Regulation of New Steps in NAD Metabolism
NAD is a co-enzyme for a variety of hydride transfer enzymes, which are required for intermediary metabolism. NAD is also a consumed substrate of sirtuins, ADPribose transfer enzymes including PARP and cyclic ADPribose synthases. Each of these enzymes links a signaling function to consumption of NAD, necessitating salvage biosynithesis (1, 2). We began our foray into NAD biology by discovering eukaryotic NAD synthetase, Qns1 (3, 4), and showing that the glutamine requirement of this enzyme is encoded by an intramolecular, glutamine amidotransferase activity related to nitrilase (5, 6). Though we initially developed assays to dissect Qns1 protein structure and function, we realized that we were in a unique position to determine whether the commonly accepted biosynthetic schemes for NAD, which had never been subjected to a rigorous genetic test, were correct and complete. Remarkably, these experiments showed that yeast can utilize nicotinamide riboside (NR), a natural product found in milk, to produce NAD in a manner that bypasses de novo biosynthesis and the previously described salvage routes to NAD through nicotinic acid and nicotinamide (7). Through these experiments, we discovered the yeast and vertebrate nicotinamide riboside kinase (Nrk) genes and began to characterize Nrk enzymes (7).
Consistent with NR as a previously unappreciated vitamin, we discovered a high affinity NR transporter (8) and uncovered a second biosynthetic route from NR to NAD (9). Significantly, NR supplementation of yeast cells extends their replicative lifespan on high glucose media in a manner that depends on presence of NR salvage enzymes (9). Surprisingly, the crystal structure of human Nrk1 bound to NR revealed a lack of features that would distinguish NR from nicotinic acid riboside (NAR). With the crystal structure of Nrk1 as a clue, we discovered that Nrk enzymes are dual specificity NR/NAR kinases (10). At this point, the table was set to use liquid chromatography-mass spectroscopy (LC-MS) to prove that NR and NAR are authentic metabolites and to determine what genes and enzymes are responsible for their intracellular production. Using LC-MS, we established the normal levels of the core NAD metabolome (11), identified 5' nucleotidases that produce NR and NAR from pyridine mononucleotides (12), and showed that two commonly used means to extend yeast lifespan, namely calorie restriction and vitamin supplementation, are metabolically distinguishable (13).
New experiments have uncovered a previously unappreciated aspect of NAD metabolism that is modulated by the availability of glucose. We aim to dissect how carbon limitation alters regulation of NAD biosynthesis in order to explain how calorie restriction extends liefspan in yeast. In addition, we aim to extend what is known about intracellular NAD metabolism to more complex systems involving cell-to-cell transmission of metabolites, and to connect regulation of NAD-biosynthesis with NAD dependent signaling through sirtuins and PARPs.
P. Bieganowski & C. Brenner, "Discoveries of Nicotinamide Riboside as a Nutrient and Conserved NRK Genes Establish a Preiss-Handler Independent Route to NAD+ in Fungi and Humans," Cell, v. 117, pp. 495-502 (2004). Download pdf reprint.
P. Belenky, F.G. Racette, K.L. Bogan, J.M. McClure, J.S. Smith & C. Brenner, "Nicotinamide Riboside Promotes Sir2 Silencing and Extends Lifespan via Nrk and Urh1/Pnp1/Meu1 Pathways to NAD+," Cell, v. 129, pp. 473-484 (2007). Download pdf reprint and supplementary data. Read Leading Edge Preview in Cell, Research Highlights in Nature and Spotlight in ACS Chemical Biology.
W. Tempel, W.M. Rabeh, K.L. Bogan, P. Belenky, M. Wojcik, H.F. Seidle, L. Nedyalkova, T. Yang, A.A. Sauve, H.-W. Park & C. Brenner, "Nicotinamide Riboside Kinase Structures Reveal New Pathways to NAD+," PLoS Biology, v. 5, issue 10, e263 (2007). View Nrk1-ADP, Nrk1-NR, Nrk1-AppNHp+NR, Nrk1-NMN, and Nrk1-tiazofurin entries at the Protein Data Bank. Download pdf reprint and supporting information.
K.L. Bogan & C. Brenner, "Nicotinic Acid, Nicotinamide, and Nicotinamide Riboside: A Molecular Evaluation of NAD+ Precursor Vitamins in Human Nutrition," Ann Review Nutrition, v. 28, pp. 115-130 (2008). Download pdf reprint.
K.L. Bogan, C. Evans, P. Belenky, P. Song, C.F. Burant, R.T. Kennedy & C. Brenner, " Identification of Isn1 and Sdt1 as Glucose and Vitamin-regulated NMN and NaMN 5'-nucleotidases Responsible for Production of Nicotinamide Riboside and Nicotinic Acid Riboside," J Biol Chem, v. 284, pp. 34861-34869 (2009). Download pdf reprint and supplementary information.
C. Evans, K.L. Bogan, P. Song, C.F. Burant, R.T. Kennedy & C. Brenner, "NAD+ Metabolite Levels as a Function of Vitamins and Calorie Restriction: Evidence for Different Mechanisms of Longevity," BMC Chem Biol, v. 10, 2 (2010). Download pdf reprint and supplementary information.
P. Belenky, R. Stebbins, K.L. Bogan, C.R. Evans & C. Brenner, "Nrt1 and Tna1-Independent Export of NAD+ Precursor Vitamins Promotes NAD+ Homeostasis and Allows Engineering of Vitamin Production," PLoS ONE, v. 6, p. e19710 (2011). Download pdf reprint.
K.L. Bogan & C. Brenner, "Biochemistry: Niacin/NAD(P)," Encyclopedia of Biological Chemistry, W.J. Lennarz & M.D. Lane, eds., v. 3, pp.172-178, (2013), Waltham, MA: Academic Press. Download pdf reprint.
S. Ghanta, R.E. Grossmann & C. Brenner, "Mitochondrial protein acetylation as a cell-intrinsic, evolutionary driver of fat storage: chemical and metabolic logic of acetyl-lysine modifications" Critical Rev Biochem & Mol Biol, v. 48, pp. 561-574, 2013. Download pdf reprint.