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Overexpressing Nicotinamidase 3 gene, and the exogenous application of its metabolite nicotinic acid (NA), enhance drought stress tolerance and increase biomass in Arabidopsis thaliana. With progressive global climatic changes, plant productivity is threatened severely by drought stress. Deciphering the molecular mechanisms regarding genes responsible for balancing plant growth and stress amelioration could imply multiple possibilities for future sustainable goals. Nicotinamide adenine dinucleotide (NAD) biosynthesis and recycling/ distribution is a crucial feature for plant growth. The current study focuses on the functional characterization of nicotinamidase 3 (NIC3) gene, which is involved in the biochemical conversion of nicotinamide (NAM) to nicotinic acid (NA) in the salvage pathway of NAD biosynthesis. Our data show that overexpression of NIC3 gene enhances drought stress tolerance and increases plant growth. NIC3-OX plants accumulated more NA as compared to WT plants. Moreover, the upregulation of several genes related to plant growth/stress tolerance indicates that regulating the NAD salvage pathway could significantly enhance plant growth and drought stress tolerance. The exogenous application of nicotinic acid (NA) showed a similar phenotype as the effect of overexpressing NIC3 gene. In short, we contemplated the role of NIC3 gene and NA application in drought stress tolerance and plant growth. Our results would be helpful in engineering plants with enhanced drought stress tolerance and increased growth potential.

Nicotinamidase (Nic) (E.C.3.5.1.19) is a representative protein of the isochorismatase superfamily from Escherichia coli. Despite showing no (+) γ-lactamase activity, its active site constellations (ASCs) are very similar to those of two other known (+) γ-lactamases (Mhpg and RutB), indicating that it could be a latent (+) γ-lactamase. In this study, the primary sequences of the five representative proteins of the isochorismatase superfamily from E. coli were aligned, and a "lid"-like unit of a six-residue loop (112GENPLV117) was established. The Nic protein was converted to a (+) γ-lactamase by eliminating the loop. A conversion mechanism was proposed in which a more compact binding pocket is formed after lid deletion. In addition, the "shrunk" binding pocket stabilized the small substrate and the catalysis intermediate, which triggered catalysis. Moreover, we identified another latent (+) γ-lactamase in the E. coli isochorismatase superfamily and successfully converted it into an active (+) γ-lactamase. In summary, the isochorismatase superfamily is potentially a good candidate for obtaining novel (+) γ-lactamases.

Nicotinamide (NAM) alters behavior in C. elegans and Drosophila, serving as an agonist of TRPV channels affecting sensory neurons and mimicking the mode of action of insecticides used to control phloem-feeding insects. The impact of NAM on green peach aphid (Myzus persicae) behaviors was assessed in artificial diet assays and foliar applications to Arabidopsis plants. Aphids feeding on artificial diets supplemented with NAM impaired stylet movement causing feeding interruptions and ultimately starvation and death. Aphid feeding behaviors were negatively impacted on NAM sprayed plants at concentrations as low as 2.5 mM leading to increased mortality. In choice assays with NAM sprayed leaves aphids showed clear preference for untreated control leaves. NAM is an intermediate in the NAD salvage pathway that should accumulate in nicotinamidase (nic) mutants. LC-MS analysis showed NAM accumulates 60-fold in nic-1-1 Arabidopsis mutants as compared with Col-0. Aphid reproductive potential was significantly decreased on nic-1-1 mutant plants, resulting in a smaller colony size and arrested population development. The results support the hypothesis that dietary NAM causes behavioral changes in aphids, including altered feeding, reduced reproduction, and increased mortality. NAM is thought to bind to TRPV channels causing overstimulation of sensory neurons in the aphid feeding apparatus.

Novel antimicrobials for treatment of Mycobacterium tuberculosis are needed. We hypothesized that nicotinamide (NAM) and nicotinic acid (NA) modulate macrophage function to restrict M. tuberculosis replication in addition to their direct antimicrobial properties. Both compounds had modest activity in 7H9 broth, but only NAM inhibited replication in macrophages. Surprisingly, in macrophages NAM and the related compound pyrazinamide restricted growth of bacille Calmette-Guérin but not wild-type Mycobacterium bovis, which both lack a functional nicotinamidase/pyrazinamidase (PncA) rendering each strain resistant to these drugs in broth culture. Interestingly, NAM was not active in macrophages infected with a virulent M. tuberculosis mutant encoding a deletion in pncA. We conclude that the differential activity of NAM and nicotinic acid on infected macrophages suggests host-specific NAM targets rather than PncA-dependent direct antimicrobial properties. These activities are sufficient to restrict attenuated BCG, but not virulent wild-type M. bovis or M. tuberculosis.

A novel Alcaligenes sp. strain P156, which can utilize nicotinamide as its sole source of carbon, nitrogen and energy, was enriched and isolated from soil in a solid waste treatment plant. Aerobic growth and degradation with nicotinamide were characterized. Seven nicotinamide degradation-related genes were obtained by sequence alignment from the genome sequence of strain P156. Four genes, designated naaA, naaD, naaE and naaF, were cloned and heterologously expressed. Nicotinamide degradation is initiated by deamination to form nicotinic acid catalyzed by the nicotinamidase NaaA, which shares highest amino acid sequence identity (27.2%) with nicotinamidase from Arabidopsis thaliana. Nicotinic acid is converted to 6-hydroxynicotinic acid, which is further oxidized to 2,5-dihydroxypyridine (2,5-DHP). 2,5-DHP is then transformed to a ring-cleavage product, N-formylmaleamic acid, by an Fe2+ dependent dioxygenase NaaD. N-formylmaleamic acid is transformed to fumaric acid through maleamic acid and maleic acid by NaaE and NaaF, respectively. To our knowledge, this is the first report of the complete microbial degradation of nicotinamide in bacteria. Nicotinamide is considered as a model compound for the study of microbial degradation of pyridinic compounds, and the nicotinamide degrading related genes in strain P156 were distributed differently from the reported similar gene clusters. Therefore, this study contribute to the knowledge on the degradation of pyridinic compounds.

Given the upsurge of drug-resistant tuberculosis worldwide, there is much focus on developing novel drug combinations allowing shorter treatment duration and a lower toxicity profile. Nicotinamide adenine dinucleotide (NAD) biosynthesis targeting is acknowledged as a promising strategy to combat drug-susceptible, drug-resistant, and latent tuberculosis (TB) infections. In this review, we describe the potential synergy of NAD biosynthesis inhibitors with several TB-drugs in prospective novel combination therapy. Despite not directly targeting the essential NAD cofactor’s biosynthesis, several TB prodrugs either require a NAD biosynthesis enzyme to be activated or form a toxic chemical adduct with NAD(H) itself. For example, pyrazinamide requires the action of nicotinamidase (PncA), often referred to as pyrazinamidase, to be converted into its active form. PncA is an essential player in NAD salvage and recycling. Since most pyrazinamide-resistant strains are PncA-defective, a combination with downstream NAD-blocking molecules may enhance pyrazinamide activity and possibly overcome the resistance mechanism. Isoniazid, ethionamide, and delamanid form NAD adducts in their active form, partly perturbing the redox cofactor metabolism. Indeed, NAD depletion has been observed in Mycobacterium tuberculosis (Mtb) during isoniazid treatment, and activation of the intracellular NAD phosphorylase MbcT toxin potentiates its effect. Due to the NAD cofactor’s crucial role in cellular energy production, additional synergistic correlations of NAD biosynthesis blockade can be envisioned with bedaquiline and other drugs targeting energy-metabolism in mycobacteria. In conclusion, future strategies targeting NAD metabolism in Mtb should consider its potential synergy with current and other forthcoming TB-drugs.

In Saccharomyces cerevisiae, replicative life span (RLS) is primarily affected by the stability of ribosomal DNA (rDNA). The stability of the highly repetitive rDNA array is maintained through transcriptional silencing by the NAD+-dependent histone deacetylase Sir2. Recently, the loss of Smi1, a protein of unknown molecular function that has been proposed to be involved in cell wall synthesis, has been demonstrated to extend RLS in S. cerevisiae, but the mechanism by which Smi1 regulates RLS has not been elucidated. In this study, we determined that the loss of Smi1 extends RLS in a Sir2-dependent manner. We observed that the smi1Δ mutation enhances transcriptional silencing at the rDNA locus and promotes rDNA stability. In the absence of Smi1, the stress-responsive transcription factor Msn2 translocates from the cytoplasm to the nucleus, and nuclear-accumulated Msn2 stimulates the expression of nicotinamidase Pnc1, which serves as an activator of Sir2. In addition, we observed that the MAP kinase Hog1 is activated in smi1Δ cells and that the activation of Hog1 induces the translocation of Msn2 into the nucleus. Taken together, our findings suggest that the loss of Smi1 leads to the nuclear accumulation of Msn2 and stimulates the expression of Pnc1, thereby enhancing Sir2-mediated rDNA stability and extending RLS in S. cerevisiae.

Entamoeba histolytica is a protozoan parasite and a major cause of dysentery and diarrheal disease in developing countries. Disease transmission from one host to another occurs via cysts which can survive in environmental extremes and are transmitted through contaminated food and water. Recent studies in our lab identified a novel transcription factor, Encystation Regulatory Motif- Binding Protein (ERM-BP), which is responsive to NAD+ and has an important role in encystation. The key residues important for ERM-BP function were demonstrated in vitro using recombinant protein. In this study we demonstrate the in vivo functional consequences of mutations in key domains and their impact on Entamoeba encystation. Our results show that mutations in the DNA binding domain (ERM-BP-DBM) and in the nicotinamidase domain (ERM-BP-C198A) lead to protein mis-localization in both trophozoites and cysts and significantly reduce encystation efficiency. Additionally, we showed that silencing of ERM-BP significantly decreased the size and number of multi-nucleated giant cells (MGC) that form during encystation, indicating that ERM-BP functions upstream of the cellular aggregation that precedes stage conversion. Dissection of epistatic interactions between ERM-BP and a second encystation-related transcription factor, NF-Y revealed that ERM-BP is upstream of NF-Y in controlling the developmental cascade and appears to be one of the earliest regulators of development identified to date in Entamoeba. We also demonstrated that ERM-BP is upregulated during heat stress in Entamoeba, another condition which increases intracellular NAD+ levels and that overexpression of ERM-BP makes E. histolytica and E. invadens parasites more resistant to heat stress. Overexpression of ERM-BP in E. histolytica also induced the formation of cyst-like quadrinucleated cells and formation of MGCs. Overall, our work has identified an important role of ERM-BP in Entamoeba stress response and links an NAD+-responsive transcription factor to both development and heat shock response. Characterization of stress and developmental cascades are important avenues to investigate for Entamoeba, an important human parasitic pathogen.

Tuberculosis is an infectious disease caused by the bacterium Mycobacterium tuberculosis and remains one of the major causes of disease and death worldwide. Pyrazinamide is a key drug used in the treatment of tuberculosis, yet its mechanism of action is not fully understood, and testing strains of M. tuberculosis for pyrazinamide resistance is not easy with the tools that are presently available. The significance of the present research is that a metallochaperone-like protein may be crucial to pyrazinamide’s mechanisms of action and of resistance. This may support the development of improved tools to detect pyrazinamide resistance, which would have significant implications for the clinical management of patients with tuberculosis: drug regimens that are appropriately tailored to the resistance profile of a patient’s individual strain lead to better clinical outcomes, reduced onward transmission of infection, and reduction of the development of resistant strains that are more challenging and expensive to treat., Mycobacterium tuberculosis nicotinamidase-pyrazinamidase (PZAse) is a metalloenzyme that catalyzes conversion of nicotinamide-pyrazinamide to nicotinic acid-pyrazinoic acid. This study investigated whether a metallochaperone is required for optimal PZAse activity. M. tuberculosis and Escherichia coli PZAses (PZAse-MT and PZAse-EC, respectively) were inactivated by metal depletion (giving PZAse-MT–Apo and PZAse-EC–Apo). Reactivation with the E. coli metallochaperone ZnuA or Rv2059 (the M. tuberculosis analog) was measured. This was repeated following proteolytic and thermal treatment of ZnuA and Rv2059. The CDC1551 M. tuberculosis reference strain had the Rv2059 coding gene knocked out, and PZA susceptibility and the pyrazinoic acid (POA) efflux rate were measured. ZnuA (200 μM) achieved 65% PZAse-EC–Apo reactivation. Rv2059 (1 μM) and ZnuA (1 μM) achieved 69% and 34.3% PZAse-MT–Apo reactivation, respectively. Proteolytic treatment of ZnuA and Rv2059 and application of three (but not one) thermal shocks to ZnuA significantly reduced the capacity to reactivate PZAse-MT–Apo. An M. tuberculosis Rv2059 knockout strain was Wayne positive and susceptible to PZA and did not have a significantly different POA efflux rate than the reference strain, although a trend toward a lower efflux rate was observed after knockout. The metallochaperone Rv2059 restored the activity of metal-depleted PZAse in vitro. Although Rv2059 is important in vitro, it seems to have a smaller effect on PZA susceptibility in vivo. It may be important to mechanisms of action and resistance to pyrazinamide in M. tuberculosis. Further studies are needed for confirmation. IMPORTANCE Tuberculosis is an infectious disease caused by the bacterium Mycobacterium tuberculosis and remains one of the major causes of disease and death worldwide. Pyrazinamide is a key drug used in the treatment of tuberculosis, yet its mechanism of action is not fully understood, and testing strains of M. tuberculosis for pyrazinamide resistance is not easy with the tools that are presently available. The significance of the present research is that a metallochaperone-like protein may be crucial to pyrazinamide’s mechanisms of action and of resistance. This may support the development of improved tools to detect pyrazinamide resistance, which would have significant implications for the clinical management of patients with tuberculosis: drug regimens that are appropriately tailored to the resistance profile of a patient’s individual strain lead to better clinical outcomes, reduced onward transmission of infection, and reduction of the development of resistant strains that are more challenging and expensive to treat.

The nicotinamidase/pyrazinamidase PncA is a member of a large family of hydrolase enzymes that catalyze the deamination of nicotinamide to nicotinic acid. PncA also functions as a pyrazinamidase in a wide variety of eubacteria and is an essential coenzyme in many cellular redox reactions in living systems. We report the crystal structure of substrate-free PncA from Bacillus subtilis (BsPncA) at 2.0 A resolution to improve our understanding of the PncA family. The structure of BsPncA consists of an α/β domain and a subdomain. The subdomain of BsPncA has a different conformation than that of PncA enzymes from other organisms. The B-factor analysis revealed a rigid structure of the α/β domain, while the subdomain is highly flexible. Both dimers and tetramers were observed in BsPncA protein crystals, but only dimers were observed in solution. Our results provide useful information that will further enhance our understanding of the molecular functions of PncA family members.

Nicotinamide adenine dinucleotide (NAD), a cofactor for hundreds of metabolic reactions in all cell types, plays an essential role in metabolism, DNA repair, and aging. However, how NAD metabolism is impacted by the environment remains unclear. Here, we report an unexpected trans-kingdom cooperation between bacteria and mammalian cells wherein bacteria contribute to host NAD biosynthesis. Bacteria confer resistance to inhibitors of NAMPT, the rate-limiting enzyme in the amidated NAD salvage pathway, in cancer cells and xenograft tumors. Mechanistically, a microbial nicotinamidase (PncA) that converts nicotinamide to nicotinic acid, a precursor in the alternative deamidated NAD salvage pathway, is necessary and sufficient for this protective effect. Using stable isotope tracing and microbiota-depleted mice, we demonstrate that this bacteria-mediated deamidation contributes substantially to the NAD-boosting effect of oral nicotinamide and nicotinamide riboside supplementation in several tissues. Collectively, our findings reveal an important role of bacteria-enabled deamidated pathway in host NAD metabolism.

Maleamate amidohydrolase (NicF) is a key enzyme in vitamin B3 metabolism that catalyzes the hydrolysis of maleamate to produce maleic acid and ammonia. Unlike most members from the amidohydrolase superfamily it does not require a metal ion. Here, we use multiscale computational enzymology to investigate the catalytic mechanism, substrate binding, oxyanion hole, and roles of key active site residues of NicF from Bordetella bronchiseptica. In particular, molecular dynamics (MD) simulations, quantum mechanics/molecular mechanics (QM/MM) and QTAIM methods have been applied. The mechanism of the NicF-catalyzed reaction proceeds by a nucleophilic addition-elimination sequence involving the formation of a thioester enzyme intermediate (IC2 in stage 1) followed by hydrolysis of the thioester bond to form the products (stage 2). Consequently, the formation of IC2 in stage 1 is the rate-limiting step with a barrier of 88.8 kJ·mol-1 relative to the reactant complex, RC. Comparisons with related metal-dependent enzymes, particularly the zinc-dependent nicotinamidase from Streptococcus pneumonia (SpNic), have also been made to further illustrate unique features of the present mechanism. Along with -NH- donor groups of the oxyanion hole (i.e., HN-Thr146, HN-Cys150), the active site β-hydroxyl of threonine (HO-βThr146) is concluded to play a role in stabilizing the carbonyl oxygen of maleamate during the mechanism.

Peroxisomes are ubiquitous cell organelles that play a central role in cellular lipid and hydrogen peroxide metabolism. These organelles also fulfill several non-metabolic functions related to stress and stress adaptation. Although they contribute to the generation of harmful reactive oxygen species (ROS), peroxisomes also possess enzymes to maintain redox balance and counteract oxidative stress. Peroxisomal defects in humans result in severe disorders which are often lethal, illustrating the importance of these organelles. The research described in this thesis aimed to identify novel peroxisomal functions in yeasts, which play an important role as simple model organisms in cell biology. We identified a novel peroxisomal protein with a functional peroxisome targeting signal (PTS). However, the absence of this protein did not affect yeast cells sensitivity to stress conditions. Next in our study, we demonstrated how nicotinamidase (Pnc1), which does not contain a PTS, is imported into the peroxisomes by piggybacking onto a PTS containing protein, glycerol-3-phosphate dehydrogenase (Gpd1). We showed that Pnc1 physically interacts with Gpd1 allowing its piggyback import to peroxisomes. We also identified a novel peroxisomal membrane protein, Pex37, which is required for peroxisome fission and segregation between the mother cell and the bud. Additionally, we explored the role of a vacuolar membrane protein, Vac8 and identified that it is required for ER-vacuole contact sites (NVJs) formation and vacuole inheritance. Identification of novel peroxisome functions will open doors to endless research possibilities to explore and provide a more coherent understanding of the role of peroxisomes in multiple cellular functions.

Background For almost a century, antimonials have remained the first-line drugs for the treatment of leishmaniasis. However, little is known about their mode of action and clinical resistance mechanisms. Objectives We have previously shown that Leishmania nicotinamidase (PNC1) is an essential enzyme for parasite NAD+ homeostasis and virulence in vivo. Here, we found that parasites lacking the pnc1 gene (Δpnc1) are hypersusceptible to the active form of antimony (SbIII) and used these mutant parasites to better understand antimony’s mode of action and the mechanisms leading to resistance. Methods SbIII-resistant WT and Δpnc1 parasites were selected in vitro by a stepwise selection method. NAD(H)/NADP(H) dosages and quantitative RT–PCR experiments were performed to explain the susceptibility differences observed between strains. WGS and a marker-free CRISPR/Cas9 base-editing approach were used to identify and validate the role of a new resistance mutation. Results NAD+-depleted Δpnc1 parasites were highly susceptible to SbIII and this phenotype could be rescued by NAD+ precursor or trypanothione precursor supplementation. Δpnc1 parasites could become resistant to SbIII by an unknown mechanism. WGS revealed a unique amino acid substitution (H451Y) in an EF-hand domain of an orphan calcium-dependent kinase, recently named SCAMK. When introduced into a WT reference strain by base editing, the H451Y mutation allowed Leishmania parasites to survive at extreme concentrations of SbIII, potentiating the rapid emergence of resistant parasites. Conclusions These results establish that Leishmania SCAMK is a new central hub of antimony’s mode of action and resistance development, and uncover the importance of drug tolerance mutations in the evolution of parasite drug resistance.

UMR BFP - Equipe Métabolisme; International audience; Central metabolism is the engine of plant biomass, supplying fruit growth with building blocks, energy, and biochemical cofactors. Among metabolic cornerstones, nicotinamide adenine dinucleotide (NAD) is particularly pivotal for electron transfer through reduction–oxidation (redox) reactions, thus participating in a myriad of biochemical processes. Besides redox functions, NAD is now assumed to act as an integral regulator of signaling cascades involved in growth and environmental responses. However, the regulation of NAD metabolism and signaling during fruit development remains poorly studied and understood. Here, we benefit from RNAseq and proteomic data obtained from nine growth stages of tomato fruit (var. Moneymaker) to dissect mRNA and protein profiles that link to NAD metabolism, including de novo biosynthesis, recycling, utilization, and putative transport. As expected for a cofactor synthesis pathway, protein profiles failed to detect enzymes involved in NAD synthesis or utilization, except for nicotinic acid phosphoribosyltransferase (NaPT) and nicotinamidase (NIC), which suggested that most NAD metabolic enzymes were poorly represented quantitatively. Further investigations on transcript data unveiled differential expression patterns during fruit development. Interestingly, among specific NAD metabolism-related genes, early de novo biosynthetic genes were transcriptionally induced in very young fruits, in association with NAD kinase, while later stages of fruit growth rather showed an accumulation of transcripts involved in later stages of de novo synthesis and in NAD recycling, which agreed with augmented NAD(P) levels. In addition, a more global overview of 119 mRNA and 78 protein significant markers for NAD(P)-dependent enzymes revealed differential patterns during tomato growth that evidenced clear regulations of primary metabolism, notably with respect to mitochondrial functions. Overall, we propose that NAD metabolism and signaling are very dynamic in the developing tomato fruit and that its differential regulation is certainly critical to fuel central metabolism linking to growth mechanisms.

Nicotinamide adenine dinucleotide (NAD), a cofactor for hundreds of metabolic reactions in all cell types, plays an essential role in diverse cellular processes including metabolism, DNA repair, and aging1. NAD metabolism is critical to maintain cellular homeostasis in response to the environment, and disruption of this homeostasis is associated with decreased cellular NAD levels in aging2. Conversely, elevated NAD synthesis is required to sustain the increased metabolic rate of cancer cells3,4. Consequently, therapeutic strategies aimed to both upregulate NAD (i.e. NAD-boosting nutriceuticals) or downregulate NAD (inhibitors of key NAD synthesis enzymes) are being actively investigated5–10. However, how this essential metabolic pathway is impacted by the environment remains unclear. Here, we report an unexpected trans-kingdom cooperation between bacteria and mammalian cells wherein bacteria contribute to host NAD biosynthesis. Bacteria confer cancer cells with the resistance to inhibitors of NAMPT, the rate limiting enzyme in the main vertebrate NAD salvage pathway. Mechanistically, a microbial nicotinamidase (PncA) that converts nicotinamide to nicotinic acid, a key precursor in the alternative deamidated NAD salvage pathway, is necessary and sufficient for this protective effect. This bacteria-enabled resistance mechanism that allows the mammalian host to bypass the drug-induced metabolic block represents a novel paradigm in drug resistance. This host-microbe metabolic interaction also enables bacteria to dramatically enhance the NAD-boosting efficiency of nicotinamide supplementationin vitroandin vivo, demonstrating a crucial role of microbes, gut microbiota in particular, in organismal NAD metabolism.

Nicotinamide is both a reaction product and an inhibitor of the conserved sirtuin family of deacetylases, which have been implicated in a broad range of cellular functions in eukaryotes from yeast to humans. Phenotypes observed following treatment with nicotinamide are most often assumed to stem from inhibition of one or more of these enzymes. Here, we used this small molecule to inhibit multiple sirtuins at once during treatment with DNA damaging agents in the Saccharomyces cerevisiae model system. Since sirtuins have been previously implicated in the DNA damage response, we were surprised to observe that nicotinamide actually increased the survival of yeast cells exposed to the DNA damage agent MMS. Remarkably, we found that enhanced resistance to MMS in the presence of nicotinamide was independent of all five yeast sirtuins. Enhanced resistance was also independent of the nicotinamide salvage pathway, which uses nicotinamide as a substrate to generate NAD+, and of a DNA damage-induced increase in the salvage enzyme Pnc1. Our data suggest a novel and unexpected function for nicotinamide that has broad implications for its use in the study of sirtuin biology across model systems.

Nicotinamide adenine dinucleotide (NAD), a cofactor for hundreds of metabolic reactions in all cell types, plays an essential role in metabolism, DNA repair, and aging. However, how NAD metabolism is impacted by the environment remains unclear. Here, we report an unexpected trans-kingdom cooperation between bacteria and mammalian cells wherein bacteria contribute to host NAD biosynthesis. Bacteria confer resistance to inhibitors of NAMPT, the rate-limiting enzyme in the amidated NAD salvage pathway, in cancer cells and xenograft tumors. Mechanistically, a microbial nicotinamidase (PncA) that converts nicotinamide to nicotinic acid, a precursor in the alternative deamidated NAD salvage pathway, is necessary and sufficient for this protective effect. Using stable isotope tracing and microbiota-depleted mice, we demonstrate that this bacteria-mediated deamidation contributes substantially to the NAD-boosting effect of oral nicotinamide and nicotinamide riboside supplementation in several tissues. Collectively, our findings reveal an important role of bacteria-enabled deamidated pathway in host NAD metabolism.

Plant pathogenic fungi are a major threat to food security and impose a severe economic burden, thus there is a continuous need to develop new strategies to manage them. NAD+ is a co-factor in numerous enzymatic activities and determines the metabolic fate of the cell. Therefore, maintenance of NAD+ concentration is important for cellular viability. Consequently, the NAD+ biosynthetic pathway and redox homeostasis was suggested as a target for antifungal development. We aimed to study how Fusarium oxysporum senses and responds to nicotinaldehyde (NA), an inhibitor of Pnc1, a key enzyme in the salvage pathway of NAD+ biosynthesis. We were able to show that NA was inhibitory in high concentrations to several fungal plant pathogens, with much milder effects on tomato growth. Under low nutrient conditions NA reduced the total amounts of NAD+ in the fungal cell, a trend that was also observed in rich media, although without statistical significance. In low and high nutrient availability NA dramatically reduced the NAD+/NADH ratio. After exposure to NA, NADH levels were increased and NAD+ levels and the biomass were greatly reduced. Cells responded to NA by up-regulation of oxidoreductases, with hardly any up-regulation of the classic response to oxidative stress. Direct measurement of oxidative stress response showed that unlike formaldehyde and hydrogen peroxide, NA caused reductive rather than oxidative stress. Surprisingly, alcohol dehydrogenases were significantly up-regulated more than any other dehydrogenases, including aldehyde dehydrogenases. We propose that conidia of F. oxysporum efficiently detoxified the aldehyde group of NA by reducing NAD+ to NADH; the high concentrations of the latter provoked the expression of alcohol dehydrogenases that in yeast can act to reduce NADH and increase NAD+ amounts, respectively. Overall, the results suggest that targeting NAD+ biosynthesis pathway and redox homeostasis can be a potential approach to manage fungal plant pathogens. Many of the natural antifungal compounds produced by bio-control agents or even the natural biome are aldehydes, and thus the results presented here predict the possible response of Fusarium to wide sources of toxicity in the environment.

DNA methylation plays an important role in diverse developmental processes in many eukaryotes, including the response to environmental stress. Abscisic acid (ABA) is a plant hormone that is up-regulated under stress. The involvement of DNA methylation in the ABA response has been reported but is poorly understood. DNA demethylation is a reverse process of DNA methylation and often induces structural changes of chromatin leading to transcriptional activation. In Arabidopsis (Arabidopsis thaliana), active DNA demethylation depends on the activity of REPRESSOR OF SILENCING 1 (ROS1), which directly excises 5-methylcytosine from DNA. Here we showed that ros1 mutants were hypersensitive to ABA during early seedling development and root elongation. Expression levels of some ABA-inducible genes were decreased in ros1 mutants, and more than 60% of their proximal regions became hypermethylated, indicating that a subset of ABA-inducible genes are under the regulation of ROS1-dependent DNA demethylation. Notable among them is NICOTINAMIDASE 3 (NIC3) that encodes an enzyme that converts nicotinamide to nicotinic acid in the NAD(+) salvage pathway. Many enzymes in this pathway are known to be involved in stress responses. The nic3 mutants display hypersensitivity to ABA, whereas overexpression of NIC3 restores normal ABA responses. Our data suggest that NIC3 is responsive to ABA but requires ROS1-mediated DNA demethylation at the promoter as a prerequisite to transcriptional activation. These findings suggest that ROS1-induced active DNA demethylation maintains the active state of NIC3 transcription in response to ABA.

Nicotinamidase is a key enzyme for the salvage pathway catalyzing the first step for the conversion of nicotinamide (NAm) to nicotinic acid (NA) required for the synthesis of Nicotinamide Adenine Dinucleotide (NAD+) in the subsequent steps. Leishmania protozoan parasites are NAD+ auxotrophs and need precursors (nicotinamide, nicotinic acid, nicotinamide riboside) from their host environment to synthesize NAD+ for their survival. Interestingly, absence of this enzyme in higher eukaryotes and its absolute requirement in the developmental cycle of Leishmania has led nicotinamidase an attractive drug target towards leishmaniasis. Hence, we report some potential inhibitors for nicotinamidase screened based on 3-D pharmacophore model consisting of "ML", "Hyd|Aro", "Acc" and "Excl vol" features. Subsequently, dynamics simulation studies validate the proposed pharmacophore model suggesting its reliability for future studies. Furthermore, these essential site-specific features will help in enhancing the inhibition of nicotinamidase activity. Results of our study suggest that blocking of active site of nicotinamidase by the identified lead inhibitor will have major impact on the infectious processes and the survival of the parasite. Furthermore, due to the structural homology in the enzyme among L. donovani, L. infantum, L. major, we anticipate that our study would help to design more potent drug candidates against leshmaniasis for these three species.

6-Aminonicotinamide (6AN) is an antimetabolite used to inhibit the NADPH-producing pentose phosphate pathway (PPP) in many cellular systems, making them more susceptible to oxidative stress. It is converted by a NAD(P)+ glycohydrolase to 6-aminoNAD and 6-aminoNADP, causing the accumulation of PPP intermediates, due to their inability to participate in redox reactions. Some parasites like Plasmodium falciparum and Coccidia are highly sensitive but not all cell types showed a strong responsiveness to 6AN, probably due to the different targeted pathway. For instance, in bacteria the main target is the Preiss-Handler salvage pathway for NAD+ biosynthesis. We were interested in testing 6AN on the kinetoplastid protozoan Leishmania as another model to clarify the mechanisms of action of 6AN, by using metabolomics. Leishmania promastigotes, the life-cycle stage residing in the sandfly, demonstrated a three order of magnitude higher EC50 (mM) compared to P. falciparum and mammalian cells (μM), although pre-treatment with 100 μM 6AN prior to sub-lethal oxidative challenge induced a supra-additive cell kill in L. infantum. By metabolomics, we did not detect 6ANAD/P suggesting that NAD+ glycohydrolases in Leishmania may not be highly efficient in catalysing transglycosidation as happens in other microorganisms. Contrariwise to the reported effect on 6AN-treated cancer cells, we did not detect 6-phosphogluconate (6 PG) accumulation, indicating that 6ANADP cannot bind with high affinity to the PPP enzyme 6 PG dehydrogenase. By contrast, 6AN caused a profound phosphoribosylpyrophosphate (PRPP) decrease and nucleobases accumulation confirming that PPP is somehow affected. More importantly, we found a decrease in nicotinate production, evidencing the interference with the Preiss-Handler salvage pathway for NAD+ biosynthesis, most probably by inhibiting the reaction catalysed by nicotinamidase. Therefore, our combined data from Leishmania strains, though confirming the interference with PPP, also showed that 6AN impairs the Preiss-Handler pathway, underlining the importance to develop compounds targeting this last route.

Many organisms possess pathways that regenerate NAD+ from its degradation products, and two pathways are known to salvage NAD+ from nicotinamide (Nm). One is a four-step pathway that proceeds through deamination of Nm to nicotinic acid (Na) by Nm deamidase and phosphoribosylation to nicotinic acid mononucleotide (NaMN), followed by adenylylation and amidation. Another is a two-step pathway that does not involve deamination and directly proceeds with the phosphoribosylation of Nm to nicotinamide mononucleotide (NMN), followed by adenylylation. Judging from genome sequence data, the hyperthermophilic archaeon Thermococcus kodakarensis is supposed to utilize the four-step pathway, but the fact that the adenylyltransferase encoded by TK0067 recognizes both NMN and NaMN also raises the possibility of a two-step salvage mechanism. Here, we examined the substrate specificity of the recombinant TK1676 protein, annotated as nicotinic acid phosphoribosyltransferase. The TK1676 protein displayed significant activity toward Na and phosphoribosyl pyrophosphate (PRPP) and only trace activity with Nm and PRPP. We further performed genetic analyses on TK0218 (quinolinic acid phosphoribosyltransferase) and TK1650 (Nm deamidase), involved in de novo biosynthesis and four-step salvage of NAD+, respectively. The ΔTK0218 mutant cells displayed growth defects in a minimal synthetic medium, but growth was fully restored with the addition of Na or Nm. The ΔTK0218 ΔTK1650 mutant cells did not display growth in the minimal medium, and growth was restored with the addition of Na but not Nm. The enzymatic and genetic analyses strongly suggest that NAD+ salvage in T. kodakarensis requires deamination of Nm and proceeds through the four-step pathway.

Temporal developmental progression is highly coordinated in Caenorhabditis elegans. However, loss of nicotinamidase PNC-1 activity slows reproductive development, uncoupling it from its typical progression relative to the soma. Using LC/MS we demonstrate that pnc-1 mutants do not salvage the nicotinamide released by NAD(+) consumers to resynthesize NAD(+), resulting in a reduction in global NAD(+) bioavailability. We manipulate NAD(+) levels to demonstrate that a minor deficit in NAD(+) availability is incompatible with a normal pace of gonad development. The NAD(+) deficit compromises NAD(+) consumer activity, but we surprisingly found no functional link between consumer activity and reproductive development. As a result we turned to a comparative metabolomics approach to identify the cause of the developmental phenotype. We reveal widespread metabolic perturbations, and using complementary pharmacological and genetic approaches, we demonstrate that a glycolytic block accounts for the slow pace of reproductive development. Interestingly, mitochondria are protected from both the deficiency in NAD(+) biosynthesis and the effects of reduced glycolytic output. We suggest that compensatory metabolic processes that maintain mitochondrial activity in the absence of efficient glycolysis are incompatible with the requirements for reproductive development, which requires high levels of cell division. In addition to demonstrating metabolic requirements for reproductive development, this work also has implications for understanding the mechanisms behind therapeutic interventions that target NAD(+) salvage biosynthesis for the purposes of inhibiting tumor growth.

Lysine acetylation has emerged as a global protein regulation system in all domains of life. Sirtuins, or Sir2-like enzymes, are a family of histone deacetylases characterized by their employing NAD+ as a co-substrate. Sirtuins can deacetylate several acetylated proteins, but a consensus substrate recognition sequence has not yet been established. Product inhibition of many eukaryotic sirtuins by nicotinamide and its analogues has been studied in vitro due to their potential role as anticancer agents. In this work, the kinetics of CobB, the main Escherichia coli deacetylase, have been characterized. To our knowledge, this is the first kinetic characterization of a sirtuin employing a fully acetylated and natively folded protein as a substrate. CobB deacetylated several acetyl-CoA synthetase acetylated lysines with a single kinetic rate. In addition, in vitro nicotinamide inhibition of CobB has been characterized, and the intracellular nicotinamide concentrations have been determined under different growth conditions. The results suggest that nicotinamide can act as a CobB regulator in vivo. A nicotinamidase deletion strain was thus phenotypically characterized, and it behaved similarly to the ΔcobB strain. The results of this work demonstrate the potential regulatory role of the nicotinamide metabolite in vivo.

NAD (NAD + ) is a cofactor related to many cellular processes. This cofactor is known to be unstable, especially at high temperatures, where it chemically decomposes to nicotinamide and ADP-ribose. Bacteria, yeast, and higher organisms possess the salvage pathway for reconstructing NAD + from these decomposition products; however, the importance of the salvage pathway for survival is not well elucidated, except for in pathogens lacking the NAD + de novo synthesis pathway. Herein, we report the importance of the NAD + salvage pathway in the thermophilic bacterium Thermus thermophilus HB8 at high temperatures. We identified the gene encoding nicotinamidase (TTHA0328), which catalyzes the first reaction of the NAD + salvage pathway. This recombinant enzyme has a high catalytic activity against nicotinamide ( K m of 17 μM, k cat of 50 s −1 , k cat / K m of 3.0 × 10 3 s −1 · mM −1 ). Deletion of this gene abolished nicotinamide deamination activity in crude extracts of T. thermophilus and disrupted the NAD + salvage pathway in T. thermophilus . Disruption of the salvage pathway led to the severe growth retardation at a higher temperature (80°C), owing to the drastic decrease in the intracellular concentrations of NAD + and NADH. IMPORTANCE NAD + and other nicotinamide cofactors are essential for cell metabolism. These molecules are unstable and decompose, even under the physiological conditions in most organisms. Thermophiles can survive at high temperatures where NAD + decomposition is, in general, more rapid. This study emphasizes that NAD + instability and its homeostasis can be one of the important factors for thermophile survival in extreme temperatures.