD+. Synthesis of GDP-ribose polymers from nicotinamide guanine dinucleotide (NGD+) was more efficient, but still significantly slower than poly(ADP-ribosyl)ation of the enzyme using NAD+. The size of the different polymers appeared to correlate with these observations. After 30 min of incubation in the presence of 1 mM substrate, polymers formed from epsilon-NAD+ or NHD+ contained up to 30 epsilon-ADP-ribose or IDP-ribose units, respectively. Using NGD+ as substrate polymers consisted of more than 60 GDP-ribose units, an amount similar to that achieved by poly(ADP-ribosyl)ation in the presence of only 0.1 mM NAD+ as substrate. These results suggest that the presence of an amino group in the purine base of NAD+ may facilitate catalysis. Substitution of the nicotinamide moiety of NAD+ with 3-acetylpyridine had no detectable effect on polymer formation. Oligomers of GDP-ribose and epsilon-ADP-ribose exhibited a slower mobility in polyacrylamide gels as compared to ADP-ribose or IDP-ribose oligomers. This feature of the two former analogs as well as their markedly attenuated polymerization by pADPRT provide valuable tools for the investigation of the enzymatic mechanism of this protein. Moreover, polymers of epsilon-ADP-ribose may be useful for studying enzymes degrading poly(ADP-ribose) owing to the fluorescence of this analog. Digestion of epsilon-ADPR polymers with snake venom phosphodiesterase was accompanied by a significant fluorescence enhancement.

Topics: Adenosine Diphosphate Ribose; Catalysis; Guanine Nucleotides; NAD; NAD+ Nucleosidase; Phosphodiesterase I; Phosphoric Diester Hydrolases; Poly(ADP-ribose) Polymerases; Polymers; Recombinant Proteins; Structure-Activity Relationship