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1.  Nrt1 and Tna1-Independent Export of NAD+ Precursor Vitamins Promotes NAD+ Homeostasis and Allows Engineering of Vitamin Production 
PLoS ONE  2011;6(5):e19710.
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.
doi:10.1371/journal.pone.0019710
PMCID: PMC3092764  PMID: 21589930
2.  Targeted, LCMS-based Metabolomics for Quantitative Measurement of NAD+ Metabolites 
Nicotinamide adenine dinucleotide (NAD+) is a coenzyme for hydride transfer reactions and a substrate for sirtuins and other NAD+-consuming enzymes. The abundance of NAD +, NAD+ biosynthetic intermediates, and related nucleotides reflects the metabolic state of cells and tissues. High performance liquid chromatography (HPLC) followed by ultraviolet-visible (UV-Vis) spectroscopic analysis of NAD+ metabolites does not offer the specificity and sensitivity necessary for robust quantification of complex samples. Thus, we developed a targeted, quantitative assay of the NAD+ metabolome with the use of HPLC coupled to mass spectrometry. Here we discuss NAD+ metabolism as well as the technical challenges required for reliable quantification of the NAD+ metabolites. The new method incorporates new separations and improves upon a previously published method that suffered from the problem of ionization suppression for particular compounds.
doi:10.5936/csbj.201301012
PMCID: PMC3962138  PMID: 24688693
3.  Dependence of Tumor Cell Lines and Patient-Derived Tumors on the NAD Salvage Pathway Renders Them Sensitive to NAMPT Inhibition with GNE-61812 
Neoplasia (New York, N.Y.)  2013;15(10):1151-1160.
Nicotinamide adenine dinucleotide (NAD) is a critical metabolite that is required for a range of cellular reactions. A key enzyme in the NAD salvage pathway is nicotinamide phosphoribosyl transferase (NAMPT), and here, we describe GNE-618, an NAMPT inhibitor that depletes NAD and induces cell death in vitro and in vivo. While cells proficient for nicotinic acid phosphoribosyl transferase (NAPRT1) can be protected from NAMPT inhibition as they convert nicotinic acid (NA) to NAD independent of the salvage pathway, this protection only occurs if NA is added before NAD depletion. We also demonstrate that tumor cells are unable to generate NAD by de novo synthesis as they lack expression of key enzymes in this pathway, thus providing a mechanistic rationale for the reliance of tumor cells on the NAD salvage pathway. Identifying tumors that are sensitive to NAMPT inhibition is one potential way to enhance the therapeutic effectiveness of an NAMPT inhibitor, and here, we show that NAMPT, but not NAPRT1, mRNA and protein levels inversely correlate with sensitivity to GNE-618 across a panel of 53 non-small cell lung carcinoma cell lines. Finally, we demonstrate that GNE-618 reduced tumor growth in a patient-derived model, which is thought to more closely represent heterogeneous primary patient tumors. Thus, we show that dependence of tumor cells on the NAD salvage pathway renders them sensitive to GNE-618 in vitro and in vivo, and our data support further evaluation of the use of NAMPT mRNA and protein levels as predictors of overall sensitivity.
PMCID: PMC3819631  PMID: 24204194
4.  High Resolution Crystal Structures of Streptococcus pneumoniae Nicotinamidase with Trapped Intermediates Provide Insights into Catalytic Mechanism and Inhibition by Aldehydes∥,‡ 
Biochemistry  2010;49(40):8803-8812.
Nicotinamidases are salvage enzymes that convert nicotinamide to nicotinic acid. These enzymes are essential for the recycling of nicotinamide into NAD+ in most prokaryotes, most single cell and multicellular eukaryotes, but not in mammals. The significance of these enzymes for nicotinamide salvage and for NAD+ homeostasis has increased interest in nicotinamidases as possible antibiotic targets. Nicotinamidases are also regulators of intracellular nicotinamide concentrations, thereby regulating signaling of downstream NAD+ consuming enzymes, such as the NAD+-dependent deacetylases (sirtuins). Here, we report several high resolution crystal structures of the nicotinamidase from Streptococcus pneumoniae (SpNic) in unliganded and ligand-bound forms. The structure of the C136S mutant in complex with nicotinamide provides details about substrate binding while a trapped nicotinoyl-thioester complexed with SpNic reveals the structure of the proposed thioester reaction intermediate. Examination of the active site of SpNic reveals several important features including a metal ion that coordinates the substrate and the catalytically relevant water molecule, and an oxyanion hole which both orients the substrate and offsets the negative charge that builds up during catalysis. Structures of this enzyme with bound nicotinaldehyde inhibitors elucidate the mechanism of inhibition and provide further details about the catalytic mechanism. In addition, we provide a biochemical analysis of the identity and role of the metal ion that orients the ligand in the active site and activates the water molecule responsible for hydrolysis of the substrate. These data provide structural evidence for several proposed reaction intermediates and allow for a more complete understanding of the catalytic mechanism of this enzyme.
doi:10.1021/bi1012436
PMCID: PMC3006156  PMID: 20853856
5.  CYB5R3: a key player in aerobic metabolism and aging? 
Aging (Albany NY)  2009;2(1):63-68.
Aging results from a complex and not completely understood chain of processes that are associated with various negative metabolic consequences and ultimately leads to senescence and death. The intracellular ratio of pyridine nucleotides (NAD+/NADH), has been proposed to be at the center stage of age-related biochemical changes in organisms, and may help to explain the observed influence of calorie restriction and energy-sensitive proteins on lifespan in model organisms. Indeed, the NAD+/NADH ratios affect the activity of a number of proteins, including sirtuins, which have gained prominence in the aging field as potential mediators of the beneficial effects of calorie restriction and mediating lifespan. Here we review the activities of a redox enzyme (NQR1 in yeast and CYB5R3 in mammals) that also influences the NAD+/NADH ratio and may play a regulatory role that connects aerobic metabolism with aging.
PMCID: PMC2837205  PMID: 20228936
cytochrome b; reductase; NAD; /NADH; NQR1; lifespan; respiration
6.  Targeting NAD+ Metabolism in the Human Malaria Parasite Plasmodium falciparum 
PLoS ONE  2014;9(4):e94061.
Nicotinamide adenine dinucleotide (NAD+) is an essential metabolite utilized as a redox cofactor and enzyme substrate in numerous cellular processes. Elevated NAD+ levels have been observed in red blood cells infected with the malaria parasite Plasmodium falciparum, but little is known regarding how the parasite generates NAD+. Here, we employed a mass spectrometry-based metabolomic approach to confirm that P. falciparum lacks the ability to synthesize NAD+ de novo and is reliant on the uptake of exogenous niacin. We characterized several enzymes in the NAD+ pathway and demonstrate cytoplasmic localization for all except the parasite nicotinamidase, which concentrates in the nucleus. One of these enzymes, the P. falciparum nicotinate mononucleotide adenylyltransferase (PfNMNAT), is essential for NAD+ metabolism and is highly diverged from the human homolog, but genetically similar to bacterial NMNATs. Our results demonstrate the enzymatic activity of PfNMNAT in vitro and demonstrate its ability to genetically complement the closely related Escherichia coli NMNAT. Due to the similarity of PfNMNAT to the bacterial enzyme, we tested a panel of previously identified bacterial NMNAT inhibitors and synthesized and screened twenty new derivatives, which demonstrate a range of potency against live parasite culture. These results highlight the importance of the parasite NAD+ metabolic pathway and provide both novel therapeutic targets and promising lead antimalarial compounds.
doi:10.1371/journal.pone.0094061
PMCID: PMC3991606  PMID: 24747974
7.  Microbial NAD Metabolism: Lessons from Comparative Genomics 
Summary: 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.
doi:10.1128/MMBR.00042-08
PMCID: PMC2738131  PMID: 19721089
8.  Nicotinamide Clearance by Pnc1 Directly Regulates Sir2-Mediated Silencing and Longevity 
Molecular and Cellular Biology  2004;24(3):1301-1312.
The Saccharomyces cerevisiae Sir2 protein is an NAD+-dependent histone deacetylase (HDAC) that functions in transcriptional silencing and longevity. The NAD+ salvage pathway protein, Npt1, regulates Sir2-mediated processes by maintaining a sufficiently high intracellular NAD+ concentration. However, another NAD+ salvage pathway component, Pnc1, modulates silencing independently of the NAD+ concentration. Nicotinamide (NAM) is a by-product of the Sir2 deacetylase reaction and is a natural Sir2 inhibitor. Pnc1 is a nicotinamidase that converts NAM to nicotinic acid. Here we show that recombinant Pnc1 stimulates Sir2 HDAC activity in vitro by preventing the accumulation of NAM produced by Sir2. In vivo, telomeric, rDNA, and HM silencing are differentially sensitive to inhibition by NAM. Furthermore, PNC1 overexpression suppresses the inhibitory effect of exogenously added NAM on silencing, life span, and Hst1-mediated transcriptional repression. Finally, we show that stress suppresses the inhibitory effect of NAM through the induction of PNC1 expression. Pnc1, therefore, positively regulates Sir2-mediated silencing and longevity by preventing the accumulation of intracellular NAM during times of stress.
doi:10.1128/MCB.24.3.1301-1312.2004
PMCID: PMC321434  PMID: 14729974
9.  Quinolinate Salvage and Insights for Targeting NAD Biosynthesis in Group A Streptococci 
Journal of Bacteriology  2013;195(4):726-732.
The essential coenzyme NAD plays important roles in metabolic reactions and cell regulation in all organisms. As such, NAD synthesis has been investigated as a source for novel antibacterial targets. Cross-species genomics-based reconstructions of NAD metabolism in group A streptococci (GAS), combined with focused experimental testing in Streptococcus pyogenes, led to a better understanding of NAD metabolism in the pathogen. The predicted niacin auxotrophy was experimentally verified, as well as the essential role of the nicotinamidase PncA in the utilization of nicotinamide (Nm). PncA is dispensable in the presence of nicotinate (Na), ruling it out as a viable antibacterial target. The function of the “orphan” NadC enzyme, which is uniquely present in all GAS species despite the absence of other genes of NAD de novo synthesis, was elucidated. Indeed, the quinolinate (Qa) phosphoribosyltransferase activity of NadC from S. pyogenes allows the organism to sustain growth when Qa is present as a sole pyridine precursor. Finally, the redundancy of functional upstream salvage pathways in GAS species narrows the choice of potential drug targets to the two indispensable downstream enzymes of NAD synthesis, nicotinate adenylyltransferase (NadD family) and NAD synthetase (NadE family). Biochemical characterization of NadD confirmed its functional role in S. pyogenes, and its potential as an antibacterial target was supported by inhibition studies with previously identified class I inhibitors of the NadD enzyme family. One of these inhibitors efficiently inhibited S. pyogenes NadD (sp.NadD) in vitro (50% inhibitory concentration [IC50], 15 μM), exhibiting a noncompetitive mechanism with a Ki of 8 μM.
doi:10.1128/JB.02002-12
PMCID: PMC3562111  PMID: 23204464
10.  A Continuous Microplate Assay for Sirtuins and Nicotinamide Producing Enzymes 
Analytical biochemistry  2009;394(1):101-109.
NAD+-dependent protein deacetylases (sirtuins) and other enzymes that produce nicotinamide are integral to many cellular processes. Yet current activity measurements involve expensive and time-consuming assays. Here, we present a spectroscopic assay that circumvents many issues of previous methods. This assay permits continuous product monitoring over time, allows determination of steady-state kinetic parameters, and is readily adaptable to high throughput screening. The methodology uses an enzyme-coupled system in which nicotinamide is converted to nicotinic acid and ammonia by nicotinamidase. The ammonia is transferred to α-ketoglutarate via glutamate dehydrogenase, yielding glutamate and the oxidation of NAD(P)H to NAD(P)+, which is measured spectrophotometrically at 340 nm. Utilizing this continuous assay with sirtuin-1 (Sirt1) and the ADP-ribosyl cyclase CD38, the resulting steady-state kinetic parameters are in excellent agreement with values obtained by other published methods. Importantly, this assay permitted determination of kcat and Km values with the native acetylated substrate acetyl-CoA synthetase-1, measurement of Sirt1, Sirt2, and Sirt3 activities from mammalian cell extracts, and determination of IC50 values of various Sirt1 inhibitors. This assay is applicable to any nicotinamide forming enzyme and will be an important tool to address many outstanding questions surrounding their regulation.
doi:10.1016/j.ab.2009.07.019
PMCID: PMC2752052  PMID: 19615966
sirtuin; Sirt1; nicotinamide; sir2; NAD+; deacetylase; acetyl; CD38; nicotinamidase; glutamate dehydrogenase
11.  Increased Life Span due to Calorie Restriction in Respiratory-Deficient Yeast 
PLoS Genetics  2005;1(5):e69.
A model for replicative life span extension by calorie restriction (CR) in yeast has been proposed whereby reduced glucose in the growth medium leads to activation of the NAD+–dependent histone deacetylase Sir2. One mechanism proposed for this putative activation of Sir2 is that CR enhances the rate of respiration, in turn leading to altered levels of NAD+ or NADH, and ultimately resulting in enhanced Sir2 activity. An alternative mechanism has been proposed in which CR decreases levels of the Sir2 inhibitor nicotinamide through increased expression of the gene coding for nicotinamidase, PNC1. We have previously reported that life span extension by CR is not dependent on Sir2 in the long-lived BY4742 strain background. Here we have determined the requirement for respiration and the effect of nicotinamide levels on life span extension by CR. We find that CR confers robust life span extension in respiratory-deficient cells independent of strain background, and moreover, suppresses the premature mortality associated with loss of mitochondrial DNA in the short-lived PSY316 strain. Addition of nicotinamide to the medium dramatically shortens the life span of wild type cells, due to inhibition of Sir2. However, even in cells lacking both Sir2 and the replication fork block protein Fob1, nicotinamide partially prevents life span extension by CR. These findings (1) demonstrate that respiration is not required for the longevity benefits of CR in yeast, (2) show that nicotinamide inhibits life span extension by CR through a Sir2-independent mechanism, and (3) suggest that CR acts through a conserved, Sir2-independent mechanism in both PSY316 and BY4742.
Synopsis
Calorie restriction slows aging and increases life span in nearly every organism studied. The mechanism by which this occurs is one of the most important unanswered questions in biogerontology. One popular theory, based on work from the budding yeast Saccharomyces cerevisiae, proposes that calorie restriction works by causing a metabolic shift toward increased mitochondrial respiration, resulting in activation of a family of proteins known as Sirtuins. This study demonstrates that life span extension by calorie restriction does not require respiration and occurs even in cells completely lacking mitochondrial DNA. Interestingly, calorie restriction protects yeast cells against a severe longevity defect associated with absence of mitochondrial DNA, suggesting the possibility that the consequences of age-associated mitochondrial dysfunction might be alleviated or prevented by calorie restriction.
doi:10.1371/journal.pgen.0010069
PMCID: PMC1287956  PMID: 16311627
12.  Increased Life Span due to Calorie Restriction in Respiratory-Deficient Yeast 
PLoS Genetics  2005;1(5):e69.
A model for replicative life span extension by calorie restriction (CR) in yeast has been proposed whereby reduced glucose in the growth medium leads to activation of the NAD+–dependent histone deacetylase Sir2. One mechanism proposed for this putative activation of Sir2 is that CR enhances the rate of respiration, in turn leading to altered levels of NAD+ or NADH, and ultimately resulting in enhanced Sir2 activity. An alternative mechanism has been proposed in which CR decreases levels of the Sir2 inhibitor nicotinamide through increased expression of the gene coding for nicotinamidase, PNC1. We have previously reported that life span extension by CR is not dependent on Sir2 in the long-lived BY4742 strain background. Here we have determined the requirement for respiration and the effect of nicotinamide levels on life span extension by CR. We find that CR confers robust life span extension in respiratory-deficient cells independent of strain background, and moreover, suppresses the premature mortality associated with loss of mitochondrial DNA in the short-lived PSY316 strain. Addition of nicotinamide to the medium dramatically shortens the life span of wild type cells, due to inhibition of Sir2. However, even in cells lacking both Sir2 and the replication fork block protein Fob1, nicotinamide partially prevents life span extension by CR. These findings (1) demonstrate that respiration is not required for the longevity benefits of CR in yeast, (2) show that nicotinamide inhibits life span extension by CR through a Sir2-independent mechanism, and (3) suggest that CR acts through a conserved, Sir2-independent mechanism in both PSY316 and BY4742.
Synopsis
Calorie restriction slows aging and increases life span in nearly every organism studied. The mechanism by which this occurs is one of the most important unanswered questions in biogerontology. One popular theory, based on work from the budding yeast Saccharomyces cerevisiae, proposes that calorie restriction works by causing a metabolic shift toward increased mitochondrial respiration, resulting in activation of a family of proteins known as Sirtuins. This study demonstrates that life span extension by calorie restriction does not require respiration and occurs even in cells completely lacking mitochondrial DNA. Interestingly, calorie restriction protects yeast cells against a severe longevity defect associated with absence of mitochondrial DNA, suggesting the possibility that the consequences of age-associated mitochondrial dysfunction might be alleviated or prevented by calorie restriction.
doi:10.1371/journal.pgen.0010069
PMCID: PMC1287956  PMID: 16311627
13.  Regulation of Yeast Sirtuins by NAD+ Metabolism and Calorie Restriction 
Biochimica et biophysica acta  2009;1804(8):1567-1575.
The Sir2 family proteins (sirtuins) are evolutionally conserved NAD+ (nicotinamide adenine dinucleotide)-dependent protein deacetylases and ADP-ribosylases, which have been shown to play important roles in the regulation of stress response, gene transcription, cellular metabolism and longevity. Recent studies have also suggested that sirtuins are downstream targets of calorie restriction (CR), which mediate CR-induced beneficial effects including life span extension in a NAD+-dependent manner. CR extends life span in many species and has been shown to ameliorate many age-associated disorders such as diabetes and cancers. Understanding the mechanisms of CR as well as the regulation of sirtuins will therefore provide insights into the molecular basis of these age-associated metabolic diseases. This review focuses on discussing advances in studies of sirtuins and NAD+ metabolism in genetically tractable model system, the budding yeast Saccharomyces cerevisiae. These studies have unraveled key metabolic longevity factors in the CR signaling and NAD+ biosynthesis pathways, which may also contribute to the regulation of sirtuin activity. Many components of the NAD+ biosynthesis pathway and CR signaling pathway are conserved in yeast and higher eukaryotes including humans. Therefore, these findings will help elucidate the mechanisms underlying age-associated metabolic disease and perhaps human aging.
doi:10.1016/j.bbapap.2009.09.030
PMCID: PMC2886167  PMID: 19818879
Sir2; NAD+; calorie restriction; longevity regulation; aging
14.  Age Related Changes in NAD+ Metabolism Oxidative Stress and Sirt1 Activity in Wistar Rats 
PLoS ONE  2011;6(4):e19194.
The cofactor nicotinamide adenine dinucleotide (NAD+) has emerged as a key regulator of metabolism, stress resistance and longevity. Apart from its role as an important redox carrier, NAD+ also serves as the sole substrate for NAD-dependent enzymes, including poly(ADP-ribose) polymerase (PARP), an important DNA nick sensor, and NAD-dependent histone deacetylases, Sirtuins which play an important role in a wide variety of processes, including senescence, apoptosis, differentiation, and aging. We examined the effect of aging on intracellular NAD+ metabolism in the whole heart, lung, liver and kidney of female wistar rats. Our results are the first to show a significant decline in intracellular NAD+ levels and NAD∶NADH ratio in all organs by middle age (i.e.12 months) compared to young (i.e. 3 month old) rats. These changes in [NAD(H)] occurred in parallel with an increase in lipid peroxidation and protein carbonyls (o- and m- tyrosine) formation and decline in total antioxidant capacity in these organs. An age dependent increase in DNA damage (phosphorylated H2AX) was also observed in these same organs. Decreased Sirt1 activity and increased acetylated p53 were observed in organ tissues in parallel with the drop in NAD+ and moderate over-expression of Sirt1 protein. Reduced mitochondrial activity of complex I–IV was also observed in aging animals, impacting both redox status and ATP production. The strong positive correlation observed between DNA damage associated NAD+ depletion and Sirt1 activity suggests that adequate NAD+ concentrations may be an important longevity assurance factor.
doi:10.1371/journal.pone.0019194
PMCID: PMC3082551  PMID: 21541336
15.  Transition state of ADP-ribosylation of acetyllysine catalyzed by Archeaglobus fulgidus Sir2 determined by kinetic isotope effects and computational approaches 
Journal of the American Chemical Society  2010;132(35):12286-12298.
Sirtuins are protein modifying enzymes distributed throughout all forms of life. These enzymes bind NAD+, a universal metabolite, and react it with acetyllysine residues to effect deacetylation of protein side chains. This NAD+-dependent deacetylation reaction has been observed for sirtuin enzymes derived from archaeal, eubacterial, yeast, metazoan and mammalian species, suggesting conserved chemical mechanisms for these enzymes. The first chemical step of deacetylation is the reaction of NAD+ with an acetyllysine residue which forms an enzyme-bound ADPR-peptidylimidate intermediate and nicotinamide. In this manuscript, the transition state for the ADP-ribosylation of acetyllysine is solved for an Archaeaglobus fulgidus sirtuin (Af2Sir2). Kinetic isotope effects (KIEs) were obtained by the competitive substrate method and were [1N-15N] = 1.024(2), [1′N-14C] = 1.014(4), [1′N-3H] = 1.300(3), [2′N-3H] =1.099(5), [4′N-3H] = 0.997(2), [5′N-3H] = 1.020(5), [4′N-18O] = 0.984(5). KIEs were calculated for candidate transition state structures using computational methods (Gaussian 03 and ISOEFF 98) in order to match computed and experimentally determined KIEs to solve the transition state. The results indicate that the enzyme stabilizes a highly dissociated oxocarbenium ion-like transition state with very low bond orders to the leaving group nicotinamide and the nucleophile acetyllysine. A concerted yet highly asynchronous substitution mechanism forms the ADPR-peptidylimidate intermediate of the sirtuin deacetylation reaction.
doi:10.1021/ja910342d
PMCID: PMC2952425  PMID: 20718419
16.  Synthesizing and Salvaging NAD+: Lessons Learned from Chlamydomonas reinhardtii 
PLoS Genetics  2010;6(9):e1001105.
The essential coenzyme nicotinamide adenine dinucleotide (NAD+) plays important roles in metabolic reactions and cell regulation in all organisms. Bacteria, fungi, plants, and animals use different pathways to synthesize NAD+. Our molecular and genetic data demonstrate that in the unicellular green alga Chlamydomonas NAD+ is synthesized from aspartate (de novo synthesis), as in plants, or nicotinamide, as in mammals (salvage synthesis). The de novo pathway requires five different enzymes: L-aspartate oxidase (ASO), quinolinate synthetase (QS), quinolate phosphoribosyltransferase (QPT), nicotinate/nicotinamide mononucleotide adenylyltransferase (NMNAT), and NAD+ synthetase (NS). Sequence similarity searches, gene isolation and sequencing of mutant loci indicate that mutations in each enzyme result in a nicotinamide-requiring mutant phenotype in the previously isolated nic mutants. We rescued the mutant phenotype by the introduction of BAC DNA (nic2-1 and nic13-1) or plasmids with cloned genes (nic1-1 and nic15-1) into the mutants. NMNAT, which is also in the de novo pathway, and nicotinamide phosphoribosyltransferase (NAMPT) constitute the nicotinamide-dependent salvage pathway. A mutation in NAMPT (npt1-1) has no obvious growth defect and is not nicotinamide-dependent. However, double mutant strains with the npt1-1 mutation and any of the nic mutations are inviable. When the de novo pathway is inactive, the salvage pathway is essential to Chlamydomonas for the synthesis of NAD+. A homolog of the human SIRT6-like gene, SRT2, is upregulated in the NS mutant, which shows a longer vegetative life span than wild-type cells. Our results suggest that Chlamydomonas is an excellent model system to study NAD+ metabolism and cell longevity.
Author Summary
Nicotinamide adenine dinucleotide (NAD+) is an essential coenzyme. NAD+ is necessary for electron transfer in many metabolic reactions. NAD+ functions as a substrate for several enzymes, one of which is sirtuin, an enzyme involved in gene regulation and aging. NAD+ can be synthesized either from amino acids (de novo) or metabolites (salvage). Given the importance of NAD+, enzymes involved in NAD+ synthesis are targets for drug discovery. In the unicellular green alga Chlamydomonas we investigated both the de novo and salvage NAD+ biosynthetic pathways. Mutations in the plant-like de novo synthesis pathway lead to a nicotinamide-requiring phenotype. We identified an insertional mutation in the first enzyme in the mammal-like salvage pathway; it has no growth defect in cells with an active de novo synthesis pathway but causes lethality when the de novo synthesis pathway is inactive. Coupled with NAD+ biosynthesis, sirtuin is involved in NAD+ consumption. Our study links upregulation of a sirtuin gene with extended life span in the nic13-1 mutant strain, which has a defective de novo synthesis pathway and suggests that Chlamydomonas is an excellent genetic model to study NAD+ metabolism and cell longevity.
doi:10.1371/journal.pgen.1001105
PMCID: PMC2936527  PMID: 20838591
17.  NAD+-Dependent Deacetylase Hst1p Controls Biosynthesis and Cellular NAD+ Levels in Saccharomyces cerevisiae 
Molecular and Cellular Biology  2003;23(19):7044-7054.
Nicotine adenine dinucleotide (NAD+) performs key roles in electron transport reactions, as a substrate for poly(ADP-ribose) polymerase and NAD+-dependent protein deacetylases. In the latter two processes, NAD+ is consumed and converted to ADP-ribose and nicotinamide. NAD+ levels can be maintained by regeneration of NAD+ from nicotinamide via a salvage pathway or by de novo synthesis of NAD+ from tryptophan. Both pathways are conserved from yeast to humans. We describe a critical role of the NAD+-dependent deacetylase Hst1p as a sensor of NAD+ levels and regulator of NAD+ biosynthesis. Using transcript arrays, we show that low NAD+ states specifically induce the de novo NAD+ biosynthesis genes while the genes in the salvage pathway remain unaffected. The NAD+-dependent deacetylase activity of Hst1p represses de novo NAD+ biosynthesis genes in the absence of new protein synthesis, suggesting a direct effect. The known Hst1p binding partner, Sum1p, is present at promoters of highly inducible NAD+ biosynthesis genes. The removal of HST1-mediated repression of the NAD+ de novo biosynthesis pathway leads to increased cellular NAD+ levels. Transcript array analysis shows that reduction in cellular NAD+ levels preferentially affects Hst1p-regulated genes in comparison to genes regulated with other NAD+-dependent deacetylases (Sir2p, Hst2p, Hst3p, and Hst4p). In vitro experiments demonstrate that Hst1p has relatively low affinity toward NAD+ in comparison to other NAD+-dependent enzymes. These findings suggest that Hst1p serves as a cellular NAD+ sensor that monitors and regulates cellular NAD+ levels.
doi:10.1128/MCB.23.19.7044-7054.2003
PMCID: PMC193940  PMID: 12972620
18.  Pyridine Nucleotide Transhydrogenase from Azotobacter vinelandii 
Journal of Bacteriology  1970;102(2):438-447.
A method is described for the partial purification of pyridine nucleotide transhydrogenase from Azotobacter vinelandii (ATCC 9104) cells. The most highly purified preparation catalyzes the reduction of 300 μmoles of nicotinamide adenine dinucleotide (NAD+) per min per mg of protein under the assay conditions employed. The enzyme catalyzes the reduction of NAD+, deamino-NAD+, and thio-NAD+ with reduced nicotinamide adenine dinucleotide phosphate (NADPH) as hydrogen donor, and the reduction of nicotinamide adenine dinucleotide phosphate (NADP+) and thio-NAD+ with reduced NAD (NADH) as hydrogen donor. The reduction of acetylpyridine AD+, pyridinealdehyde AD+, acetylpyridine deamino AD+, and pyridinealdehydedeamino AD+ with NADPH as hydrogen donor was not catalyzed. The enzyme catalyzes the transfer of hydrogen more readily from NADPH than from NADH with different hydrogen acceptors. The transfer of hydrogen from NADH to NADP+ and thio-NAD+ was markedly stimulated by 2′-adenosine monophosphate (2′-AMP) and inhibited by adenosine diphosphate (ADP), adenosine triphosphate (ATP), and phosphate ions. The transfer of hydrogen from NADPH to NAD+ was only slightly affected by phosphate ions and 2′-AMP, except at very high concentrations of the latter reagent. In addition, the transfer of hydrogen from NADPH to thio-NAD+ was only slightly influenced by 2′-AMP, ADP, ATP, and other nucleotides. The kinetics of the transhydrogenase reactions which utilized thio-NAD+ as hydrogen acceptor and NADH or NADPH as hydrogen donor were studied in some detail. The results suggest that there are distinct binding sites for NADH and NAD+ and perhaps a third regulator site for NADP+ or 2′-AMP. The heats of activation for the transhydrogenase reactions were determined. The properties of this enzyme are compared with those of other partially purified transhydrogenases with respect to the regulatory functions of 2′-AMP and other nucleotides on the direction of flow of hydrogen between NAD+ and NADP+.
PMCID: PMC247569  PMID: 4392895
19.  Small molecule activators of SIRT1 as therapeutics for the treatment of type 2 diabetes 
Nature  2007;450(7170):712-716.
Calorie restriction extends lifespan and produces a metabolic profile desirable for treating diseases of ageing such as type 2 diabetes1,2. SIRT1, an NAD+-dependent deacetylase, is a principal modulator of pathways downstream of calorie restriction that produce beneficial effects on glucose homeostasis and insulin sensitivity3–9. Resveratrol, a polyphenolic SIRT1 activator, mimics the anti-ageing effects of calorie restriction in lower organisms and in mice fed a high-fat diet ameliorates insulin resistance, increases mitochondrial content, and prolongs survival10–14. Here we describe the identification and characterization of small molecule activators of SIRT1 that are structurally unrelated to, and 1,000-fold more potent than, resveratrol. These compounds bind to the SIRT1 enzyme—peptide substrate complex at an allosteric site amino-terminal to the catalytic domain and lower the Michaelis constant for acetylated substrates. In diet-induced obese and genetically obese mice, these compounds improve insulin sensitivity, lower plasma glucose, and increase mitochondrial capacity. In Zucker fa/fa rats, hyperinsulinaemic-euglycaemic clamp studies demonstrate that SIRT1 activators improve whole-body glucose homeostasis and insulin sensitivity in adipose tissue, skeletal muscle and liver. Thus, SIRT1 activation is a promising new therapeutic approach for treating diseases of ageing such as type 2 diabetes.
doi:10.1038/nature06261
PMCID: PMC2753457  PMID: 18046409
20.  NAD+ metabolism, a therapeutic target for age-related metabolic disease 
Critical reviews in biochemistry and molecular biology  2013;48(4):10.3109/10409238.2013.789479.
Nicotinamide adenine dinucleotide (NAD) is a central metabolic cofactor by virtue of its redox capacity, and as such regulates a wealth of metabolic transformations. However, the identification of the longevity protein Sir2, the founding member of the sirtuin protein family, as being NAD+-dependent reignited interest in this metabolite. The sirtuins (SIRT1-7 in mammals) utilize NAD+ to deacetylate proteins in different subcellular compartments with a variety of functions, but with a strong convergence on optimizing mitochondrial function. Since cellular NAD+ levels are limiting for sirtuin activity, boosting its levels is a powerful means to activate sirtuins as a potential therapy for mitochondrial, often age-related, diseases. Indeed, supplying excess precursors, or blocking its utilization by PARP enzymes or CD38/CD157, boosts NAD+ levels, activates sirtuins and promotes healthy aging. Here, we discuss the current state of knowledge of NAD+ metabolism, primarily in relation to sirtuin function. We highlight how NAD+ levels change in diverse physiological conditions, and how this can be employed as a pharmacological strategy.
doi:10.3109/10409238.2013.789479
PMCID: PMC3858599  PMID: 23742622
Aging; Metabolism; Mitochondria; PARPs; Sirtuins
21.  Sirtuin/Sir2 Phylogeny, Evolutionary Considerations and Structural Conservation 
Molecules and cells  2009;28(5):407-415.
The sirtuins are a protein family named after the first identified member, S. cerevisiae Sir2p. Sirtuins are protein deacetylases whose activity is dependent on NAD+ as a cosubstrate. They are structurally defined by two central domains that together form a highly conserved catalytic center, which catalyzes the transfer of an acetyl moiety from acetyllysine to NAD+, yielding nicotinamide, the unique metabolite O-acetyl-ADP-ribose and deacetylated lysine. One or more sirtuins are present in virtually all species from bacteria to mammals. Here we describe a phylogenetic analysis of sirtuins. Based on their phylogenetic relationship, sirtuins can be grouped into over a dozen classes and subclasses. Humans, like most vertebrates, have seven sirtuins: SIRT1-SIRT7. These function in diverse cellular pathways, regulating transcriptional repression, aging, metabolism, DNA damage responses and apoptosis. We show that these seven sirtuins arose early during animal evolution. Conserved residues cluster around the catalytic center of known sirtuin family members.
doi:10.1007/s10059-009-0169-x
PMCID: PMC3710699  PMID: 19936627
deacetylase; evolution; molecular phylogeny; SIR2; sirtuin
22.  Role of Sirtuins in Lifespan Regulation is Linked to Methylation of Nicotinamide 
Nature chemical biology  2013;9(11):693-700.
Sirtuins, a family of histone deacetylases, have a fiercely debated role in regulating lifespan. Contrasting recent observations, we here find that overexpression of sir-2.1, the orthologue of mammalian SirT1, does extend C. elegans lifespan. Sirtuins mandatorily convert NAD+ into nicotinamide (NAM). We here find that NAM and its metabolite, 1-methylnicotinamide (MNA), extend C. elegans lifespan, even in the absence of sir-2.1. We identify anmt-1 to encode a C. elegans orthologue of nicotinamide-N-methyltransferase (NNMT), the enzyme that methylates NAM to generate MNA. Disruption and overexpression of anmt-1 have opposing effects on lifespan independent of sirtuins, with loss of anmt-1 fully inhibiting sir-2.1-mediated lifespan extension. MNA serves as a substrate for a newly identified aldehyde oxidase, GAD-3, to generate hydrogen peroxide acting as a mitohormetic ROS signal to promote C. elegans longevity. Taken together, sirtuin-mediated lifespan extension depends on methylation of NAM, providing an unexpected mechanistic role for sirtuins beyond histone deacetylation.
doi:10.1038/nchembio.1352
PMCID: PMC4076143  PMID: 24077178
23.  Cross-Feeding of Escherichia coli Mutants Defective in the Biosynthesis of Nicotinamide Adenine Dinucleotide 
Journal of Bacteriology  1973;115(3):982-986.
Mutants of Escherichia coli defective in the biosynthesis of nicotinamide adenine dinucleotide (NAD) are able to grow in a Casamino Acids medium lacking NAD and its immediate precursors, nicotinic acid and nicotinamide. This property has allowed the development of a system to measure cross-feeding between a nadA and a nadB mutant. This system provides a means of isolating the intermediate, prequinolinic acid, as well as a biological assay for the compound. The nadB mutant feeds the nadA mutant, indicating that the nadA enzyme occurs first in the pathway and the nadB enzyme second. No cross-feeding was detected between nadA and nadC or between nadB and nadC.
PMCID: PMC246345  PMID: 4353874
24.  The NAD+ precursor nicotinamide riboside enhances oxidative metabolism and protects against high-fat diet induced obesity 
Cell metabolism  2012;15(6):838-847.
SUMMARY
As NAD+ is a rate-limiting co-substrate for the sirtuin enzymes, its modulation is emerging as a valuable tool to regulate sirtuin function and, consequently, oxidative metabolism. In line with this premise, decreased activity of PARP-1 or CD38 —both NAD+ consumers— increases NAD+ bioavailability, resulting in SIRT1 activation and protection against metabolic disease. Here we evaluated whether similar effects could be achieved by increasing the supply of nicotinamide riboside (NR), a recently described natural NAD+ precursor with the ability to increase NAD+ levels, Sir2-dependent gene silencing and replicative lifespan in yeast. We show that NR supplementation in mammalian cells and mouse tissues increases NAD+ levels and activates SIRT1 and SIRT3, culminating in enhanced oxidative metabolism and protection against high fat diet-induced metabolic abnormalities. Consequently, our results indicate that the natural vitamin, NR, could be used as a nutritional supplement to ameliorate metabolic and age-related disorders characterized by defective mitochondrial function.
doi:10.1016/j.cmet.2012.04.022
PMCID: PMC3616313  PMID: 22682224
25.  Periplasmic localization of nicotinate phosphoribosyltransferase in Escherichia coli. 
Journal of Bacteriology  1978;133(3):1108-1112.
Nicotinate phosphoribosyltransferase (NAPRTase) in Escherichia coli mediates the formation of nicotinate mononucleotide, a direct precursor of nicotinamide adenine dinucleotide (NAD), from nicotinate and 5-phosphoribosyl-1-pyrophosphate. Specifically, NAPRTase contributes to NAD synthesis by utilizing intracellular nicotinate formed from NAD degradation products, which are recycled by NAD cycle enzymes and exogenous nicotinate when it is available. In previous studies, it has been tacitly assumed that almost all NAD cycle enzymes are localized in the cytoplasm of E. coli. The results of this investigation provide evidence that NAPRTase is a periplasmic (extracytoplasmic) enzyme. The osmotic shock of exponential-phase cells of E. coli K-12 and ML 308-225 resulted in the release of 63 to 72% and 42 to 48%, respectively, of the NAPRTase into the shock medium. In addition, when exponential cells of strains K-12 and ML 308-225 were converted into spheroplasts, 75 to 84% and 54 to 68%, respectively, of the enzyme was released into the spheroplast medium. Since previous estimates of the effective levels of NAPRTase present in putative repressed and derepressed E. coli cells appeared to be very low, a more convenient and accurate alternative method for the evaluation of NAPRTase in whole cells was developed. The results show that NAPRTase is subject only to a modest degree of enzyme repression. In addition, no evidence was found for the presence of a protein or low-molecular-weight inhibitor of the enzyme in repressed cells.
PMCID: PMC222140  PMID: 346557

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