[show abstract][hide abstract] ABSTRACT: NAD(+) is both a co-enzyme for hydride transfer enzymes and a substrate of sirtuins and other NAD(+) consuming enzymes. NAD(+) biosynthesis is required for two different regimens that extend lifespan in yeast. NAD(+) is synthesized from tryptophan and the three vitamin precursors of NAD(+): nicotinic acid, nicotinamide and nicotinamide riboside. Supplementation of yeast cells with NAD(+) precursors increases intracellular NAD(+) levels and extends replicative lifespan. Here we show that both nicotinamide riboside and nicotinic acid are not only vitamins but are also exported metabolites. We found that the deletion of the nicotinamide riboside transporter, Nrt1, leads to increased export of nicotinamide riboside. This discovery was exploited to engineer a strain to produce high levels of extracellular nicotinamide riboside, which was recovered in purified form. We further demonstrate that extracellular nicotinamide is readily converted to extracellular nicotinic acid in a manner that requires intracellular nicotinamidase activity. Like nicotinamide riboside, export of nicotinic acid is elevated by the deletion of the nicotinic acid transporter, Tna1. The data indicate that NAD(+) metabolism has a critical extracellular element in the yeast system and suggest that cells regulate intracellular NAD(+) metabolism by balancing import and export of NAD(+) precursor vitamins.
PLoS ONE 01/2011; 6(5):e19710. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: NAD+ is a coenzyme for hydride transfer enzymes and a substrate for sirtuins and other NAD+-dependent ADPribose transfer enzymes. In wild-type Saccharomyces cerevisiae, calorie restriction accomplished by glucose limitation extends replicative lifespan in a manner that depends on Sir2 and the NAD+ salvage enzymes, nicotinic acid phosphoribosyl transferase and nicotinamidase. Though alterations in the NAD+ to nicotinamide ratio and the NAD+ to NADH ratio are anticipated by models to account for the effects of calorie restriction, the nature of a putative change in NAD+ metabolism requires analytical definition and quantification of the key metabolites.
Hydrophilic interaction chromatography followed by tandem electrospray mass spectrometry were used to identify the 12 compounds that constitute the core NAD+ metabolome and 6 related nucleosides and nucleotides. Whereas yeast extract and nicotinic acid increase net NAD+ synthesis in a manner that can account for extended lifespan, glucose restriction does not alter NAD+ or nicotinamide levels in ways that would increase Sir2 activity.
The results constrain the possible mechanisms by which calorie restriction may regulate Sir2 and suggest that provision of vitamins and calorie restriction extend lifespan by different mechanisms.
BMC Chemical Biology 02/2010; 10:2. · 1.60 Impact Factor
[show abstract][hide abstract] ABSTRACT: 5′-Nucleotidase (EC 22.214.171.124) designates a set of enzymes, which catalyze the hydrolysis of ribonucleoside and deoxyribonucleoside monophosphates into the corresponding nucleosides plus orthophosphate. 5′-Nucleotidases are classified according to subcellular localization, nucleobase specificity and their ability to hydrolyze deoxynucleoside monophosphate substrates. Membrane-bound 5′-nucleotidases are ectoenzymes principally involved in salvage of extracellular nucleosides, and often display a preference toward adenosine monophosphate, thereby modulating signal transduction cascades involving purinergic receptors. Cytosolic 5′-nucleotidases are members of the haloacid dehalogenase superfamily of enzymes, which are two-domain proteins containing a modified Rossman fold as the core and a variable cap structure. Extracellular and intracellular 5′-nucleotidase activities participate in purine and pyrimidine salvage to support balanced synthesis of nucleotides, which is critical for maintaining high fidelity DNA replication. While the production of ribonucleosides from ribonucleotides by 5′-nucleotidases remains the most well studied function, it appears that the physiological functions of these activities are more broad. Indeed, Sdt1, previously termed a pyrimidine-specific 5′-nucleotidase, and Isn1, previously termed an inosine monophosphate (IMP)-specific 5′-nucleotidase, have recently been implicated in catabolic processes in nicotinamide adenine dinucleotide (NAD+) metabolism, and are regulated by the NAD+ precursor vitamin nicotinic acid, glucose and phosphate availability in the medium. In addition, Usha, Pho5, Sdt1 and Phm8 are phosphate starvation-induced 5′-nucleotidases with diverse substrate specificities that liberate phosphate under phosphate starvation conditions. Here we review 5′-nucleotidase enzyme structure, catalytic mechanism and substrate specificity and focus on new biological roles for these enzymes in nucleotide, NAD+ and phosphate metabolism.
New Journal of Chemistry 01/2010; 34(5). · 2.97 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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, are 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.
Journal of Biological Chemistry 10/2009; 284(50):34861-9. · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: NAD is a coenzyme for redox reactions and a substrate of NAD-consuming enzymes, including ADP-ribose transferases, Sir2-related protein lysine deacetylases, and bacterial DNA ligases. Microorganisms that synthesize NAD from as few as one to as many as five of the six identified biosynthetic precursors have been identified. De novo NAD synthesis from aspartate or tryptophan is neither universal nor strictly aerobic. Salvage NAD synthesis from nicotinamide, nicotinic acid, nicotinamide riboside, and nicotinic acid riboside occurs via modules of different genes. Nicotinamide salvage genes nadV and pncA, found in distinct bacteria, appear to have spread throughout the tree of life via horizontal gene transfer. Biochemical, genetic, and genomic analyses have advanced to the point at which the precursors and pathways utilized by a microorganism can be predicted. Challenges remain in dissecting regulation of pathways.
Microbiology and molecular biology reviews: MMBR 10/2009; 73(3):529-41, Table of Contents. · 12.59 Impact Factor
[show abstract][hide abstract] ABSTRACT: NAD+ is a co-enzyme for hydride transfer enzymes and an essential substrate of ADP-ribose transfer enzymes and sirtuins, the type III protein lysine deacetylases related to yeast Sir2. Supplementation of yeast cells with nicotinamide riboside extends replicative lifespan and increases Sir2-dependent gene silencing by virtue of increasing net NAD+ synthesis. Nicotinamide riboside elevates NAD+ levels via the nicotinamide riboside kinase pathway and by a pathway initiated by splitting the nucleoside into a nicotinamide base followed by nicotinamide salvage. Genetic evidence has established that uridine hydrolase, purine nucleoside phosphorylase, and methylthioadenosine phosphorylase are required for Nrk-independent utilization of nicotinamide riboside in yeast. Here we show that mammalian purine nucleoside phosphorylase but not methylthioadenosine phosphorylase is responsible for mammalian nicotinamide riboside kinase-independent nicotinamide riboside utilization. We demonstrate that so-called uridine hydrolase is 100-fold more active as a nicotinamide riboside hydrolase than as a uridine hydrolase and that uridine hydrolase and mammalian purine nucleoside phosphorylase cleave nicotinic acid riboside, whereas the yeast phosphorylase has little activity on nicotinic acid riboside. Finally, we show that yeast nicotinic acid riboside utilization largely depends on uridine hydrolase and nicotinamide riboside kinase and that nicotinic acid riboside bioavailability is increased by ester modification.
Journal of Biological Chemistry 12/2008; 284(1):158-64. · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: The Arabidopsis thaliana VTC2 gene encodes an enzyme that catalyzes the conversion of GDP-L-galactose to L-galactose 1-phosphate in the first committed step of the Smirnoff-Wheeler pathway to plant vitamin C synthesis. Mutations in VTC2 had previously been found to lead to only partial vitamin C deficiency. Here we show that the Arabidopsis gene At5g55120 encodes an enzyme with high sequence identity to VTC2. Designated VTC5, this enzyme displays substrate specificity and enzymatic properties that are remarkably similar to those of VTC2, suggesting that it may be responsible for residual vitamin C synthesis in vtc2 mutants. The exact nature of the reaction catalyzed by VTC2/VTC5 is controversial because of reports that kiwifruit and Arabidopsis VTC2 utilize hexose 1-phosphates as phosphorolytic acceptor substrates. Using liquid chromatography-mass spectroscopy and a VTC2-H238N mutant, we provide evidence that the reaction proceeds through a covalent guanylylated histidine residue within the histidine triad motif. Moreover, we show that both the Arabidopsis VTC2 and VTC5 enzymes catalyze simple phosphorolysis of the guanylylated enzyme, forming GDP and L-galactose 1-phosphate from GDP-L-galactose and phosphate, with poor reactivity of hexose 1-phosphates as phosphorolytic acceptors. Indeed, the endogenous activities from Japanese mustard spinach, lemon, and spinach have the same substrate requirements. These results show that Arabidopsis VTC2 and VTC5 proteins and their homologs in other plants are enzymes that guanylylate a conserved active site His residue with GDP-L-galactose, forming L-galactose 1-phosphate for vitamin C synthesis, and regenerate the enzyme with phosphate to form GDP.
Journal of Biological Chemistry 08/2008; 283(27):18483-92. · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: Despite the common occurrence of forkhead associated (FHA) phosphopeptide-binding domains and really interesting new gene (RING) E3 ubiquitin ligase domains, gene products containing both an N-terminal FHA domain and C-terminal RING domain constitute a highly distinctive intersection. Characterized FHA-RING ligases include the two vertebrate proteins, Checkpoint with FHA and RING (Chfr) and RING finger 8 (Rnf8), as well as three fungal proteins, Defective in mitosis (Dma1), Chf1 and Chf2. These FHA-RING ligases play roles in negative regulation of the cell division cycle, apparently by coupling protein phosphorylation events to specific ubiquitylation of target proteins. Here, the available data on upstream and downstream regulation of and by FHA-RING ligases are reviewed.
Cellular and Molecular Life Sciences CMLS 08/2008; 65(21):3458-66. · 5.62 Impact Factor
[show abstract][hide abstract] ABSTRACT: Although baseline requirements for nicotinamide adenine dinucleotide (NAD+) synthesis can be met either with dietary tryptophan or with less than 20 mg of daily niacin, which consists of nicotinic acid and/or nicotinamide, there is growing evidence that substantially greater rates of NAD+ synthesis may be beneficial to protect against neurological degeneration, Candida glabrata infection, and possibly to enhance reverse cholesterol transport. The distinct and tissue-specific biosynthetic and/or ligand activities of tryptophan, nicotinic acid, nicotinamide, and the newly identified NAD+ precursor, nicotinamide riboside, reviewed herein, are responsible for vitamin-specific effects and side effects. Because current data suggest that nicotinamide riboside may be the only vitamin precursor that supports neuronal NAD+ synthesis, we present prospects for human nicotinamide riboside supplementation and propose areas for future research.
Annual Review of Nutrition 05/2008; 28:115-30. · 9.16 Impact Factor
[show abstract][hide abstract] ABSTRACT: NAD(+) is an essential coenzyme for hydride transfer enzymes and a substrate of sirtuins and other NAD(+)-consuming enzymes. Nicotinamide riboside is a recently discovered eukaryotic NAD(+) precursor converted to NAD(+) via the nicotinamide riboside kinase pathway and by nucleosidase activity and nicotinamide salvage. Nicotinamide riboside supplementation of yeast extends replicative life span on high glucose medium. The molecular basis for nicotinamide riboside uptake was unknown in any eukaryote. Here, we show that deletion of a single gene, YOR071C, abrogates nicotinamide riboside uptake without altering nicotinic acid or nicotinamide import. The gene, which is negatively regulated by Sum1, Hst1, and Rfm1, fully restores nicotinamide riboside import and utilization when resupplied to mutant yeast cells. The encoded polypeptide, Nrt1, is a predicted deca-spanning membrane protein related to the thiamine transporter, which functions as a pH-dependent facilitator with a K(m) for nicotinamide riboside of 22 microm. Nrt1-related molecules are conserved in particular fungi, suggesting a similar basis for nicotinamide riboside uptake.
Journal of Biological Chemistry 04/2008; 283(13):8075-9. · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: Checkpoint with forkhead-associated and RING (Chfr) is a ubiquitin ligase (E3) that establishes an antephase or prometaphase checkpoint in response to mitotic stress. Though ubiquitination is essential for checkpoint function, the sites, linkages and ubiquitin conjugating enzyme (E2) specificity are controversial. Here we dissect the function of the two Chfr homologs in S. cerevisiae, Chf1 and Chf2, overexpression of which retard cell cycle at both G(1) and G(2). Using a genetic assay, we establish that Ubc4 is required for Chf2-dependent G(1) cell cycle delay and Chf protein turnover. In contrast, Ubc13/Mms2 is required for G(2) delay and does not contribute to Chf protein turnover. By reconstituting cis and trans-ubiquitination activities of Chf proteins in purified systems and characterizing sites modified and linkages formed by tandem mass spectrometry, we discovered that Ubc13/Mms2- dependent modifications are a distinct subset of those catalyzed by Ubc4. Mutagenesis of Lys residues identified in vitro indicates that site-specific Ubc4-dependent Chf protein autoubiquitination is responsible for Chf protein turnover. Thus, combined genetic and biochemical analyses indicate that Chf proteins have dual E2 specificity accounting for different functions in the cell cycle.
[show abstract][hide abstract] ABSTRACT: The eukaryotic nicotinamide riboside kinase (Nrk) pathway, which is induced in response to nerve damage and promotes replicative life span in yeast, converts nicotinamide riboside to nicotinamide adenine dinucleotide (NAD+) by phosphorylation and adenylylation. Crystal structures of human Nrk1 bound to nucleoside and nucleotide substrates and products revealed an enzyme structurally similar to Rossmann fold metabolite kinases and allowed the identification of active site residues, which were shown to be essential for human Nrk1 and Nrk2 activity in vivo. Although the structures account for the 500-fold discrimination between nicotinamide riboside and pyrimidine nucleosides, no enzyme feature was identified to recognize the distinctive carboxamide group of nicotinamide riboside. Indeed, nicotinic acid riboside is a specific substrate of human Nrk enzymes and is utilized in yeast in a novel biosynthetic pathway that depends on Nrk and NAD+ synthetase. Additionally, nicotinic acid riboside is utilized in vivo by Urh1, Pnp1, and Preiss-Handler salvage. Thus, crystal structures of Nrk1 led to the identification of new pathways to NAD+.
[show abstract][hide abstract] ABSTRACT: The first committed step in the biosynthesis of L-ascorbate from D-glucose in plants requires conversion of GDP-L-galactose to L-galactose 1-phosphate by a previously unidentified enzyme. Here we show that the protein encoded by VTC2, a gene mutated in vitamin C-deficient Arabidopsis thaliana strains, is a member of the GalT/Apa1 branch of the histidine triad protein superfamily that catalyzes the conversion of GDP-L-galactose to L-galactose 1-phosphate in a reaction that consumes inorganic phosphate and produces GDP. In characterizing recombinant VTC2 from A. thaliana as a specific GDP-L-galactose/GDP-D-glucose phosphorylase, we conclude that enzymes catalyzing each of the ten steps of the Smirnoff-Wheeler pathway from glucose to ascorbate have been identified. Finally, we identify VTC2 homologs in plants, invertebrates, and vertebrates, suggesting that a similar reaction is used widely in nature.
Journal of Biological Chemistry 07/2007; 282(26):18879-85. · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: Although NAD(+) biosynthesis is required for Sir2 functions and replicative lifespan in yeast, alterations in NAD(+) precursors have been reported to accelerate aging but not to extend lifespan. In eukaryotes, nicotinamide riboside is a newly discovered NAD(+) precursor that is converted to nicotinamide mononucleotide by specific nicotinamide riboside kinases, Nrk1 and Nrk2. In this study, we discovered that exogenous nicotinamide riboside promotes Sir2-dependent repression of recombination, improves gene silencing, and extends lifespan without calorie restriction. The mechanism of action of nicotinamide riboside is totally dependent on increased net NAD(+) synthesis through two pathways, the Nrk1 pathway and the Urh1/Pnp1/Meu1 pathway, which is Nrk1 independent. Additionally, the two nicotinamide riboside salvage pathways contribute to NAD(+) metabolism in the absence of nicotinamide-riboside supplementation. Thus, like calorie restriction in the mouse, nicotinamide riboside elevates NAD(+) and increases Sir2 function.
[show abstract][hide abstract] ABSTRACT: Nicotinamide adenine dinucleotide (NAD(+)) is both a coenzyme for hydride-transfer enzymes and a substrate for NAD(+)-consuming enzymes, which include ADP-ribose transferases, poly(ADP-ribose) polymerases, cADP-ribose synthases and sirtuins. Recent results establish protective roles for NAD(+) that might be applicable therapeutically to prevent neurodegenerative conditions and to fight Candida glabrata infection. In addition, the contribution that NAD(+) metabolism makes to lifespan extension in model systems indicates that therapies to boost NAD(+) might promote some of the beneficial effects of calorie restriction. Nicotinamide riboside, the recently discovered nucleoside precursor of NAD(+) in eukaryotic systems, might have advantages as a therapy to elevate NAD(+) without inhibiting sirtuins, which is associated with high-dose nicotinamide, or incurring the unpleasant side-effects of high-dose nicotinic acid.
Trends in Biochemical Sciences 02/2007; 32(1):12-9. · 13.08 Impact Factor
[show abstract][hide abstract] ABSTRACT: Glutamine-dependent NAD(+) synthetase, Qns1, utilizes a glutamine aminotransferase domain to supply ammonia for amidation of nicotinic acid adenine dinucleotide (NaAD(+)) to NAD(+). Earlier characterization of Qns1 suggested that glutamine consumption exceeds NAD(+) production by 40%. To explore whether Qns1 is systematically wasteful or whether additional features account for this behavior, we performed a careful kinetic and molecular genetic analysis. In fact, Qns1 possesses remarkable properties to reduce waste. The glutaminase active site is stimulated by NaAD(+) more than 50-fold such that glutamine is not appreciably consumed in the absence of NaAD(+). Glutamine consumption exceeds NAD(+) production over the whole range of glutamine and NaAD(+) substrate concentrations with greatest efficiency occurring at saturation of both substrates. Kinetic data coupled with site-directed mutagenesis of amino acids in the predicted ammonia channel indicate that NaAD(+) stimulates the glutaminase active site in the k(cat) term by a synergistic mechanism that does not require ammonia utilization by the NaAD(+) substrate. Six distinct classes of Qns1 mutants that fall within the glutaminase domain and the synthetase domain selectively inhibit components of the coordinated reaction.
Journal of Biological Chemistry 12/2006; 281(44):33395-402. · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: Production of NADP and NADPH depends on activity of NAD and NADH kinases. Here we characterized all combinations of mutants in yeast NAD and NADH kinases to determine their physiological roles. We constructed a diploid strain heterozygous for disruption of POS5, encoding mitochondrial NADH kinase, UTR1, cytosolic NAD kinase, and YEF1, a UTR1-homologous gene we characterized as encoding a low specific activity cytosolic NAD kinase. pos5 utr1 is a synthetic lethal combination rescued by plasmid-borne copies of the POS5 or UTR1 genes or by YEF1 driven by the ADH1 promoter. Respiratory-deficient and oxidative damage-sensitive defects in pos5 mutants were not made more deleterious by yef1 deletion, and a quantitative growth phenotype of pos5 and its arginine auxotrophy were repaired by plasmid-borne POS5 but not UTR1 or ADH1-driven YEF1. utr1 haploids have a slow growth phenotype on glucose not exacerbated by yef1 deletion but reversed by either plasmid-borne UTR1 or ADH1-driven YEF1. The defect in fermentative growth of utr1 mutants renders POS5 but not POS5-dependent mitochondrial genome maintenance essential because rho-utr1 derivatives are viable. Purified Yef1 has similar nucleoside triphosphate specificity but substantially lower specific activity and less discrimination in favor of NAD versus NADH phosphorylation than Utr1. Low expression and low intrinsic NAD kinase activity of Yef1 and the lack of phenotype associated with yef1 suggest that Utr1 and Pos5 are responsible for essentially all NAD/NADH kinase activity in vivo. The data are compatible with a model in which there is no exchange of NADP, NADPH, or cytoplasmic NAD/NADH kinase between nucleocytoplasmic and mitochondrial compartments, but the cytoplasm is exposed to mitochondrial NAD/NADH kinase during the transit of the molecule.
Journal of Biological Chemistry 09/2006; 281(32):22439-45. · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: Hints, histidine triad nucleotide-binding proteins, are adenosine monophosphate-lysine hydrolases of uncertain biological function. Here we report the characterization of human Hint2.
Tissue distribution was determined by real-time quantitative polymerase chain reaction and immunoblotting, cellular localization by immunocytochemistry, and transfection with green fluorescent protein constructs. Enzymatic activities for protein kinase C and adenosine phosphoramidase in the presence of Hint2 were measured. HepG2 cell lines with Hint2 overexpressed or knocked down were established. Apoptosis was assessed by immunoblotting for caspases and by flow cytometry. Tumor growth was measured in SCID mice. Expression in human tumors was investigated by microarrays.
Hint2 was predominantly expressed in liver and pancreas. Hint2 was localized in mitochondria. Hint2 hydrolyzed adenosine monophosphate linked to an amino group (AMP-pNA; k(cat):0.0223 s(-1); Km:128 micromol/L). Exposed to apoptotic stress, fewer HepG2 cells overexpressing Hint2 remained viable (32.2 +/- 0.6% vs 57.7 +/- 4.6%), and more cells displayed changes of the mitochondrial membrane potential (87.8 +/- 2.35 vs 49.7 +/- 1.6%) with more cleaved caspases than control cells. The opposite was observed in HepG2 cells with knockdown expression of Hint2. Subcutaneous injection of HepG2 cells overexpressing Hint2 in SCID mice resulted in smaller tumors (0.32 +/- 0.13 g vs 0.85 +/- 0.35 g). Microarray analyses revealed that HINT2 messenger RNA is downregulated in hepatocellular carcinomas (-0.42 +/- 0.58 log2 vs -0.11 +/- 0.28 log2). Low abundance of HINT2 messenger RNA was associated with poor survival.
Hint2 defines a novel class of mitochondrial apoptotic sensitizers down-regulated in hepatocellular carcinoma.