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1.  Quantification of Protein Copy Number in Yeast: The NAD+ Metabolome 
PLoS ONE  2014;9(9):e106496.
Saccharomyces cerevisiae is calorie-restricted by lowering glucose from 2% to 0.5%. Under low glucose conditions, replicative lifespan is extended in a manner that depends on the NAD+-dependent protein lysine deacetylase Sir2 and NAD+ salvage enzymes. Because NAD+ is required for glucose utilization and Sir2 function, it was postulated that glucose levels alter the levels of NAD+ metabolites that tune Sir2 function. Though NAD+ precursor vitamins, which increase the levels of all NAD+ metabolites, can extend yeast replicative lifespan, glucose restriction does not significantly change the levels or ratios of intracellular NAD+ metabolites. To test whether glucose restriction affects protein copy numbers, we developed a technology that combines the measurement of Urh1 specific activity and quantification of relative expression between Urh1 and any other protein. The technology was applied to obtain the protein copy numbers of enzymes involved in NAD+ metabolism in rich and synthetic yeast media. Our data indicated that Sir2 and Pnc1, two enzymes that sequentially convert NAD+ to nicotinamide and then to nicotinic acid, are up-regulated by glucose restriction in rich media, and that Pnc1 alone is up-regulated in synthetic media while levels of all other enzymes are unchanged. These data suggest that production or export of nicotinic acid might be a connection between NAD+ and calorie restriction-mediated lifespan extension in yeast.
doi:10.1371/journal.pone.0106496
PMCID: PMC4154715  PMID: 25188219
2.  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
3.  Calorie Restriction-Mediated Replicative Lifespan Extension in Yeast Is Non-Cell Autonomous 
PLoS Biology  2015;13(1):e1002048.
Calorie-restriction extends lifespan in many multicellular organisms; here substances secreted by calorie-restricted yeast are found to induce longer life in other yeast cells, suggesting that cellular communication is a component of this phenomenon even in a single-celled organism.
In laboratory yeast strains with Sir2 and Fob1 function, wild-type NAD+ salvage is required for calorie restriction (CR) to extend replicative lifespan. CR does not significantly alter steady state levels of intracellular NAD+ metabolites. However, levels of Sir2 and Pnc1, two enzymes that sequentially convert NAD+ to nicotinic acid (NA), are up-regulated during CR. To test whether factors such as NA might be exported by glucose-restricted mother cells to survive later generations, we developed a replicative longevity paradigm in which mother cells are moved after 15 generations on defined media. The experiment reveals that CR mother cells lose the longevity benefit of CR when evacuated from their local environment to fresh CR media. Addition of NA or nicotinamide riboside (NR) allows a moved mother to maintain replicative longevity despite the move. Moreover, conditioned medium from CR-treated cells transmits the longevity benefit of CR to moved mother cells. Evidence suggests the existence of a longevity factor that is dialyzable but is neither NA nor NR, and indicates that Sir2 is not required for the longevity factor to be produced or to act. Data indicate that the benefit of glucose-restriction is transmitted from cell to cell in budding yeast, suggesting that glucose restriction may benefit neighboring cells and not only an individual cell.
Author Summary
Though calorie restriction extends lifespan and healthspan in multiple model organisms, the intrinsic mechanisms remain unclear. In budding yeast Saccharomyces cerevisiae, manipulation of nicotinamide adenine dinucleotide (NAD+)—a central metabolic cofactor—can restrict or extend replicative lifespan, suggesting that NAD+-dependent targets might be mediators of extended longevity. However, although treating cells with the NAD+ precursor nicotinamide riboside extends lifespan, intracellular NAD+ metabolites levels are not altered by glucose restriction. This suggests the potential involvement of extracellular factors in replicative lifespan extension. Here we show that though yeast cells display a longevity benefit upon glucose restriction, these cells surprisingly lose the longevity benefit if moved from their local environment to fresh glucose-restricted media. They are, however, able to regain the longevity benefit, despite the change in environment, if the new environment is supplemented with conditioned medium from glucose restricted cells. Our results suggest that calorie restriction-induced longevity is not cell autonomous and, instead, appears to be transmitted from cell to cell in S. cerevisiae via a dialyzable extracellular factor.
doi:10.1371/journal.pbio.1002048
PMCID: PMC4310591  PMID: 25633578
4.  Nicotinamide Riboside Kinase Structures Reveal New Pathways to NAD+ 
PLoS Biology  2007;5(10):e263.
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+.
Author Summary
Biosynthesis of nicotinamide adenine dinucleotide (NAD+) is fundamental to cells, because NAD+ is an essential co-factor for metabolic and gene regulatory pathways that control life and death. Two vitamin precursors of NAD+ were discovered in 1938. We recently discovered nicotinamide riboside (NR) as a third vitamin precursor of NAD+ in eukaryotes, which extends yeast life span without caloric restriction and protects damaged dorsal root ganglion neurons from degeneration. Biosynthesis of NAD+ from NR requires enzyme activities in either of two pathways. In one pathway, specific NR kinases, including human Nrk1 and Nrk2, phosphorylate NR to nicotinamide mononucleotide. A second and Nrk-independent pathway is initiated by yeast nucleoside-splitting enzymes, Urh1 and Pnp1. We solved five crystal structures of human Nrk1 and, on the basis of co-crystal structures with substrates, suggested that the enzyme might be able to phosphorylate a novel compound, nicotinic acid riboside (NaR). We then demonstrated that human Nrk enzymes have dual specificity as NR/NaR kinases in vitro, and we established the ability of NaR to be used as a vitamin precursor of NAD+ via pathways initiated by Nrk1, Urh1, and Pnp1 in living yeast cells. Thus, starting from the structure of human Nrk1, we discovered a synthetic vitamin precursor of NAD+ and suggest the possibility that NaR is a normal NAD+ metabolite.
Eukaryotic nicotinamide riboside kinase (Nrk) converts nicotinamide riboside to NAD+ by phosphorylation and adenylylation. The structures of this enzyme bound to several substrates lead to identification of new pathways to NAD+
doi:10.1371/journal.pbio.0050263
PMCID: PMC1994991  PMID: 17914902
5.  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
6.  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
7.  Yeast Tdh3 (Glyceraldehyde 3-Phosphate Dehydrogenase) Is a Sir2-Interacting Factor That Regulates Transcriptional Silencing and rDNA Recombination 
PLoS Genetics  2013;9(10):e1003871.
Sir2 is an NAD+-dependent histone deacetylase required to mediate transcriptional silencing and suppress rDNA recombination in budding yeast. We previously identified Tdh3, a glyceraldehyde 3-phosphate dehydrogenase (GAPDH), as a high expression suppressor of the lethality caused by Sir2 overexpression in yeast cells. Here we show that Tdh3 interacts with Sir2, localizes to silent chromatin in a Sir2-dependent manner, and promotes normal silencing at the telomere and rDNA. Characterization of specific TDH3 alleles suggests that Tdh3's influence on silencing requires nuclear localization but does not correlate with its catalytic activity. Interestingly, a genetic assay suggests that Tdh3, an NAD+-binding protein, influences nuclear NAD+ levels; we speculate that Tdh3 links nuclear Sir2 with NAD+ from the cytoplasm.
Author Summary
Cells respond to changing signals or environmental conditions by altering the expression of their genes. For instance, our cells respond to the presence of glucose or insulin in the bloodstream by regulating the expression of genes involved in basic cell metabolism. The sirtuin family of proteins has been proposed to serve as a link between a cell's metabolic state and gene expression, although the molecular mechanisms that connect metabolic status with Sir2 activity remain unclear. The expression of genes is controlled in part by the structural organization of the local chromatin region within which they reside. The yeast sirtuin protein, Sir2, mediates repression (“silencing”) of sets of genes by modulating the structural organization of specific chromatin regions. In this study we describe a novel link between a key metabolic enzyme and Sir2 function. We show that a yeast GAPDH protein, which plays a central role in glucose metabolism, also associates with Sir2 in the nucleus and promotes Sir2-dependent gene silencing. Sirtuin activity requires a small molecule, NAD+, whose availability may fluctuate depending on the metabolic state of the cell. Based on our data, we suggest that Tdh3 may promote silencing by maintaining sufficient levels of NAD+ available to Sir2 within the nucleus.
doi:10.1371/journal.pgen.1003871
PMCID: PMC3798266  PMID: 24146631
8.  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
9.  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
10.  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
11.  SIR-2.1 integrates metabolic homeostasis with the reproductive neuromuscular excitability in early aging male Caenorhabditis elegans 
eLife  2014;3:e01730.
The decline of aging C. elegans male’s mating behavior is correlated with the increased excitability of the cholinergic circuitry that executes copulation. In this study, we show that the mating circuits’ functional durability depends on the metabolic regulator SIR-2.1, a NAD+-dependent histone deacetylase. Aging sir-2.1(0) males display accelerated mating behavior decline due to premature hyperexcitability of cholinergic circuits used for intromission and ejaculation. In sir-2.1(0) males, the hypercontraction of the spicule-associated muscles pinch the vas deferens opening, thus blocking sperm release. The hyperexcitability is aggravated by reactive oxygen species (ROS). Our genetic, pharmacological, and behavioral analyses suggest that in sir-2.1(0) and older wild-type males, enhanced catabolic enzymes expression, coupled with the reduced expression of ROS-scavengers contribute to the behavioral decline. However, as a compensatory response to reduce altered catabolism/ROS production, anabolic enzymes expression levels are also increased, resulting in higher gluconeogenesis and lipid synthesis.
DOI: http://dx.doi.org/10.7554/eLife.01730.001
eLife digest
Although the signs of aging are clear to us all, precisely why we age is less well understood. One possibility is that as cells use oxygen to fuel the breakdown of large molecules into smaller ones to release energy, they also generate by-products called reactive oxygen species that can damage DNA. As we get older, this damage gets worse. Consistent with this idea, it has been shown that a reduced calorie intake can reduce oxidative damage in certain species, in addition to extending lifespan.
Many experiments on aging have been performed on worms belonging to the species C. elegans. Male worms of this species live for an average of 11–12 days, but begin to show signs of aging—for example, a reduced ability to mate—as early as day 3 of their adult lives. Now, Guo and García have revealed that a protein called SIR-2.1, which regulates metabolism in worms, also helps to protect the animals from the effects of aging.
Male worms in which the gene for this protein has been ‘knocked out’ have a normal lifespan, but show signs of aging earlier than normal males. They are also more susceptible to the damaging effects of reactive oxygen species, suggesting that SIR-2.1 may offer protection against oxidative damage. Indeed, levels of ATP—the molecule used to move energy around inside cells—are increased in knockout worms. This suggests that certain metabolic processes and the production of reactive oxygen species, are increased in the knockout worms, which speeds up the aging process.
While the link between metabolism and aging is well known, the work of Guo and García offers insights into some of the molecular mechanisms that may form the basis of this relationship.
DOI: http://dx.doi.org/10.7554/eLife.01730.002
doi:10.7554/eLife.01730
PMCID: PMC3989601  PMID: 24755287
behavioral aging; reactive oxygen species (ROS); metabolism; cell excitability; PEPCK; compensatory mechanism; C. elegans
12.  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
13.  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
14.  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
15.  Identification of LDH-A as a therapeutic target for cancer cell killing via (i) p53/NAD(H)-dependent and (ii) p53-independent pathways 
Oncogenesis  2014;3(5):e102-.
Most cancer cells use aerobic glycolysis to fuel their growth. The enzyme lactate dehydrogenase-A (LDH-A) is key to cancer's glycolytic phenotype, catalysing the regeneration of nicotinamide adenine dinucleotide (NAD+) from reduced nicotinamide adenine dinucleotide (NADH) necessary to sustain glycolysis. As such, LDH-A is a promising target for anticancer therapy. Here we ask if the tumour suppressor p53, a major regulator of cellular metabolism, influences the response of cancer cells to LDH-A suppression. LDH-A knockdown by RNA interference (RNAi) induced cancer cell death in p53 wild-type, mutant and p53-null human cancer cell lines, indicating that endogenous LDH-A promotes cancer cell survival irrespective of cancer cell p53 status. Unexpectedly, however, we uncovered a novel role for p53 in the regulation of cancer cell NAD+ and its reduced form NADH. Thus, LDH-A silencing by RNAi, or its inhibition using a small-molecule inhibitor, resulted in a p53-dependent increase in the cancer cell ratio of NADH:NAD+. This effect was specific for p53+/+ cancer cells and correlated with (i) reduced activity of NAD+-dependent deacetylase sirtuin 1 (SIRT1) and (ii) an increase in acetylated p53, a known target of SIRT1 deacetylation activity. In addition, activation of the redox-sensitive anticancer drug EO9 was enhanced selectively in p53+/+ cancer cells, attributable to increased activity of NAD(P)H-dependent oxidoreductase NQO1 (NAD(P)H quinone oxidoreductase 1). Suppressing LDH-A increased EO9-induced DNA damage in p53+/+ cancer cells, but importantly had no additive effect in non-cancer cells. Our results identify a unique strategy by which the NADH/NAD+ cellular redox status can be modulated in a cancer-specific, p53-dependent manner and we show that this can impact upon the activity of important NAD(H)-dependent enzymes. To summarise, this work indicates two distinct mechanisms by which suppressing LDH-A could potentially be used to kill cancer cells selectively, (i) through induction of apoptosis, irrespective of cancer cell p53 status and (ii) as a part of a combinatorial approach with redox-sensitive anticancer drugs via a novel p53/NAD(H)-dependent mechanism.
doi:10.1038/oncsis.2014.16
PMCID: PMC4035693  PMID: 24819061
cancer metabolism; LDH-A; NAD+/NADH; p53; apoptosis; combinatorial anticancer therapy
16.  Sir2 Protein Deacetylases: Evidence for Chemical Intermediates and Functions of a Conserved Histidine† 
Biochemistry  2006;45(1):272-282.
Sir2 NAD+-dependent protein deacetylases are implicated in a variety of cellular processes such as apoptosis, gene silencing, life-span regulation, and fatty acid metabolism. In spite of this, there have been relatively few investigations into the detailed chemical mechanism. Sir2 proteins (sirtuins) catalyze the chemical conversion of NAD+ and acetylated-lysine to nicotinamide, deacetylated-lysine, and 2’-O-acetyl-ADP-ribose (OAADPr). In this study, Sir2-catalyzed reactions are shown to transfer an 18O-label from the peptide acetyl group to the ribose 1’-position of OAADPr, providing direct evidence for the formation of a covalent α-1’-O-alkylamidate, whose existence is further supported by the observed methanolysis of the α-1’-O-alkylamidate intermediate to yield β-1’-O-methyl-ADP-ribose in a Sir2 histidine-to-alanine mutant. This conserved histidine (His-135 in HST2) activates the ribose 2’-hydroxyl for attack on the α-1’-O-alkylamidate. The histidine mutant is stalled at the intermediate, allowing water and other alcohols to compete kinetically with the attacking 2’-hydroxyl. Measurement of the pH dependence of kcat and kcat/Km values for both wild-type and histidine-to-alanine mutant enzymes confirms roles of this residue in NAD+-binding and in general-base activation of the 2’-hydroxyl. Also, transfer of an 18O-label from water to the carbonyl oxygen of the acetyl group in OAADPr is consistent with water addition to the proposed 1’-2’cyclic intermediate formed after 2’-hydroxyl attack on the α-1’-O-alkylamidate. The effect of pH and of solvent viscosity on the kcat values suggests that final product release is rate-limiting in the wild-type enzyme. Implications of this new evidence on the mechanisms of deacetylation and possible ADP-ribosylation catalyzed by Sir2 deacetylases are discussed.
doi:10.1021/bi052014t
PMCID: PMC2519119  PMID: 16388603
SIR2; sirtuin; Deacetylation; NAD; Histone
17.  Energy metabolism, altered proteins, sirtuins and ageing: converging mechanisms? 
Biogerontology  2007;9(1):49-55.
The predominant molecular symptom of ageing is the accumulation of altered gene products. Nutritional studies show that ageing in animals can be significantly influenced by dietary restriction. Genetics has revealed that ageing may be controlled by changes in intracellular NAD/NADH ratio regulating sirtuin activity. Physiological and other approaches indicate that mitochondria may also regulate ageing. A mechanism is proposed which links diet, exercise and mitochondria-dependent changes in NAD/NADH ratio to intracellular generation of altered proteins. It is suggested that ad libitum feeding conditions decrease NAD availability which also decreases metabolism of the triose phosphate glycolytic intermediates, glyceraldehyde-3-phosphate and dihydroxyacetone-phosphate, which can spontaneously decompose into methylglyoxal (MG). MG is a highly toxic glycating agent and a major source of protein advanced-glycosylation end-products (AGEs). MG and AGEs can induce mitochondrial dysfunction and formation of reactive oxygen species (ROS), as well as affect gene expression and intracellular signalling. In dietary restriction–induced fasting, NADH would be oxidised and NAD regenerated via mitochondrial action. This would not only activate sirtuins and extend lifespan but also suppress MG formation. This proposal can also explain the apparent paradox whereby increased aerobic activity suppresses formation of glycoxidized proteins and extends lifespan. Variation in mitochondrial DNA composition and consequent mutation rate, arising from dietary-controlled differences in DNA precursor ratios, could also contribute to tissue differences in age-related mitochondrial dysfunction.
doi:10.1007/s10522-007-9110-x
PMCID: PMC2174522  PMID: 17929190
NAD; NADH; Glycolysis; Methylglyoxal; Dietary restriction; Altered proteins; Deacetylases; Ageing
18.  The Critical Role of the Sirtuin Pathway in Aging and Alzheimer Disease: Mechanistic and Therapeutic Considerations 
Lancet neurology  2011;10(3):275-279.
Background
Advancements in gerontology have revealed key insights into the molecular and biochemical aspects of the aging process. The sirtuin pathway, most notable for its association with the anti-aging effects of calorie restriction, has received particular attention, and pharmacologic or transgenic upregulation of the sirtuin pathway has demonstrated some very promising results in laboratory models of aging. Alzheimer disease (AD), the leading cause of senile dementia, is a devastating neurodegenerative condition that is imposing an increasing burden on society. The lack of therapeutics currently available for the disease provides strong incentive for the development of an effective treatment strategy and, interestingly, research has uncovered a novel mechanism of action of the sirtuin pathway that offers significant potential as such.
Recent Developments
Sirt1, one of the seven mammalian homologues of the sirtuin family of NAD+-dependent deacetylases, has recently been demonstrated to attenuate amyloidogenic processing of amyloid-β protein precursor (AβPP) in cell culture studies in vitro and transgenic mouse models of AD. Mechanistically, Sirt1 increases α-secretase production and activity through activation of the α-secretase gene ADAM10. Since α-secretase is the critical enzyme responsible for the non-amyloidogenic cleavage of AβPP, upregulation of α-secretase shifts AβPP processing to reduce the pathological accumulation of the presumptive toxic Aβ species that results from β- and γ-secretase activity. Interestingly, a recent study of the spatial patterns of Aβ deposition in the brain indicates a strong correlation with an increased utilization of aerobic glycolysis in those regions. Aerobic glycolysis depletes cellular levels of NAD+ (via decreased NAD+/NADH ratio), and it is possible that a corresponding downregulation of the NAD+-dependent sirtuin pathway is partly responsible for the amyloidogenic processing of AβPP.
Where Next?
The specific inhibition of Aβ generation by Sirt1 coupled with the link between aerobic glycolysis, NAD+ depletion, and amyloidogenesis via the sirtuin pathway has translational implications. On the one hand, the likely underlying role of the sirtuin pathway in AD onset and development may enlighten our understanding of this devastating condition. On the other, therapeutic upregulation of Sirt1 may provide opportunities for the amelioration of AD-type neuropathology through an inhibition of amyloidogenesis, among other things (i.e., regulation of cellular metabolism or inhibition of tau pathology — see below). Ultimately, further analysis into both aspects is necessary if any progress is to be made.
doi:10.1016/S1474-4422(11)70013-8
PMCID: PMC3163839  PMID: 21349442
19.  Molecular Links between Caloric Restriction and Sir2/SIRT1 Activation 
Diabetes & Metabolism Journal  2014;38(5):321-329.
Ageing is the most significant risk factor for a range of prevalent diseases, including cancer, cardiovascular disease, and diabetes. Accordingly, interventions are needed for delaying or preventing disorders associated with the ageing process, i.e., promotion of healthy ageing. Calorie restriction is the only nongenetic and the most robust approach to slow the process of ageing in evolutionarily divergent species, ranging from yeasts, worms, and flies to mammals. Although it has been known for more than 80 years that calorie restriction increases lifespan, a mechanistic understanding of this phenomenon remains elusive. Yeast silent information regulator 2 (Sir2), the founding member of the sirtuin family of protein deacetylases, and its mammalian homologue Sir2-like protein 1 (SIRT1), have been suggested to promote survival and longevity of organisms. SIRT1 exerts protective effects against a number of age-associated disorders. Caloric restriction increases both Sir2 and SIRT1 activity. This review focuses on the mechanistic insights between caloric restriction and Sir2/SIRT1 activation. A number of molecular links, including nicotinamide adenine dinucleotide, nicotinamide, biotin, and related metabolites, are suggested to be the most important conduits mediating caloric restriction-induced Sir2/SIRT1 activation and lifespan extension.
doi:10.4093/dmj.2014.38.5.321
PMCID: PMC4209345  PMID: 25349818
Biotin; Dietary restriction; NAD; Nicotinamide; Lifespan; Sirtuins
20.  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
21.  Dietary restriction involves NAD+-dependent mechanisms and a shift toward oxidative metabolism 
Aging Cell  2014;13(6):1075-1085.
Interventions that slow aging and prevent chronic disease may come from an understanding of how dietary restriction (DR) increases lifespan. Mechanisms proposed to mediate DR longevity include reduced mTOR signaling, activation of the NAD+-dependent deacylases known as sirtuins, and increases in NAD+ that derive from higher levels of respiration. Here, we explored these hypotheses in Caenorhabditis elegans using a new liquid feeding protocol. DR lifespan extension depended upon a group of regulators that are involved in stress responses and mTOR signaling, and have been implicated in DR by some other regimens [DAF-16 (FOXO), SKN-1 (Nrf1/2/3), PHA-4 (FOXA), AAK-2 (AMPK)]. Complete DR lifespan extension required the sirtuin SIR-2.1 (SIRT1), the involvement of which in DR has been debated. The nicotinamidase PNC-1, a key NAD+ salvage pathway component, was largely required for DR to increase lifespan but not two healthspan indicators: movement and stress resistance. Independently of pnc-1, DR increased the proportion of respiration that is coupled to ATP production but, surprisingly, reduced overall oxygen consumption. We conclude that stress response and NAD+-dependent mechanisms are each critical for DR lifespan extension, although some healthspan benefits do not require NAD+ salvage. Under DR conditions, NAD+-dependent processes may be supported by a DR-induced shift toward oxidative metabolism rather than an increase in total respiration.
doi:10.1111/acel.12273
PMCID: PMC4244309  PMID: 25257342
aging; dietary restriction; C. elegans; stress response; sirtuins; NAD+
22.  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
23.  MSN2 and MSN4 Link Calorie Restriction and TOR to Sirtuin-Mediated Lifespan Extension in Saccharomyces cerevisiae 
PLoS Biology  2007;5(10):e261.
Calorie restriction (CR) robustly extends the lifespan of numerous species. In the yeast Saccharomyces cerevisiae, CR has been proposed to extend lifespan by boosting the activity of sirtuin deacetylases, thereby suppressing the formation of toxic repetitive ribosomal DNA (rDNA) circles. An alternative theory is that CR works by suppressing the TOR (target of rapamycin) signaling pathway, which extends lifespan via mechanisms that are unknown but thought to be independent of sirtuins. Here we show that TOR inhibition extends lifespan by the same mechanism as CR: by increasing Sir2p activity and stabilizing the rDNA locus. Further, we show that rDNA stabilization and lifespan extension by both CR and TOR signaling is due to the relocalization of the transcription factors Msn2p and Msn4p from the cytoplasm to the nucleus, where they increase expression of the nicotinamidase gene PNC1. These findings suggest that TOR and sirtuins may be part of the same longevity pathway in higher organisms, and that they may promote genomic stability during aging.
Author Summary
There are only a few techniques that reliably promote longevity in multiple, distantly related species. Perhaps the best known, caloric restriction (CR), was first shown to promote lifespan in rodents in the 1930s and has since been shown to work in most species it has been tested on. We and others have previously proposed that CR extends lifespan in budding yeast by boosting the activity of sirtuin deacetylases, which work to extend lifespan by suppressing genomic instability. A competing theory is that CR works by suppressing the TOR (target of rapamycin) signaling pathway, which has recently been discovered to extend the lifespan of yeast and worms, but the downstream players are not yet known. We show that TOR inhibition and sirtuins are part of the same CR pathway that extends yeast lifespan by stabilizing the genome. CR and TOR inhibition promote longevity by relocalizing two transcription factors, Msn2p and Msn4p, from the cytoplasm to the nucleus, where they increase expression of the nicotinamidase gene PNC1, a regulator of sirtuin activity. We propose that TOR signaling and sirtuins may also be part of the same CR pathway in mammals.
Both caloric restriction and suppression of the TOR signaling pathway are known to extend lifespan in yeast. Here, both manipulations are shown to act via the same mechanism.
doi:10.1371/journal.pbio.0050261
PMCID: PMC1994990  PMID: 17914901
24.  Malate and Fumarate Extend Lifespan in Caenorhabditis elegans 
PLoS ONE  2013;8(3):e58345.
Malate, the tricarboxylic acid (TCA) cycle metabolite, increased lifespan and thermotolerance in the nematode C. elegans. Malate can be synthesized from fumarate by the enzyme fumarase and further oxidized to oxaloacetate by malate dehydrogenase with the accompanying reduction of NAD. Addition of fumarate also extended lifespan, but succinate addition did not, although all three intermediates activated nuclear translocation of the cytoprotective DAF-16/FOXO transcription factor and protected from paraquat-induced oxidative stress. The glyoxylate shunt, an anabolic pathway linked to lifespan extension in C. elegans, reversibly converts isocitrate and acetyl-CoA to succinate, malate, and CoA. The increased longevity provided by malate addition did not occur in fumarase (fum-1), glyoxylate shunt (gei-7), succinate dehydrogenase flavoprotein (sdha-2), or soluble fumarate reductase F48E8.3 RNAi knockdown worms. Therefore, to increase lifespan, malate must be first converted to fumarate, then fumarate must be reduced to succinate by soluble fumarate reductase and the mitochondrial electron transport chain complex II. Reduction of fumarate to succinate is coupled with the oxidation of FADH2 to FAD. Lifespan extension induced by malate depended upon the longevity regulators DAF-16 and SIR-2.1. Malate supplementation did not extend the lifespan of long-lived eat-2 mutant worms, a model of dietary restriction. Malate and fumarate addition increased oxygen consumption, but decreased ATP levels and mitochondrial membrane potential suggesting a mild uncoupling of oxidative phosphorylation. Malate also increased NADPH, NAD, and the NAD/NADH ratio. Fumarate reduction, glyoxylate shunt activity, and mild mitochondrial uncoupling likely contribute to the lifespan extension induced by malate and fumarate by increasing the amount of oxidized NAD and FAD cofactors.
doi:10.1371/journal.pone.0058345
PMCID: PMC3589421  PMID: 23472183
25.  Characterization of Nicotinamidases: Steady-State Kinetic Parameters, Class-wide Inhibition by Nicotinaldehydes and Catalytic Mechanism† 
Biochemistry  2010;49(49):10421-10439.
Nicotinamidases are metabolic enzymes that hydrolyze nicotinamide to nicotinic acid. These enzymes are widely distributed across biology, with examples found encoded in the genomes of Mycobacteria, Archaea, Eubacteria, Protozoa, yeast and invertebrates but there are none found in mammals. Although recent structural work has improved understanding of these enzymes, their catalytic mechanism is still not well understood. Recent data shows that nicotinamidases are required for growth and virulence of several pathogenic microbes. The enzymes of Saccharomyces cerevisiae, Drosophila melanogaster and Caenorhabditis elegans regulate lifespan in their respective organisms, consistent with proposed roles in the regulation of NAD+ metabolism and organismal aging. In this manuscript, the steady state kinetic parameters of nicotinamidase enzymes from C. elegans, S. cerevisiae, Streptococcus pneumoniae (a pathogen responsible for human pneumonia), Borrelia burgdorferi (the pathogen that causes Lyme Disease) and Plasmodium falciparum (responsible for most human malaria) are reported. Nicotinamidases are generally efficient catalysts with steady state kcat values typically exceeding 1 s−1. The Km values for nicotinamide are low and are in the range from 2 – 110 µM. Nicotinaldehyde was determined to be a potent competitive inhibitor of these enzymes, binding in the low µM to low nM range for all nicotinamidases tested. A variety of nicotinaldehyde derivatives were synthesized and evaluated as inhibitors in kinetic assays. Inhibitions are consistent with reaction of the universally conserved catalytic Cys on each enzyme with the aldehyde carbonyl carbon to form a thiohemiacetal complex which is stabilized by a conserved oxyanion hole. The S. pneumoniae nicotinamidase can catalyse exchange of 18O into the carboxy oxygens of nicotinic acid with 18O-water. The collected data, along with kinetic analysis of several mutants, allowed us to propose a catalytic mechanism that explains nicotinamidase and nicotinic acid 18O exchange chemistry for the S. pneumoniae enzyme involving key catalytic residues, a catalytic transition metal ion and the intermediacy of a thioester intermediate.
doi:10.1021/bi1012518
PMCID: PMC3010197  PMID: 20979384

Results 1-25 (705518)