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.     

Increasing NAD Synthesis in Muscle via Nicotinamide Phosphoribosyltransferase Is Not Sufficient to Promote Oxidative Metabolism

The NAD biosynthetic precursors nicotinamide mononucleotide and nicotinamide riboside are reported to confer resistance to metabolic defects induced by high fat feeding in part by promoting oxidative metabolism in skeletal muscle. Similar effects are obtained by germ line deletion of major NAD-consuming enzymes, suggesting that the bioavailability of NAD is limiting for maximal oxidative capacity. However, because of their systemic nature, the degree to which these interventions exert cell- or tissue-autonomous effects is unclear. Here, we report a tissue-specific approach to increase NAD biosynthesis only in muscle by overexpressing nicotinamide phosphoribosyltransferase, the rate-limiting enzyme in the salvage pathway that converts nicotinamide to NAD (mNAMPT mice). These mice display a ∼50% increase in skeletal muscle NAD levels, comparable with the effects of dietary NAD precursors, exercise regimens, or loss of poly(ADP-ribose) polymerases yet surprisingly do not exhibit changes in muscle mitochondrial biogenesis or mitochondrial function and are equally susceptible to the metabolic consequences of high fat feeding. We further report that chronic elevation of muscle NAD in vivo does not perturb the NAD/NADH redox ratio. These studies reveal for the first time the metabolic effects of tissue-specific increases in NAD synthesis and suggest that critical sites of action for supplemental NAD precursors reside outside of the heart and skeletal muscle.     

Cloning and Characterization of a Novel Drosophila Stress Induced DNase

Drosophila melanogaster flies mount an impressive immune response to a variety of pathogens with an efficient system comprised of both humoral and cellular responses. The fat body is the main producer of the anti-microbial peptides (AMPs) with anti-pathogen activity. During bacterial infection, an array of secreted peptidases, proteases and other enzymes are involved in the dissolution of debris generated by pathogen clearance. Although pathogen destruction should result in the release a large amount of nucleic acids, the mechanisms for its removal are still not known. In this report, we present the characterization of a nuclease gene that is induced not only by bacterial infection but also by oxidative stress. Expression of the identified protein has revealed that it encodes a potent nuclease that has been named Stress Induced DNase (SID). SID belongs to a family of evolutionarily conserved cation-dependent nucleases that degrade both single and double-stranded nucleic acids. Down-regulation of sid expression via RNA interference leads to significant reduction of fly viability after bacterial infection and oxidative stress. Our results indicate that SID protects flies from the toxic effects of excess DNA/RNA released by pathogen destruction and from oxidative damage.     

Sequence divergence and diversity suggests ongoing functional diversification of vertebrate NAD metabolism

NAD is not only an important cofactor in redox reactions but has also received attention in recent years because of its physiological importance in metabolic regulation, DNA repair and signaling. In contrast to the redox reactions, these regulatory processes involve degradation of NAD and therefore necessitate a constant replenishment of its cellular pool. NAD biosynthetic enzymes are common to almost all species in all clades, but the number of NAD degrading enzymes varies substantially across taxa. In particular, vertebrates, including humans, have a manifold of NAD degrading enzymes which require a high turnover of NAD. As there is currently a lack of a systematic study of how natural selection has shaped enzymes involved in NAD metabolism we conducted a comprehensive evolutionary analysis based on intraspecific variation and interspecific divergence. We compare NAD biosynthetic and degrading enzymes in four eukaryotic model species and subsequently focus on human NAD metabolic enzymes and their orthologs in other vertebrates. We find that the majority of enzymes involved in NAD metabolism are subject to varying levels of purifying selection. While NAD biosynthetic enzymes appear to experience a rather high level of evolutionary constraint, there is evidence for positive selection among enzymes mediating NAD-dependent signaling. This is particularly evident for members of the PARP family, a diverse protein family involved in DNA damage repair and programmed cell death. Based on haplotype information and substitution rate analysis we pinpoint sites that are potential targets of positive selection. We also link our findings to a three dimensional structure, which suggests that positive selection occurs in domains responsible for DNA binding and polymerization rather than the NAD catalytic domain. Taken together, our results indicate that vertebrate NAD metabolism is still undergoing functional diversification.     

Metabolic Profiling of Alternative NAD Biosynthetic Routes in Mouse Tissues

NAD plays essential redox and non-redox roles in cell biology. In mammals, its de novo and recycling biosynthetic pathways encompass two independent branches, the “amidated” and “deamidated” routes. Here we focused on the indispensable enzymes gating these two routes, i.e. nicotinamide mononucleotide adenylyltransferase (NMNAT), which in mammals comprises three distinct isozymes, and NAD synthetase (NADS). First, we measured the in vitro activity of the enzymes, and the levels of all their substrates and products in a number of tissues from the C57BL/6 mouse. Second, from these data, we derived in vivo estimates of enzymes''rates and quantitative contributions to NAD homeostasis. The NMNAT activity, mainly represented by nuclear NMNAT1, appears to be high and nonrate-limiting in all examined tissues, except in blood. The NADS activity, however, appears rate-limiting in lung and skeletal muscle, where its undetectable levels parallel a relative accumulation of the enzyme''s substrate NaAD (nicotinic acid adenine dinucleotide). In all tissues, the amidated NAD route was predominant, displaying highest rates in liver and kidney, and lowest in blood. In contrast, the minor deamidated route showed higher relative proportions in blood and small intestine, and higher absolute values in liver and small intestine. Such results provide the first comprehensive picture of the balance of the two alternative NAD biosynthetic routes in different mammalian tissues under physiological conditions. This fills a gap in the current knowledge of NAD biosynthesis, and provides a crucial information for the study of NAD metabolism and its role in disease.     

Effects of lethal exposure to hyperoxia and to hydrogen peroxide on NAD(H) and ATP pools in Chinese hamster ovary cells

Cell death by oxidative stress has been proposed to be based on suicidal NAD depletion, typically followed by ATP depletion, caused by the NAD-consuming enzyme poly(ADP)ribose polymerase, which becomes activated by the presence of excessive DNA-strand breaks. In this study NAD+, NADH and ATP levels as well as DNA-strand breaks (assayed by alkaline elution) were determined in Chinese hamster ovary (CHO) cells treated with either H2O2 or hyperoxia to a level of more than 80% clonogenic cell killing. With H2O2 extensive DNA damage and NAD depletion were observed, while at a higher H2O2 dosage ATP also became depleted. In agreement with results of others, the poly(ADP)ribose polymerase inhibitor 3-aminobenzamide completely prevented NAD depletion. However, both H2O2-induced ATP depletion and cell killing were unaffected by the inhibitor, suggesting that ATP depletion may be a more critical factor than NAD depletion in H2O2-induced killing of CHO cells. With hyperoxia, only moderate DNA damage (2 X background) and no NAD depletion were observed, whereas ATP became largely (70%) depleted. We conclude that (1) there is no direct relation between ATP and NAD depletion in CHO cells subjected to toxic doses of H2O2 or hyperoxia; (2) H2O2-induced NAD depletion is not by itself sufficient to kill CHO cells; (3) killing of CHO cells by hyperoxia is not due to NAD depletion, but may be due to depletion of ATP.     

Integrated Proteomics and Metabolomics of Arabidopsis Acclimation to Gene-Dosage Dependent Perturbation of Isopropylmalate Dehydrogenases

Maintaining metabolic homeostasis is critical for plant growth and development. Here we report proteome and metabolome changes when the metabolic homeostasis is perturbed due to gene-dosage dependent mutation of Arabidopsis isopropylmalate dehydrogenases (IPMDHs). By integrating complementary quantitative proteomics and metabolomics approaches, we discovered that gradual ablation of the oxidative decarboxylation step in leucine biosynthesis caused imbalance of amino acid homeostasis, redox changes and oxidative stress, increased protein synthesis, as well as a decline in photosynthesis, which led to rearrangement of central metabolism and growth retardation. Disruption of IPMDHs involved in aliphatic glucosinolate biosynthesis led to synchronized increase of both upstream and downstream biosynthetic enzymes, and concomitant repression of the degradation pathway, indicating metabolic regulatory mechanisms in controlling glucosinolate biosynthesis.     

Identification of novel components of NAD-utilizing metabolic pathways and prediction of their biochemical functions

Nicotinamide adenine dinucleotide (NAD) is a ubiquitous cofactor participating in numerous redox reactions. It is also a substrate for regulatory modifications of proteins and nucleic acids via the addition of ADP-ribose moieties or removal of acyl groups by transfer to ADP-ribose. In this study, we use in-depth sequence, structure and genomic context analysis to uncover new enzymes and substrate-binding proteins in NAD-utilizing metabolic and macromolecular modification systems. We predict that Escherichia coli YbiA and related families of domains from diverse bacteria, eukaryotes, large DNA viruses and single strand RNA viruses are previously unrecognized components of NAD-utilizing pathways that probably operate on ADP-ribose derivatives. Using contextual analysis we show that some of these proteins potentially act in RNA repair, where NAD is used to remove 2'-3' cyclic phosphodiester linkages. Likewise, we predict that another family of YbiA-related enzymes is likely to comprise a novel NAD-dependent ADP-ribosylation system for proteins, in conjunction with a previously unrecognized ADP-ribosyltransferase. A similar ADP-ribosyltransferase is also coupled with MACRO or ADP-ribosylglycohydrolase domain proteins in other related systems, suggesting that all these novel systems are likely to comprise pairs of ADP-ribosylation and ribosylglycohydrolase enzymes analogous to the DraG-DraT system, and a novel group of bacterial polymorphic toxins. We present evidence that some of these coupled ADP-ribosyltransferases/ribosylglycohydrolases are likely to regulate certain restriction modification enzymes in bacteria. The ADP-ribosyltransferases found in these, the bacterial polymorphic toxin and host-directed toxin systems of bacteria such as Waddlia also throw light on the evolution of this fold and the origin of eukaryotic polyADP-ribosyltransferases and NEURL4-like ARTs, which might be involved in centrosomal assembly. We also infer a novel biosynthetic pathway that might be involved in the synthesis of a nicotinate-derived compound in conjunction with an asparagine synthetase and AMPylating peptide ligase. We use the data derived from this analysis to understand the origin and early evolutionary trajectories of key NAD-utilizing enzymes and present targets for future biochemical investigations.     

DEAD box 1 (DDX1) protein binds to and protects cytoplasmic stress response mRNAs in cells exposed to oxidative stress

The integrated stress response is a network of highly orchestrated pathways activated when cells are exposed to environmental stressors. While global repression of translation is a well-recognized hallmark of the integrated stress response, less is known about the regulation of mRNA stability during stress. DEAD box proteins are a family of RNA unwinding/remodeling enzymes involved in every aspect of RNA metabolism. We previously showed that DEAD box 1 (DDX1) protein accumulates at DNA double-strand breaks during genotoxic stress and promotes DNA double-strand break repair via homologous recombination. Here, we examine the role of DDX1 in response to environmental stress. We show that DDX1 is recruited to stress granules (SGs) in cells exposed to a variety of environmental stressors, including arsenite, hydrogen peroxide, and thapsigargin. We also show that DDX1 depletion delays resolution of arsenite-induced SGs. Using RNA immunoprecipitation sequencing, we identify RNA targets bound to endogenous DDX1, including RNAs transcribed from genes previously implicated in stress responses. We show the amount of target RNAs bound to DDX1 increases when cells are exposed to stress, and the overall levels of these RNAs are increased during stress in a DDX1-dependent manner. Even though DDX1’s RNA-binding property is critical for maintenance of its target mRNA levels, we found RNA binding is not required for localization of DDX1 to SGs. Furthermore, DDX1 knockdown does not appear to affect RNA localization to SGs. Taken together, our results reveal a novel role for DDX1 in maintaining cytoplasmic mRNA levels in cells exposed to oxidative stress.     

Control of Redox Balance by the Stringent Response Regulatory Protein Promotes Antioxidant Defenses of Salmonella

We report herein a critical role for the stringent response regulatory DnaK suppressor protein (DksA) in the coordination of antioxidant defenses. DksA helps fine-tune the expression of glutathione biosynthetic genes and discrete steps in the pentose phosphate pathway and tricarboxylic acid cycle that are associated with the generation of reducing power. Control of NAD(P)H/NAD(P)⁺ redox balance by DksA fuels downstream antioxidant enzymatic systems in nutritionally starving Salmonella. Conditional expression of the glucose-6-phosphate dehydrogenase-encoding gene zwf, shown here to be under DksA control, increases both the NADPH pool and antioxidant defenses of dksA mutant Salmonella. The DksA-mediated coordination of redox balance boosts the antioxidant defenses of stationary phase bacteria. Not only does DksA increase resistance of Salmonella against hydrogen peroxide (H₂O₂), but it also promotes fitness of this intracellular pathogen when exposed to oxyradicals produced by the NADPH phagocyte oxidase in an acute model of infection. Given the role of DksA in the adjustment of gene expression in most bacteria undergoing nutritional deprivation, our findings raise the possibility that the control of central metabolic pathways by this regulatory protein maintains redox homeostasis essential for antioxidant defenses in phylogenetically diverse bacterial species.     

The response of Paracoccidioides spp. to nitrosative stress

Paracoccidioidomycosis (PCM) is an endemic disease in Latin America caused by species belonging to the genus Paracoccidioides. During infection, immune cells present a variety of defense mechanisms against pathogens. One of these defensive strategies is the production and release of nitric oxide (NO) and S-nitroso thiols (e.g., S-nitrosoglutathione, GSNO), which produce reactive nitrogen species (RNS). This results in damage to DNA and membranes, inhibition of respiration and inactivation of cellular enzymes. In response to nitrosative stress, human pathogenic fungi possess defense mechanisms to prevent the adverse effects of NO, which helps them survive during initial contact with the host immune system. To understand how Paracoccidioides spp. respond to nitrosative stress, we conducted this study to identify genes and proteins that might contribute to this response. The results of proteomic analysis demonstrated that nitrosative stress induced a reduction in the expression of proteins related to the mitochondrial electron transport chain. This hypothesis was supported by the reduced mitochondrial activity observed in the presence of GSNO. Additionally, lipids and branched chain amino acid metabolism enzymes were altered. The role played by enzymes acting in oxidative stress in the RNS response was remarkable. This interface among enzymes acting in both stress responses was confirmed by using a RNA approach to silence the ccp gene in Paracoccidioides. It was observed that mutants with low expression of the ccp gene were more sensitive to nitrosative stress.     

Nicotinamide phosphoribosyl transferase/pre-B cell colony-enhancing factor/visfatin is required for lymphocyte development and cellular resistance to genotoxic stress

Nicotinamide phosphoribosyl transferase (Nampt)/pre-B cell colony-enhancing factor (PBEF)/visfatin is a protein displaying multiple functional properties. Originally described as a cytokine-like protein able to regulate B cell development, apoptosis, and glucose metabolism, this protein also plays an important role in NAD biosynthesis. To gain insight into its physiological role, we have generated a mouse strain expressing a conditional Nampt allele. Lack of Nampt expression strongly affects development of both T and B lymphocytes. Analysis of hemizygous cells and in vitro cell lines expressing distinct levels of Nampt illustrates the critical role of this protein in regulating intracellular NAD levels. Consequently, a clear relationship was found between intracellular Nampt levels and cell death in response to the genotoxic agent MNNG (N-methyl-N'-nitro-N-nitrosoguanidine), confirming that this enzyme represents a key regulator of cell sensitivity to NAD-consuming stress secondary to poly(ADP-ribose) polymerases overactivation. By using mutant forms of this protein and a well-characterized pharmacological inhibitor (FK866), we unequivocally demonstrate that the ability of the Nampt to regulate cell viability during genotoxic stress requires its enzymatic activity. Collectively, these data demonstrate that Nampt participates in cellular resistance to genotoxic/oxidative stress, and it may confer to cells of the immune system the ability to survive during stressful situations such as inflammation.     

Xenobiotic stress induces hepatomegaly and liver tumors via the nuclear receptor constitutive androstane receptor

The constitutive androstane receptor (CAR, NR1I3) is a central regulator of xenobiotic metabolism. CAR activation induces hepatic expression of detoxification enzymes and transporters and increases liver size. Here we show that CAR-mediated hepatomegaly is a transient, adaptive response to acute xenobiotic stress. In contrast, chronic CAR activation results in hepatocarcinogenesis. In both acute and chronic xenobiotic responses, hepatocyte DNA replication is increased and apoptosis is decreased. These effects are absent in CAR null mice, which are completely resistant to tumorigenic effects of chronic xenobiotic stress. In the acute response, direct up-regulation of Mdm2 expression by CAR contributes to both increased DNA replication and inhibition of p53-mediated apoptosis. These results demonstrate an essential role for CAR in regulating both liver homeostasis and tumorigenesis in response to xenobiotic stresses, and they also identify a specific molecular mechanism linking chronic environmental stress and tumor formation.     

Identification of a novel human nicotinamide mononucleotide adenylyltransferase

The enzyme nicotinamide mononucleotide adenylyltransferase is an ubiquitous enzyme catalyzing an essential step in NAD (NADP) biosynthetic pathway. In human cells, the nuclear enzyme, which we will now call NMNAT-1, has been the only known enzyme of this type for over 10 years. Here we describe the cloning and expression of a human cDNA encoding a novel 34.4kDa protein, that shares significant homology with the 31.9kDa NMNAT-1. We propose to call this enzyme NMNAT-2. Purified recombinant NMNAT-2 is endowed with NMN and nicotinic acid mononucleotide adenylyltransferase activities, but differs from NMNAT-1 with regard to chromosomal and cellular localization, tissue-specificity of expression, and molecular properties, supporting the idea that the two enzymes might play distinct physiological roles in NAD homeostasis.     

Structural variations between small alarmone hydrolase dimers support different modes of regulation of the stringent response

The bacterial stringent response involves wide-ranging metabolic reprogramming aimed at increasing long-term survivability during stress conditions. One of the hallmarks of the stringent response is the production of a set of modified nucleotides, known as alarmones, which affect a multitude of cellular pathways in diverse ways. Production and degradation of these molecules depend on the activity of enzymes from the RelA/SpoT homologous family, which come in both bifunctional (containing domains to both synthesize and hydrolyze alarmones) and monofunctional (consisting of only synthetase or hydrolase domain) variants, of which the structure, activity, and regulation of the bifunctional RelA/SpoT homologs have been studied most intensely. Despite playing an important role in guanosine nucleotide homeostasis in particular, mechanisms of regulation of the small alarmone hydrolases (SAHs) are still rather unclear. Here, we present crystal structures of SAH enzymes from Corynebacterium glutamicum (RelH_Cg) and Leptospira levettii (RelH_Ll) and show that while being highly similar, structural differences in substrate access and dimer conformations might be important for regulating their activity. We propose that a varied dimer form is a general property of the SAH family, based on current structural information as well as prediction models for this class of enzymes. Finally, subtle structural variations between monofunctional and bifunctional enzymes point to how these different classes of enzymes are regulated.     

Cytosolic monodehydroascorbate reductase gene affects stress adaptation and grain yield under paddy field conditions in <Emphasis Type="Italic">Oryza sativa L. japonica</Emphasis>

Monodehydroascorbate reductase (MDHAR), which is responsible for growth, development and stress response in plants, is a key enzyme in the maintenance of the ascorbate (AsA) pool through the AsA–glutathione (AsA–GSH) cycle and is induced by abiotic stresses. It has highly conserved regions containing FAD- and NAD(P)H-binding domains. In particular, NAD(P)H is a significant electron donor in the AsA–GSH pathway. In this context, we introduced RNA interference (RNAi) to determine the functional role of Oryza sativa L. japonica MDHAR isoform 3 (OsMDHAR3) and developed transgenic (mdhar3) rice plants in which the NAD(P)H domain was silenced. The mdhar3 rice plants were more sensitive to salt stress than the wild-type (WT) plants. In addition, the mdhar3 rice plants showed decreased ability for environmental adaptation because of an imbalance in the redox homeostasis and reduced AsA pool. These plants showed increased hydroperoxide levels and ion leakage, and decreased chlorophyll content and ascorbate/dehydroascorbate ratio under the paddy field conditions; they also exhibited a reduction in the total biomass and grain yield. Furthermore, the activity of a purified E196A mutant of the OsMDHAR protein decreased to approximately 70% of the activity of the WT protein. These results suggest that OsMDHAR3 plays a critical role in the intrinsic resistance, as well as in the sensitivity of seed maturation and productivity, of rice plants to environmental stresses, thereby indicating the functional importance of NADH in MDHAR activity, in vivo and in vitro.