NAD+ is essential for life in all organisms, both as a coenzyme for oxidoreductases and as a source of ADPribosyl groups used in various reactions, including those that retard aging in experimental systems. Nicotinic acid and nicotinamide were defined as the vitamin precursors of NAD+ in Elvehjem's classic discoveries of the 1930s. The accepted view of eukaryotic NAD+ biosynthesis, that all anabolism flows through nicotinic acid mononucleotide, was challenged experimentally and revealed that nicotinamide riboside is an unanticipated NAD+ precursor in yeast. Nicotinamide riboside kinases from yeast and humans essential for this pathway were identified and found to be highly specific for phosphorylation of nicotinamide riboside and the cancer drug tiazofurin. Nicotinamide riboside was discovered as a nutrient in milk, suggesting that nicotinamide riboside is a useful compound for elevation of NAD+ levels in humans.

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The histidine triad superfamily of nucleotide hydrolases and nucleotide transferases consists of a branch of proteins related to Hint and Aprataxin, a branch of Fhitrelated hydrolases, and a branch of galactose-1-phosphate uridylyltransferase (GalT)-related transferases. Although substrates of Fhit and GalT are known and consequences of mutations in Aprataxin, Fhit, and GalT are known, good substrates had not been reported for any member of the Hint branch, and mutational consequences were unknown for Hint orthologs, which are the most ancient and widespread proteins in the Hint branch and in the histidine triad superfamily. Here we show that rabbit and yeast Hint hydrolyze the natural product adenosine-5-monophosphoramidate (AMPNH 2 ) in an active-site-dependent manner at second order rates exceeding 1,000,000 M ؊1 s ؊1 . Yeast strains constructed with specific loss of the Hnt1 active site fail to grow on galactose at elevated temperatures. Loss of Hnt1 enzyme activity also leads to hypersensitivity to mutations in Ccl1, Tfb3, and Kin28, which constitute the TFIIK kinase subcomplex of general transcription factor TFIIH and to mutations in Cak1, which phosphorylates Kin28. The target of Hnt1 regulation in this pathway was shown to be downstream of Cak1 and not to affect stability of Kin28 monomers. Functional complementation of all Hnt1 phenotypes was provided by rabbit Hint, which is only 22% identical to yeast Hnt1 but has very similar adenosine monophosphoramidase activity. Histidine triad (HIT)1 proteins are a superfamily of nucleotide-binding proteins named for a near C-terminal HXHXHXX motif (X is a hydrophobic amino acid) positioned at the ␣-phosphate of nucleotide substrates (1). The first branch of the superfamily is named for rabbit Hint, which had been purified as an abundant protein from cardiac cytosol by adenosine affinity chromatography (2) and shown to have homologs in all forms of life (1). Recently, Aprataxin, a gene located at 9p13 that is inactivated in ataxia with oculomotor apraxia, the second most common of the autosomal recessive ataxias, was identified as a member of the Hint branch of the HIT superfamily (3, 4). Human Fhit (5), which functions as a tumor suppressor protein in human (6 -9) and murine (10, 11) epithelial tissues, is the prototypical member of the second branch of the HIT superfamily. Fhit homologs have been found in fungi (12) and animals (13-16) and exhibit diadenosine polyphosphate hydrolase activity. A third branch of the HIT superfamily contains more distantly related nucleotide transferases including galactose-1-phosphate uridylyltransferase, which is the enzyme deficient in galactosemics (1), budding yeast diadenosine tetraphosphate phosphorylases ApaI and Apa2 (17), and adenylylsulfate:phosphate adenylyltransferase (18). Ironically, although Hint is the most ancient and widespread of the HIT proteins, reasonable Hint substrates remained unidentified, and the consequences of mutations in Hint or Hint orthologs were unknown (19).Recently, human Hint was identified as...

Nit monomers possess a new alpha-beta-beta-alpha sandwich fold with a presumptive Cys-Glu-Lys catalytic triad. Nit assembles into a tetrameric, 52-stranded beta box that binds Fhit dimers at opposite poles and displays Nit active sites around the middle of the complex. The most carboxy-terminal beta strand of each Nit monomer exits the core of the Nit tetramer and interacts with Fhit. Residence in the NitFhit complex does not alter the nucleotide specificity of Fhit dimers, which are oriented with ApppA-binding surfaces away from Nit.