SUMMARY

A major cause of cell death caused by genotoxic stress is thought to be due to the depletion of NAD+ from the nucleus and the cytoplasm. Here we show that NAD+ levels in mitochondria remain at physiological levels following genotoxic stress and can maintain cell viability even when nuclear and cytoplasmic pools of NAD+ are depleted. Rodents fasted for 48 hr show increased levels of the NAD+ biosynthetic enzyme Nampt and a concomitant increase in mitochondrial NAD+. Increased Nampt provides protection against cell death and requires an intact mitochondrial NAD+ salvage pathway as well as the mitochondrial NAD+-dependent deacetylases SIRT3 and SIRT4. We discuss the relevance of these findings to understanding how nutrition modulates physiology and to the evolution of apoptosis.

Summary

SIRT1 regulates energy homeostasis by controlling the acetylation status and activity of a number of enzymes and transcriptional regulators. The fact that NAD+ levels control SIRT1 activity confers a hypothetical basis for the design of new strategies to activate SIRT1 by increasing NAD+ availability. Here we show that the deletion of the poly(ADP-ribose) polymerase-1 (PARP-1) gene, encoding a major NAD+-consuming enzyme, increases NAD+ content and SIRT1 activity in brown adipose tissue and muscle. PARP-1−/− mice phenocopied many aspects of SIRT1 activation, such as a higher mitochondrial content, increased energy expenditure, and protection against metabolic disease. Also, the pharmacologic inhibition of PARP in vitro and in vivo increased NAD+ content, SIRT1 activity and enhanced oxidative metabolism. These data show how PARP-1 inhibition has strong metabolic implications through the modulation of SIRT1 activity, a property that not only could be useful in the management of metabolic diseases but also of cancer.

Summary

SIRT1 is a NAD+-dependent enzyme that affects metabolism by deacetylating key transcriptional regulators of energy expenditure. Here we tested whether deletion of PARP-2, an alternative NAD+ consuming enzyme, impacts on NAD+ bioavailability and SIRT1 activity. Our results indicate that PARP-2 deficiency increases SIRT1 activity in cultured myotubes. However, this increase was not due to changes in NAD+ levels, but to an increase in SIRT1 expression, as PARP-2 acts as a direct negative regulator of the SIRT1 promoter. PARP-2 deletion in mice increases SIRT1 levels, promotes energy expenditure, and increases mitochondrial content. Furthermore, PARP-2−/− mice were protected against diet-induced obesity. Despite being insulin sensitized, PARP-2−/− mice were glucose intolerant due to a defective pancreatic function. Hence, while inhibition of PARP activity promotes oxidative metabolism through SIRT1 activation, the use of PARP inhibitors for metabolic purposes will require further understanding of the specific functions of different PARP family members.

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.

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.

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.

The ability to probe for catalytic activities of enzymes and to detect their abundance in complex biochemical contexts has traditionally relied on a combination of kinetic assays and techniques such as western blots that use expensive reagents such as antibodies. The ability to simultaneously detect activity and isolate a protein catalyst from a mixture is even more difficult and currently impossible in most cases. In this manuscript we describe a chemical approach that achieves this goal for a unique family of enzymes called sirtuins using novel chemical tools, enabling rapid detection of activity and isolation of these protein catalysts. Sirtuin deacetylases are implicated in the regulation of many physiological functions including energy metabolism, DNA-damage response, and cellular stress resistance. We synthesized an aminooxy-derivatized NAD+ and a pan-sirtuin inhibitor that reacts on sirtuin active sites to form a chemically stable complex that can subsequently be crosslinked to an aldehyde-substituted biotin. Subsequent retrieval of the biotinylated sirtuin complexes on streptavidin beads followed by gel electrophoresis enabled simultaneous detection of active sirtuins, isolation and molecular weight determination. We show that these tools are cross reactive against a variety of human sirtuin isoforms including SIRT1, SIRT2, SIRT3, SIRT5, SIRT6 and can react with microbial derived sirtuins as well. Finally, we demonstrate the ability to simultaneously detect multiple sirtuin isoforms in reaction mixtures with this methodology, establishing proof of concept tools for chemical studies of sirtuins in complex biological samples.

Sirtuins are ancient proteins widely distributed in all lifeforms of earth. These proteins are universally able to bind NAD+, and activate it to effect ADP-ribosylation of cellular nucleophiles. The most commonly observed sirtuin reaction, is the ADPribosylation of acetyllysine, which leads to NAD+-dependent deacetylation. Other types of ADP-ribosylation have also been observed, including protein ADP-ribosylation, NAD+ solvolysis and ADP-ribosyltransfer to 5,6-dimethylbenzimidazole, a reaction involved in eubacterial cobalamin biosynthesis. This review broadly surveys the chemistries and chemical mechanisms of these enzymes.

The sodium salt of [immucillin-A–CO2H]− (Imm-A), namely catena-poly[[[triaqua­disodium(I)](μ-aqua)[μ-(1S)-N-car­box­yl­ato-1-(9-deaza­adenin-9-yl)-1,4-dide­oxy-1,4-imino-d-ribi­tol][triaqua­disodium(I)][μ-(1S)-N-carboxyl­ato-1-(9-deaza­aden­in-9-yl)-1,4-dide­oxy-1,4-imino-d-ribitol]] tetra­hydrate], {[Na2(C12H13N4O6)2(H2O)7]·4H2O}n, (I), forms a polymeric chain via Na+—O inter­actions involving the carboxyl­ate and keto O atoms of two independent Imm-A mol­ecules. Extensive N,O—H⋯O hydrogen bonding utilizing all water H atoms, including four waters of crystallization, provides crystal packing. The structural definition of this novel compound was made possible through the use of synchrotron radiation utilizing a minute fragment (volume ∼2.4 × 10−5 mm−3) on a beamline optimized for protein data collection. A summary of intra-ring conformations for immucillin structures indicates considerable flexibility while retaining similar intra-ring orientations.

Methods to construct 2’-deoxy-2’-fluoro-nucleosides have undergone limited improvement in the last twenty years in spite of substantially increased value of these compounds as pharmaceuticals and as tools for studying biological processes. We herein describe a consolidated approach to synthesize precursors to these commercially and scientifically valuable compounds via diastereocontrolled fluorination of the readily available precursor 2-deoxy-d-ribonolactone. With employment of appropriate sterically bulky silyl protecting groups at 3 and 5 positions, controlled electrophilic fluorination of the Li-ribonolactone enolate by N-fluorodibenezenesulfonamide yielded the corresponding 2-deoxy-2-fluoro-arabino-lactone in high isolated yield (72 %). The protected 2-deoxy-2, 2-difluoro-ribonolactone was obtained similarly in high yield from a second round of electrophilic fluorination (2 steps, 51% from protected ribonolactone starting material). Accomplishment of the difficult ribo-fluorination of the lactone was achieved by the directive effects of a diastereoselectively installed α-trimethylsilyl group. Electrophilic fluorination of a protected 2-deoxy-2-trimethylsilyl-arabino-lactone via enolate generation provided the protected 2-deoxy-2-fluoro-ribo-lactone as the exclusive fluorinated product. The reaction also yielded the starting material, the desilylated protected 2-deoxy-ribonolactone, which was recycled to provide a 38% chemical yield of the fluorinated product (versus initial protected ribonolactone) after consecutive silylation and fluorination cycles. Using our fluorinated sugar precursors we prepared the 2’-fluoro-arabino-, 2’-fluoro-ribo- and 2’,2’-difluoro-nicotinamide adenine dinucleotides (NAD+) of potential biological interest. These syntheses provide the most consolidated and efficient methods for production of sugar precursors of 2’-deoxy-2’-fluoronucleosides and have the advantage of utilizing an air-stable electrophilic fluorinating agent. The fluorinated NAD+s are anticipated to be useful for studying a variety of cellular metabolic and signaling processes.

Sirtuins are NAD+-dependent enzymes that deacetylate a variety of cellular proteins and in some cases catalyze protein ADP-ribosyltransfer. The catalytic mechanism of deacetylation is proposed to involve an ADPR-peptidylimidate, whereas the mechanism of ADP-ribosyltransfer to proteins is undetermined. Herein we characterize a Plasmodium falciparum sirtuin that catalyzes deacetylation of histone peptide sequences. Interestingly, the enzyme can also hydrolyze NAD+. Two mechanisms of hydrolysis were identified and characterized. One is independent of acetyllysine substrate and produces α-stereochemistry as established by reaction of methanol which forms α-1-O-methyl-ADPR. This reaction is insensitive to nicotinamide inhibition. The second solvolytic mechanism is dependent on acetylated peptide and is proposed to involve the imidate to generate β-stereochemistry. Stereochemistry was established by isolation of β-1-O-methyl-ADPR when methanol was added as a co-solvent. This solvolytic reaction was inhibited by nicotinamide, suggesting that nicotinamide and solvent compete for the imidate. These findings establish new reactions of wildtype sirtuins and suggest possible mechanisms for ADP-ribosylation to proteins. These findings also illustrate the potential utility of nicotinamide as a probe for mechanisms of sirtuin catalyzed ADP-ribosyltransfer.

SUMMARY

It is intuitive to speculate that nutrient availability may influence differentiation of mammalian cells. Nonetheless, a comprehensive complement of the molecular determinants involved in this process has not been elucidated yet. Here, we have investigated how nutrients (glucose) affect skeletal myogenesis. Glucose restriction (GR) impaired differentiation of skeletal myoblasts and was associated with activation of the AMP-activated protein kinase (AMPK). Activated AMPK was required to promote GR-induced transcription of the NAD+ biosynthetic enzyme Nampt. Indeed, GR augmented the Nampt activity, which consequently modified the intracellular [NAD+]/[NADH] ratio and nicotinamide levels, and mediated inhibition of skeletal myogenesis. Skeletal myoblasts derived from SIRT1+/− heterozygous mice were resistant to the effects of either GR or AMPK activation. These experiments reveal that AMPK, Nampt, and SIRT1 are the molecular components of a functional signaling pathway that allows skeletal muscle cells to sense and react to nutrient availability.

Sirtuins are recently discovered NAD+-dependent deacetylases that remove acetyl groups from acetyllysine-modified proteins, thereby regulating the biological function of their targets. Sirtuins have been shown to increase organism and tissue survival in diverse organisms, ranging from yeast to mammals. Evidence indicates that NAD+ metabolism and sirtuins contribute to mechanisms that influence cell survival under conditions of stress and toxicity. For example, recent work has shown that sirtuins and increased NAD+ biosynthesis provide protection against neuron axonal degeneration initiated by genotoxicity or trauma. In light of their protective effects, sirtuins and NAD+ metabolism could represent therapeutic targets for treatment of acute and chronic neurodegenerative conditions. Our work has focused on elucidating the enzymatic functions of sirtuins and quantifying perturbations of cellular NAD+ metabolism. We have developed mass spectrometry methods to quantitate cellular NAD+ and nicotinamide. These methods allow the quantitation of changes in the amounts of these metabolites in cells caused by chemical and genetic interventions. Characterization of the biochemical properties of sirtuins and investigations of NAD+ metabolism are likely to provide new insights into mechanisms by which NAD+ metabolism regulates sirtuin activities in cells. To develop new strategies to improve cell stress resistance, we have initiated proof of concept studies on pharmacological approaches that target sirtuins and NAD+ metabolism, with the goal of enhancing cell protection against genotoxicity.

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+

The NAD-dependent histone deacetylase Sir2 plays a key role in connecting cellular metabolism with gene silencing and aging. The androgen receptor (AR) is a ligand-regulated modular nuclear receptor governing prostate cancer cellular proliferation, differentiation, and apoptosis in response to androgens, including dihydrotestosterone (DHT). Here, SIRT1 antagonists induce endogenous AR expression and enhance DHT-mediated AR expression. SIRT1 binds and deacetylates the AR at a conserved lysine motif. Human SIRT1 (hSIRT1) repression of DHT-induced AR signaling requires the NAD-dependent catalytic function of hSIRT1 and the AR lysine residues deacetylated by SIRT1. SIRT1 inhibited coactivator-induced interactions between the AR amino and carboxyl termini. DHT-induced prostate cancer cellular contact-independent growth is also blocked by SIRT1, providing a direct functional link between the AR, which is a critical determinant of progression of human prostate cancer, and the sirtuins.