Nicotinamide adenine dinucleotide (NAD+) is classically known as an essential coenzyme for hydride transfer reactions in metabolism. More recently, it has been recognized as a consumed substrate of ADPribose transfer enzymes including sirtuins, class III protein-lysine deacetylases. Although key biosynthetic enzymes and vitamin precursors of NAD+ were described by the early 1900s, we have recently discovered additional precursors, pathways and regulation of NAD+ biosynthesis. We have shown that nicotinamide riboside (NR) and nicotinic acid riboside (NAR) are vitamin and synthetic precursors of NAD+, respectively, that are utilized through two pathways involving distinct enzymes. In addition, NR is specifically transported into yeast cells where it extends replicative lifespan. In this work, we show that, in addition to being vitamins, NR and NAR are endogenous intracellular metabolites in budding yeast, and by virtue of a novel bioassay we show that NR is also a secreted metabolite. Using a newly developed LC-MS method, I measured the NAD+ metabolome in cells lacking NR salvage pathways, nrk1 urh1 pnp1, and determined that these pathways are critical for maintaining normal metabolite levels. Using a biochemical genomic screen and metabolic analysis, I identified three enzymes, Isn1, Sdt1 and Phm8, which can produce NR by dephosphorylation of nicotinamide mononucleotide (NMN) in vitro, and verified an in vivo role for two, Isn1 and Sdt1. Thus, Isn1 and Sdt1, originally classified as an IMP-specific 5'-nucleotidase and a pyrimidine nucleotide specific 5'-nucleotidase, respectively, are also NMN/NaMN 5'-nucleotidases, responsible for the dephosphorylation of NMN and NaMN to NR and NAR. Analysis of expression revealed that Isn1 is positively regulated by the presence of nicotinic acid and glucose in the medium, and Sdt1 is transcriptionally induced by inorganic phosphate in the environment. These results also revealed that Sdt1 and Phm8, which was also identified in the genomic screen, are components of the phosphate acquisition system. We show that these additional and unanticipated steps in NAD+ metabolism are highly regulated and are necessary for altering the NAD+ metabolome under different environmental conditions.
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