Nicotinamide Riboside and Nicotinic Acid Riboside Salvage in Fungi and Mammals

Abstract

NAD؉ is a co-enzyme for hydride transfer enzymes and an essential substrate of ADP-ribose transfer enzymes and sirtuins, the type III protein lysine deacetylases related to yeast Sir2. Supplementation of yeast cells with nicotinamide riboside extends replicative lifespan and increases Sir2-dependent gene silencing by virtue of increasing net NAD ؉ synthesis. Nicotinamide riboside elevates NAD ؉ levels via the nicotinamide riboside kinase pathway and by a pathway initiated by splitting the nucleoside into a nicotinamide base followed by nicotinamide salvage. Genetic evidence has established that uridine hydrolase, purine nucleoside phosphorylase, and methylthioadenosine phosphorylase are required for Nrk-independent utilization of nicotinamide riboside in yeast. Here we show that mammalian purine nucleoside phosphorylase but not methylthioadenosine phosphorylase is responsible for mammalian nicotinamide riboside kinase-independent nicotinamide riboside utilization. We demonstrate that so-called uridine hydrolase is 100-fold more active as a nicotinamide riboside hydrolase than as a uridine hydrolase and that uridine hydrolase and mammalian purine nucleoside phosphorylase cleave nicotinic acid riboside, whereas the yeast phosphorylase has little activity on nicotinic acid riboside. Finally, we show that yeast nicotinic acid riboside utilization largely depends on uridine hydrolase and nicotinamide riboside kinase and that nicotinic acid riboside bioavailability is increased by ester modification. NADϩ and its phosphorylated and reduced derivatives are essential co-enzymes for hydride transfer enzymes central to intermediary metabolism. NAD ϩ is also a consumed substrate of three classes of enzymes, which produce ADP-ribosyl products plus nicotinamide (Nam) 4 (1). Sirtuins utilize the ADPribose moiety of NAD ϩ to accept the acetyl modification of lysine, thereby producing a deacetylated protein plus Nam and a mixture of 2Ј-and 3Ј-acetylated ADP-ribose (2-4). Such reactions are important for chromatin silencing (5) and regulation of transcription factors and enzymes, thereby controlling a variety of genomic transactions (6), metabolic switches (7,8), and lifespan (9 -11). ADP-ribose transferases and polyADP-ribose polymerases utilize NAD ϩ to add ADP-ribose as a posttranslational modification and/or to form ADP-ribose polymers (12, 13). Finally, cyclic ADP-ribose synthases produce and hydrolyze the calcium-mobilizing compound, cADP-ribose (14, 15). Thus, via pleiotropic ways and means, NAD ϩ is a central mediator of cellular and organismal metabolism and signaling.Although co-enzymatic NAD ϩ functions do not necessitate ongoing NAD ϩ synthesis, the activities of the NAD ϩ -consuming enzymes mandate either ongoing de novo or salvage synthesis (see Fig. 1). In yeast, de novo synthesis from tryptophan maintains intracellular NAD ϩ at ϳ0.8 mM, at which concentration cells grow well but perform Sir2-dependent gene silencing poorly and have relatively short replicative life spans (16). However, supplementation with 10 M n...

Identification of Isn1 and Sdt1 as Glucose- and Vitamin-regulated Nicotinamide Mononucleotide and Nicotinic Acid Mononucleotide 5′-Nucleotidases Responsible for Production of Nicotinamide Riboside and Nicotinic Acid Riboside

“…By screening for such an activity, we identified three candidate genes, two of which, ISN1 and SDT1, are essential for production of NR and NAR in vivo. The identification of Isn1 and Sdt1, previously thought to function exclusively in purine and pyrimidine metabolism, as enzymes with roles in NAD ϩ metabolism is reminiscent of recent discoveries with respect to Urh1 and Pnp1 (1,10,11). Indeed, the multifunctionality of Isn1 and Sdt1 in nucleotide-nucleoside transactions follows a recurrent theme in NAD ϩ biosynthesis (38).…”

“…Fig. 1A, previous studies have defined the roles of exogenously supplied NR and NAR and defined the Nrk1-dependent and -independent pathways through which these nucleosides are utilized as NAD ϩ precursors (1,7,8,10,11). The vitamin properties of exogenously supplied NR include promotion of Sir2-dependent silencing and lifespan extension in glucose-replete medium (1).…”

“…Recently, we have shown that Urh1 is more effective as an NR hydrolase than as a Urd hydrolase and that Pnp1 functions as an NR phosphorylase secondary to its known function as a purine nucleoside phosphorylase (10). Given those in vitro data, we wished to test whether deletion of NR/NAR salvage enzymes would affect the levels of a set of purine and pyrimidine nucleosides and nucleotides.…”

“…Exogenously supplied NR is transported into cells (7) and is either phosphorylated by the NR kinase, Nrk1, to form nicotinamide mononucleotide (NMN) (8) or converted to Nam by the hydrolase and phosphorylase activity of Urh1 and Pnp1, respectively (9). Finally, although NAR is poorly imported (10), it can also be utilized by Nrk1 (11) and Urh1 (10) to form nicotinic acid mononucleotide (NaMN) or NA, respectively (Fig. 1A).…”

Pathways and Subcellular Compartmentation of NAD Biosynthesis in Human Cells

“…The control of NAD ϩ synthesis by the initial steps thus suggests that combination of various NAD ϩ precursors could be beneficial to enhance cellular NAD ϩ levels. Cellular Uptake of NAD ϩ Precursors-The demonstration of NR and NAR as intermediates in the NAD ϩ metabolome (17)(18)(19)(20) has added an important aspect to cellular NAD ϩ homeostasis and extended the scope of potential extracellular NAD ϩ precursors. NAD ϩ itself has been proposed to be taken up by human cell lines, including those used in this study (38).…”