Abstract
Recently, we discovered that nicotinamide riboside and nicotinic acid riboside are biosynthetic precursors of NAD(+), which are utilized through two pathways consisting of distinct enzymes. In addition, we have shown that exogenously supplied nicotinamide riboside is imported into yeast cells by a dedicated transporter, and it extends replicative lifespan on high glucose medium. Here, we show that nicotinamide riboside and nicotinic acid riboside are authentic intracellular metabolites in yeast. Secreted nicotinamide riboside was detected with a biological assay, and intracellular levels of nicotinamide riboside, nicotinic acid riboside, and other NAD(+) metabolites were determined by a liquid chromatography-mass spectrometry method. A biochemical genomic screen indicated that three yeast enzymes possess nicotinamide mononucleotide 5'-nucleotidase activity in vitro. Metabolic profiling of knock-out mutants established that Isn1 and Sdt1 are responsible for production of nicotinamide riboside and nicotinic acid riboside in cells. Isn1, initially classified as an IMP-specific 5'-nucleotidase, and Sdt1, initially classified as a pyrimidine 5'-nucleotidase, are additionally responsible for dephosphorylation of pyridine mononucleotides. Sdt1 overexpression is growth-inhibitory to cells in a manner that depends on its active site and correlates with reduced cellular NAD(+). Expression of Isn1 protein is positively regulated by the availability of nicotinic acid and glucose. These results reveal unanticipated and highly regulated steps in NAD(+) metabolism.
Highlights
We discovered that nicotinamide riboside and nicotinic acid riboside are biosynthetic precursors of NAD؉, which are utilized through two pathways consisting of distinct enzymes
Secreted nicotinamide riboside was detected with a biological assay, and intracellular levels of nicotinamide riboside, nicotinic acid riboside, and other NAD؉ metabolites were determined by a liquid chromatography-mass spectrometry method
nicotinamide riboside (NR) and nicotinic acid riboside (NAR) Are Intracellular Metabolites—As shown in 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 NADϩ precursors [1, 7, 8, 10, 11]
Summary
Nicotinamide; NA, nicotinic acid; NaAD, nicotinic acid adenine dinucleotide; NaMN, nicotinic acid mononucleotide; NAR, nicotinic acid riboside; NMN, nicotinamide mononucleotide; NR, nicotinamide riboside; CR, calorie restriction; HPLC, high pressure liquid chromatography; LC-MS, liquid chromatography mass spectrometry; SDC, synthetic dextrose complete; SGC, synthetic galactose complete; TAP, tandem affinity protein. Isn and Sdt Produce NR and NAR from NMN and NaMN sized that CR increases NADϩ biosynthetic flux without altering steady-state NADϩ levels [17]. It is of interest that CR might alter NADϩ metabolic flux in ways that increase Sir function. Beginning with the premise that the intracellular biogenesis of NR and NAR is the most significant remaining problem in establishing the intracellular wiring diagram for NADϩ metabolism in yeast, we hypothesized that the production of these nucleosides would be dependent on specific 5Ј-nucleotidases. Using genetic and metabolomic assays, we show that NR and NAR are normal cellular metabolites produced by the activities of 5Ј-nucleotidases, Isn and Sdt, previously defined as IMP- [18] and pyrimidine nucleotide-specific 5Ј-nucleotidases [19], respectively. The substantial levels of NMN and NaMN suggest that modulation of the expression and/or activities of NaMN/ NMN adenylyltransferases and NaMN/NMN 5Ј-nucleotidases may drive pyridine nucleotide metabolism “forward” to dinucleotides or “backward” to nucleosides
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
