The Leishmania nicotinamidase is essential for NAD+ production and parasite proliferation

NAD+ is a central cofactor that plays important roles in cellular metabolism and energy production in all living cells. Genomics-based reconstruction of NAD+ metabolism revealed that Leishmania protozoan parasites are NAD+ auxotrophs. Consequently, these parasites require assimilating NAD+ precursors (nicotinamide, nicotinic acid, nicotinamide riboside) from their host environment to synthesize NAD+ by a salvage pathway. Nicotinamidase is a key enzyme of this salvage pathway that catalyses conversion of nicotinamide (NAm) to nicotinic acid (Na), and that is absent in higher eukaryotes. We present here the biochemical and functional characterizations of the Leishmania infantum nicotinamidase (LiPNC1). Generation of Lipnc1 null mutants leads to a decrease in NAD+ content, associated with a metabolic shutdown-like phenotype with an extensive lag phase of growth. Both phenotypes could be rescued by an add-back construct or by addition of exogenous Na. In addition, Lipnc1 null mutants were unable to establish a sustained infection in a murine experimental model. Altogether, these results illustrate that NAD+ homeostasis is a fundamental component of Leishmania biology and virulence, and that NAm constitutes its main NAD+ source in the mammalian host. The crystal structure of LiPNC1 we solved allows now the design of rational inhibitors against this new promising therapeutic target.     

Eukaryotic NAD+ synthetase Qns1 contains an essential,obligate intramolecular thiol glutamine amidotransferase domain related to nitrilase

NAD+ is an essential co-enzyme for redox reactions and is consumed in lysine deacetylation and poly(ADP-ribosyl)ation. NAD+ synthetase catalyzes the final step in NAD+ synthesis in the well characterized de novo, salvage, and import pathways. It has been long known that eukaryotic NAD+ synthetases use glutamine to amidate nicotinic acid adenine dinucleotide while many purified prokaryotic NAD+ synthetases are ammonia-dependent. Earlier, we discovered that glutamine-dependent NAD+ synthetases contain N-terminal domains that are members of the nitrilase superfamily and hypothesized that these domains function as glutamine amidotransferases for the associated synthetases. Here we show yeast glutamine-dependent NAD+ synthetase Qns1 requires both the nitrilase-related active-site residues and the NAD+ synthetase active-site residues for function in vivo. Despite failure to complement the lethal phenotype of qns1 disruption, the former mutants retain ammonia-dependent NAD+ synthetase activity in vitro, whereas the latter mutants retain basal glutaminase activity. Moreover, the two classes of mutants fail to trans-complement despite forming a stable heteromultimer in vivo. These data indicate that the nitrilase-related domain in Qns1 is the fourth independently evolved glutamine amidotransferase domain to have been identified in nature and that glutamine-dependence is an obligate phenomenon involving intramolecular transfer of ammonia over a predicted distance of 46 A from one active site to another within Qns1 monomers.     

Phosphate-responsive signaling pathway is a novel component of NAD+ metabolism in Saccharomyces cerevisiae

Nicotinamide adenine dinucleotide (NAD(+)) is an essential cofactor involved in various cellular biochemical reactions. To date the signaling pathways that regulate NAD(+) metabolism remain unclear due to the dynamic nature and complexity of the NAD(+) metabolic pathways and the difficulty of determining the levels of the interconvertible pyridine nucleotides. Nicotinamide riboside (NmR) is a key pyridine metabolite that is excreted and re-assimilated by yeast and plays important roles in the maintenance of NAD(+) pool. In this study we establish a NmR-specific reporter system and use it to identify yeast mutants with altered NmR/NAD(+) metabolism. We show that the phosphate-responsive signaling (PHO) pathway contributes to control NAD(+) metabolism. Yeast strains with activated PHO pathway show increases in both the release rate and internal concentration of NmR. We further identify Pho8, a PHO-regulated vacuolar phosphatase, as a potential NmR production factor. We also demonstrate that Fun26, a homolog of human ENT (equilibrative nucleoside transporter), localizes to the vacuolar membrane and establishes the size of the vacuolar and cytosolic NmR pools. In addition, the PHO pathway responds to depletion of cellular nicotinic acid mononucleotide (NaMN) and mediates nicotinamide mononucleotide (NMN) catabolism, thereby contributing to both NmR salvage and phosphate acquisition. Therefore, NaMN is a putative molecular link connecting the PHO signaling and NAD(+) metabolic pathways. Our findings may contribute to the understanding of the molecular basis and regulation of NAD(+) metabolism in higher eukaryotes.     

Nicotinamidase participates in the salvage pathway of NAD biosynthesis in Arabidopsis

Nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP), which is derived from NAD, have important roles as a redox carriers in metabolism. A combination of de novo and salvage pathways contribute to the biosynthesis of NAD in all organisms. The pathways and enzymes of the NAD salvage pathway in yeast and animals, which diverge at nicotinamide, have been extensively studied. Yeast cells convert nicotinamide to nicotinic acid, while mammals lack the enzyme nicotinamidase and instead convert nicotinamide to nicotinamide mononucleotide. Here we show that Arabidopsis thaliana gene At2g22570 encodes a nicotinamidase, which is expressed in all tissues, with the highest levels observed in roots and stems. The 244-residue protein, designated AtNIC1, converts nicotinamide to nicotinic acid and has a Km value of 118 +/- 17 microM and a Kcat value of 0.93 +/- 0.13 sec(-1). Plants homozygous for a null AtNIC1 allele, nic1-1, have lower levels of NAD and NADP under normal growth conditions, indicating that AtNIC1 participates in a yeast-type NAD salvage pathway. Mutant plants also exhibit hypersensitivity to treatments of abscisic acid and NaCl, which is correlated with their inability to increase the cellular levels of NAD(H) under these growth conditions, as occurs in wild-type plants. We also show that the growth of the roots of wild-type but not nic1-1 mutant plants is inhibited and distorted by nicotinamide.     

Compartmentation of NAD+-dependent signalling

NAD(+) plays central roles in energy metabolism as redox carrier. Recent research has identified important signalling functions of NAD(+) that involve its consumption. Although NAD(+) is synthesized mainly in the cytosol, nucleus and mitochondria, it has been detected also in vesicular and extracellular compartments. Three protein families that consume NAD(+) in signalling reactions have been characterized on a molecular level: ADP-ribosyltransferases (ARTs), Sirtuins (SIRTs), and NAD(+) glycohydrolases (NADases). Members of these families serve important regulatory functions in various cellular compartments, e.g., by linking the cellular energy state to gene expression in the nucleus, by regulating nitrogen metabolism in mitochondria, and by sensing tissue damage in the extracellular compartment. Distinct NAD(+) pools may be crucial for these processes. Here, we review the current knowledge about the compartmentation and biochemistry of NAD(+)-converting enzymes that control NAD(+) signalling.     

Comparative genomics of NAD(P) biosynthesis and novel antibiotic drug targets

NAD(P) is an indispensable cofactor for all organisms and its biosynthetic pathways are proposed as promising novel antibiotics targets against pathogens such as Mycobacterium tuberculosis. Six NAD(P) biosynthetic pathways were reconstructed by comparative genomics: de novo pathway (Asp), de novo pathway (Try), NmR pathway I (RNK‐dependent), NmR pathway II (RNK‐independent), Niacin salvage, and Niacin recycling. Three enzymes pivotal to the key reactions of NAD(P) biosynthesis are shared by almost all organisms, that is, NMN/NaMN adenylyltransferase (NMN/NaMNAT), NAD synthetase (NADS), and NAD kinase (NADK). They might serve as ideal broad spectrum antibiotic targets. Studies in M. tuberculosis have in part tested such hypothesis. Three regulatory factors NadR, NiaR, and NrtR, which regulate NAD biosynthesis, have been identified. M. tuberculosis NAD(P) metabolism and regulation thereof, potential drug targets and drug development are summarized in this paper. J. Cell. Physiol. 226: 331–340, 2011. © 2010 Wiley‐Liss, Inc.     

Pathway analysis of NAD+ metabolism

NAD(+) is well known as a crucial cofactor in the redox balance of metabolism. Moreover, NAD(+) is degraded in ADP-ribosyl transfer reactions, which are important components of multitudinous signalling reactions. These include reactions linked to DNA repair and aging. In the present study, using the concept of EFMs (elementary flux modes), we established all of the potential routes in a network describing NAD(+) biosynthesis and degradation. All known biosynthetic pathways, which include de novo synthesis starting from tryptophan as well as the classical Preiss-Handler pathway and NAD(+) synthesis from other vitamin precursors, were detected as EFMs. Moreover, several EFMs were found that degrade NAD(+), represent futile cycles or have other functionalities. The systematic analysis and comparison of the networks specific for yeast and humans document significant differences between species with regard to the use of precursors, biosynthetic routes and NAD(+)-dependent signalling.     

Metabolism of NAD+ in the nuclei of human placenta

It has long been known that the major function of NAD+ is as an electron carrier in various biological oxidation-reduction systems. From many papers it is evident that NAD+ is involved as substrate in ADP-ribosylation reactions. We have focused our attention on two chromatin enzymes: NMN-adenylyltransferase that catalyzes reversible synthesis of NAD+ utilizing ATP and NMN, and poly(ADP-ribose)polymerase that covalently modifies nucleosomal proteins through poly ADP-ribosylation reactions. Here we provided evidence of these activities in a system of isolated nuclei from human placenta. The data presented in this report show that purified nuclei might be useful to study the nuclear location of these enzymes and their reciprocal interactions.     

微生物NAD合成酶及其抑制剂的研究进展

NAD(P)生物代谢在能量代谢,维持氧化还原稳态以及调节细胞寿命等许多细胞进程中有重要作用。因此,NAD生物合成途径的关键酶的抑制剂就成为备受关注的候选新药,如NAD合成酶抑制剂。本文对微生物中的NAD合成酶的催化活性特征,晶体结构,调控因子以及基于晶体结构的抑制剂设计方面进行了综述,以期为基于NAD的治疗领域打开新的思路。     

The reported human NADsyn2 is ammonia-dependent NAD synthetase from a pseudomonad

Nicotinamide-adenine dinucleotide (NAD+) synthetases catalyze the last step in NAD+ metabolism in the de novo, import, and salvage pathways that originate from tryptophan (or aspartic acid), nicotinic acid, and nicotinamide, respectively, and converge on nicotinic acid mononucleotide. NAD+ synthetase converts nicotinic acid adenine dinucleotide to NAD+ via an adenylylated intermediate. All of the known eukaryotic NAD+ synthetases are glutamine-dependent, hydrolyzing glutamine to glutamic acid to provide the attacking ammonia. In the prokaryotic world, some NAD+ synthetases are glutamine-dependent, whereas others can only use ammonia. Earlier, we noted a perfect correlation between presence of a domain related to nitrilase and glutamine dependence and then proved in the accompanying paper (Bieganowski, P., Pace, H. C., and Brenner, C. (2003) J. Biol. Chem. 278, 33049-33055) that the nitrilase-related domain is an essential, obligate intramolecular, thiol-dependent glutamine amidotransferase in the yeast NAD+ synthetase, Qns1. Independently, human NAD+ synthetase was cloned and shown to depend on Cys-175 for glutamine-dependent but not ammonia-dependent NAD+ synthetase activity. Additionally, it was claimed that a 275 amino acid open reading frame putatively amplified from human glioma cell line LN229 encodes a human ammonia-dependent NAD+ synthetase and this was speculated largely to mediate NAD+ synthesis in human muscle tissues. Here we establish that the so-called NADsyn2 is simply ammonia-dependent NAD+ synthetase from Pseudomonas, which is encoded on an operon with nicotinic acid phosphoribosyltransferase and, in some Pseudomonads, with nicotinamidase.     

Microbial NAD Metabolism: Lessons from Comparative Genomics

Summary: NAD is a coenzyme for redox reactions and a substrate of NAD-consuming enzymes, including ADP-ribose transferases, Sir2-related protein lysine deacetylases, and bacterial DNA ligases. Microorganisms that synthesize NAD from as few as one to as many as five of the six identified biosynthetic precursors have been identified. De novo NAD synthesis from aspartate or tryptophan is neither universal nor strictly aerobic. Salvage NAD synthesis from nicotinamide, nicotinic acid, nicotinamide riboside, and nicotinic acid riboside occurs via modules of different genes. Nicotinamide salvage genes nadV and pncA, found in distinct bacteria, appear to have spread throughout the tree of life via horizontal gene transfer. Biochemical, genetic, and genomic analyses have advanced to the point at which the precursors and pathways utilized by a microorganism can be predicted. Challenges remain in dissecting regulation of pathways.     

NAD metabolism in aging and cancer

NAD⁺ and its derivatives NADH, NADP⁺, and NADPH are essential cofactors in redox reactions and electron transport pathways. NAD serves also as substrate for an extensive series of regulatory enzymes including cyclic ADP-ribose hydrolases, mono(ADP-ribosyl)transferases, poly(ADP-ribose) polymerases, and sirtuin deacetylases which are O-acetyl-ADP-ribosyltransferases. As a result of the numerous and diverse enzymes that utilize NAD as well as depend on its synthesis and concentration, significant interest has developed in its role in a variety of physiologic and pathologic processes, and therapeutic initiatives have focused both on augmenting its levels as well as inhibiting some of its pathways. In this article, we examine the biosynthesis of NAD, metabolic processes in which it is involved, and its role in aging, cancer, and other age-associated comorbidities including neurodegenerative, cardiovascular, and metabolic disorders. Therapeutic interventions to augment and/or inhibit these processes are also discussed.Impact statementNAD is a central metabolite connecting energy balance and organismal growth with genomic integrity and function. It is involved in the development of malignancy and has a regulatory role in the aging process. These processes are mediated by a diverse series of enzymes whose common focus is either NAD’s biosynthesis or its utilization as a redox cofactor or enzyme substrate. These enzymes include dehydrogenases, cyclic ADP-ribose hydrolases, mono(ADP-ribosyl)transferases, poly(ADP-ribose) polymerases, and sirtuin deacetylases. This article describes the manifold pathways that comprise NAD metabolism and promotes an increased awareness of how perturbations in these systems may be important in disease prevention and/or progression.     

NAD(P) synthesis and pyridine nucleotide cycling in plants and their potential importance in stress conditions

Pyridine nucleotides are key redox carriers in the soluble phase of all living cells, and both NAD and NADP play crucial roles in pro-oxidant and antioxidant metabolism. Recent data also suggest a number of non-redox mechanisms by which these nucleotides could influence cell function. In cases where these mechanisms involve NAD(P) consumption, resynthesis must occur to maintain nucleotide pools. Important information on the pathways involved in NAD synthesis in plants is beginning to appear, but many outstanding questions remain. This work provides an overview of the current state of knowledge on NAD synthesis pathways in plants and other organisms, analyses plant sequences for the first two enzymes of the de novo synthesis of NAD, proposes a preliminary model for the intracellular distribution of NAD synthesis, presents plant homologues of recently identified yeast mitochondrial NAD transporters, and discusses factors likely to be important in the regulation of NAD synthesis and contents in plants, with particular reference to stress conditions.     

Small molecule regulation of Sir2 protein deacetylases

The Sir2 family of histone/protein deacetylases (sirtuins) is comprised of homologues found across all kingdoms of life. These enzymes catalyse a unique reaction in which NAD+ and acetylated substrate are converted into deacetylated product, nicotinamide, and a novel metabolite O-acetyl ADP-ribose. Although the catalytic mechanism is well conserved across Sir2 family members, sirtuins display differential specificity toward acetylated substrates, which translates into an expanding range of physiological functions. These roles include control of gene expression, cell cycle regulation, apoptosis, metabolism and ageing. The dependence of sirtuin activity on NAD+ has spearheaded investigations into how these enzymes respond to metabolic signals, such as caloric restriction. In addition, NAD+ metabolites and NAD+ salvage pathway enzymes regulate sirtuin activity, supporting a link between deacetylation of target proteins and metabolic pathways. Apart from physiological regulators, forward chemical genetics and high-throughput activity screening has been used to identify sirtuin inhibitors and activators. This review focuses on small molecule regulators that control the activity and functions of this unusual family of protein deacetylases.     

Validation of NAD synthase inhibitors for inhibiting the cell viability of Leishmania donovani: In silico and in vitro approach

Abstract

NAD (nicotinamide adenine dinucleotide) synthase catalyses the biochemical synthesis of NAD, from nicotinic acid adenine dinucleotide (NAAD). NAD may be synthesized through the de novo pathways and/or the salvage pathways in cells. However, in Leishmania parasite, the synthesis of NAD solely depends on the salvage pathways. NAD synthetase is widely explored as a drug target in various microorganisms. In Bacillus anthracis, a group of sulphonamides 5599, 5617 and 5824 and complex amide 5833 were reported to have activity at micromolar range against NAD synthetase. Hence, in the present study, the same group of sulphonamides and complex amide were validated through in silico and in vitro studies for its efficiency towards Leishmania donovani NAD synthase. In silico study revealed the ligands 5824 and 5833 to have better docking score. Molecular dynamics simulation for a duration of 50 ns of all the ligand–protein complexes suggested that the complexes with the ligands 5824 and 5833 were stable and interacting. In vitro and ex vivo studies have shown that 5824 and 5833 inhibit the cell viability of the organism at a lower concentration than 5599 and 5617. Hence, with further in vivo validation, 5824 (or its synthetic analogues) and 5833 could be the choice that may work synergistically with other potential drugs in treating drug-resistant cases of leishmaniasis.

Communicated by Ramaswamy H. Sarma     

NAD+ metabolism in health and disease

Nicotinamide adenine dinucleotide (NAD(+)) is both a coenzyme for hydride-transfer enzymes and a substrate for NAD(+)-consuming enzymes, which include ADP-ribose transferases, poly(ADP-ribose) polymerases, cADP-ribose synthases and sirtuins. Recent results establish protective roles for NAD(+) that might be applicable therapeutically to prevent neurodegenerative conditions and to fight Candida glabrata infection. In addition, the contribution that NAD(+) metabolism makes to lifespan extension in model systems indicates that therapies to boost NAD(+) might promote some of the beneficial effects of calorie restriction. Nicotinamide riboside, the recently discovered nucleoside precursor of NAD(+) in eukaryotic systems, might have advantages as a therapy to elevate NAD(+) without inhibiting sirtuins, which is associated with high-dose nicotinamide, or incurring the unpleasant side-effects of high-dose nicotinic acid.