In this study, we show that nicotinamide riboside, which was known to be an NAD+ precursor in bacteria such as Haemophilus influenza (Gingrich and Schlenk 1944, Leder and Handler 1951, Shifrine and Biberstein 1960) that lack the enzymes of the de novo and Preiss-Handler pathways (Fleischmann et al., 1995), is an NAD+ precursor in a previously unknown but apparently conserved eukaryotic NAD+ biosynthetic pathway. We identify yeast nicotinamide riboside kinase, Nrk1, and both human Nrk enzymes and demonstrate their specific functions in NAD+ metabolism biochemically and genetically. Their specificity suggests additionally that they are the long-sought tiazofurin kinases that perform the first step in converting cancer drugs such as tiazofurin and benzamide riboside into toxic NAD+ analogs (Cooney et al., 1983). Finally, we utilize yeast mutants of defined genotype to hunt for vitamins in a pathway-specific manner and show that milk is a source of nicotinamide riboside.

Results

Recently, we characterized the S. cerevisiae QNS1 gene encoding glutamine-dependent NAD+ synthetase and showed that mutation of either the glutaminase active site or the NAD+ synthetase active site resulted in inviable cells (Bieganowski et al., 2003). Possession of strains containing the qns1 deletion and a plasmid-borne QNS1 gene allowed us to test whether the canonical de novo, import, and salvage pathways for NAD+ as depicted in Figure 1 (Panozzo et al. 2002, Sandmeier et al. 2002, Bitterman et al. 2002, Anderson et al. 2003, Gallo et al. 2004) are a complete representation of the metabolic pathways to NAD+ in S. cerevisiae. This scheme makes two specific predictions. First, because nicotinamide is deamidated to nicotinic acid before the pyridine ring is salvaged to make more NAD+, supplementation with nicotinamide should not rescue qns1 mutants by shunting nicotinamide-containing precursors through the pathway. Second, because QNS1 is common to the three pathways, there should be no NAD+ precursor that rescues qns1 mutants. As shown in Figure 2A, consistent with the scheme's implicit assumptions about nicotinamide metabolism, nicotinamide does not rescue qns1 mutants even at 1 or 10 mM. However, apart from any intermediate or enzymatic transformation depicted in the scheme, nicotinamide riboside, which was characterized as an NAD+ precursor in bacteria such as Haemophilus influenza (Gingrich and Schlenk 1944, Leder and Handler 1951, Shifrine and Biberstein 1960), functions as a vitamin form of NAD+ at 10 μM.