Identification of Isn1 and Sdt1 as Glucose- and Vitamin-regulated Nicotinamide Mononucleotide and Nicotinic Acid Adenine Dinucleotide 5′-Nucleotidases Responsible for Production of Nicotinamide Riboside and Nicotinic Acid Riboside
Identification of Isn1 and Sdt1 as Glucose- and Vitamin-regulated Nicotinamide Mononucleotide and Nicotinic Acid Adenine Dinucleotide 5′-Nucleotidases Responsible for Production of Nicotinamide Riboside and Nicotinic Acid Riboside
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DOI：
10.1074/jbc.m109.056689
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发表时间：
2009-12-11
影响因子：
4.8
通讯作者：
Brenner, Charles
中科院分区：
文献类型：
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作者：
Bogan, Katrina L.;Evans, Charles;Brenner, Charles
最近，我们发现烟酰胺核苷和烟酸核苷是NAD（+）的生物合成前体，其通过由不同酶组成的两条途径被利用。此外，我们已经表明，外源性提供的烟酰胺核苷是由一个专门的转运蛋白输入到酵母细胞，它延长了复制寿命的高葡萄糖培养基。在这里，我们表明，烟酰胺核苷和烟酸核苷是真实的酵母细胞内代谢产物。用生物测定法检测分泌的烟酰胺核苷，用液相色谱-质谱法测定烟酰胺核苷、烟酸核苷和其他NAD（+）代谢物的细胞内水平。生化基因组筛选表明，三种酵母酶具有烟酰胺单核苷酸5 '-核苷酸酶活性在体外。敲除突变体的代谢谱确定Isn 1和Sdt 1负责细胞中烟酰胺核苷和烟酸核苷的产生。Isn 1最初被分类为IMP特异性5 '-核苷酸酶，Sdt 1最初被分类为嘧啶5'-核苷酸酶，另外负责吡啶单核苷酸的去磷酸化。Sdt 1过表达以依赖于其活性位点的方式抑制细胞生长，并与细胞NAD（+）减少相关。Isn 1蛋白的表达受烟酸和葡萄糖的可用性正调控。这些结果揭示了NAD（+）代谢中的非预期和高度调节步骤。
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, area 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.