The stereospecificity of nicotinamide-adenine dinucleotide-dependent oxidoreductases from plants.
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ABSTRACT:
SUBMITTER: Davies DD
PROVIDER: S-EPMC1178593 | biostudies-other | 1972 Apr
REPOSITORIES: biostudies-other
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Project description:The ultraviolet resonance Raman spectra of the adenine-containing enzymatic redox cofactors nicotinamide adenine dinucleotide and flavin adenine dinucleotide in aqueous solution of physiological concentration are compared with the aim of distinguishing between them and their building block adenine in potential co-occurrence in biological materials. At an excitation wavelength of 266 nm, the spectra are dominated by the strong resonant contribution from adenine; nevertheless, bands assigned to vibrational modes of the nicotinamide and the flavin unit are found to appear at similar signal strength. Comparison of spectra measured at pH 7 with data obtained pH 10 and pH 3 shows characteristic changes when pH is increased or lowered, mainly due to deprotonation of the flavin and nicotinamide moieties, and protonation of the adenine, respectively.
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Project description:Nicotinamide adenine dinucleotide (NAD+) is a key redox compound in all living cells responsible for energy transduction, genomic integrity, life-span extension, and neuromodulation. Here, we report a new function of NAD+ as a molecular photocatalyst in addition to the biological roles. Our spectroscopic and electrochemical analyses reveal light absorption and electronic properties of two π-conjugated systems of NAD+. Furthermore, NAD+ exhibits a robust photostability under UV-Vis-NIR irradiation. We demonstrate photocatalytic redox reactions driven by NAD+, such as O2 reduction, H2O oxidation, and the formation of metallic nanoparticles. Beyond the traditional role of NAD+ as a cofactor in redox biocatalysis, NAD+ executes direct photoactivation of oxidoreductases through the reduction of enzyme prosthetic groups. Consequently, the synergetic integration of biocatalysis and photocatalysis using NAD+ enables solar-to-chemical conversion with the highest-ever-recorded turnover frequency and total turnover number of 1263.4 hour-1 and 1692.3, respectively, for light-driven biocatalytic trans-hydrogenation.
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Project description:Nicotinamide adenine dinucleotide (NAD) is an important coenzyme that regulates various metabolic pathways, including glycolysis, β-oxidation, and oxidative phosphorylation. Additionally, NAD serves as a substrate for poly(ADP-ribose) polymerase (PARP), sirtuin, and NAD glycohydrolase, and it regulates DNA repair, gene expression, energy metabolism, and stress responses. Many studies have demonstrated that NAD metabolism is deeply involved in aging and aging-related diseases. Previously, we demonstrated that nicotinamide guanine dinucleotide (NGD) and nicotinamide hypoxanthine dinucleotide (NHD), which are analogs of NAD, are significantly increased in Nmnat3-overexpressing mice. However, there is insufficient knowledge about NGD and NHD in vivo. In the present study, we aimed to investigate the metabolism and biochemical properties of these NAD analogs. We demonstrated that endogenous NGD and NHD were found in various murine tissues, and their synthesis and degradation partially rely on Nmnat3 and CD38. We have also shown that NGD and NHD serve as coenzymes for alcohol dehydrogenase (ADH) in vitro, although their affinity is much lower than that of NAD. On the other hand, NGD and NHD cannot be used as substrates for SIRT1, SIRT3, and PARP1. These results reveal the basic metabolism of NGD and NHD and also highlight their biological function as coenzymes.
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Project description:The regenerative capacity of the liver is essential for recovery from surgical resection or injuries induced by trauma or toxins. During liver regeneration, the concentration of nicotinamide adenine dinucleotide (NAD) falls, at least in part due to metabolic competition for precursors. To test whether NAD availability restricts the rate of liver regeneration, we supplied nicotinamide riboside (NR), an NAD precursor, in the drinking water of mice subjected to partial hepatectomy. NR increased DNA synthesis, mitotic index, and mass restoration in the regenerating livers. Intriguingly, NR also ameliorated the steatosis that normally accompanies liver regeneration. To distinguish the role of hepatocyte NAD levels from any systemic effects of NR, we generated mice overexpressing nicotinamide phosphoribosyltransferase, a rate-limiting enzyme for NAD synthesis, specifically in the liver. Nicotinamide phosphoribosyltransferase overexpressing mice were mildly hyperglycemic at baseline and, similar to mice treated with NR, exhibited enhanced liver regeneration and reduced steatosis following partial hepatectomy. Conversely, mice lacking nicotinamide phosphoribosyltransferase in hepatocytes exhibited impaired regenerative capacity that was completely rescued by administering NR.ConclusionNAD availability is limiting during liver regeneration, and supplementation with precursors such as NR may be therapeutic in settings of acute liver injury. (Hepatology 2017;65:616-630).
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Project description:Both hereditary factors (e.g., BRCA1) and nicotinamide adenine dinucleotide (NAD)-dependent metabolic pathways are implicated in the initiation and progression of ovarian cancer. However, whether crosstalk exists between BRCA1 and NAD metabolism remains largely unknown. Here, we showed that: (i) BRCA1 inactivation events (mutation and promoter methylation) were accompanied by elevated levels of NAD; (ii) the knockdown or overexpression of BRCA1 was an effective way to induce an increase or decrease of nicotinamide phosphoribosyltransferase (Nampt)-related NAD synthesis, respectively; and (iii) BRCA1 expression patterns were inversely correlated with NAD levels in human ovarian cancer specimens. In addition, it is worth noting that: (i) NAD incubation induced increased levels of BRCA1 in a concentration-dependent manner; (ii) Nampt knockdown-mediated reduction in NAD levels was effective at inhibiting BRCA1 expression; and (iii) the overexpression of Nampt led to higher NAD levels and a subsequent increase in BRCA1 levels in primary ovarian cancer cells and A2780, HO-8910 and ES2 ovarian cancer cell lines. These results highlight a novel link between BRCA1 and NAD. Our findings imply that genetic (e.g., BRCA1 inactivation) and NAD-dependent metabolic pathways are jointly involved in the malignant progression of ovarian cancer.
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Project description:Cellular necrosis has long been regarded as an incidental and uncontrolled form of cell death. However, a regulated form of cell death termed necroptosis has been identified recently. Necroptosis can be induced by extracellular cytokines, pathogens and several pharmacological compounds, which share the property of triggering the formation of a RIPK3-containing molecular complex supporting cell death. Of interest, most ligands known to induce necroptosis (including notably TNF and FASL) can also promote apoptosis, and the mechanisms regulating the decision of cells to commit to one form of cell death or the other are still poorly defined. We demonstrate herein that intracellular nicotinamide adenine dinucleotide (NAD(+)) has an important role in supporting cell progression to necroptosis. Using a panel of pharmacological and genetic approaches, we show that intracellular NAD(+) promotes necroptosis of the L929 cell line in response to TNF. Use of a pan-sirtuin inhibitor and shRNA-mediated protein knockdown led us to uncover a role for the NAD(+)-dependent family of sirtuins, and in particular for SIRT2 and SIRT5, in the regulation of the necroptotic cell death program. Thus, and in contrast to a generally held view, intracellular NAD(+) does not represent a universal pro-survival factor, but rather acts as a key metabolite regulating the choice of cell demise in response to both intrinsic and extrinsic factors.
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Project description:Mitochondrial NAD levels influence fuel selection, circadian rhythms, and cell survival under stress. It has alternately been argued that NAD in mammalian mitochondria arises from import of cytosolic nicotinamide (NAM), nicotinamide mononucleotide (NMN), or NAD itself. We provide evidence that murine and human mitochondria take up intact NAD. Isolated mitochondria preparations cannot make NAD from NAM, and while NAD is synthesized from NMN, it does not localize to the mitochondrial matrix or effectively support oxidative phosphorylation. Treating cells with nicotinamide riboside that is isotopically labeled on the nicotinamide and ribose moieties results in the appearance of doubly labeled NAD within mitochondria. Analogous experiments with doubly labeled nicotinic acid riboside (labeling cytosolic NAD without labeling NMN) demonstrate that NAD(H) is the imported species. Our results challenge the long-held view that the mitochondrial inner membrane is impermeable to pyridine nucleotides and suggest the existence of an unrecognized mammalian NAD (or NADH) transporter.
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Project description:Universal and ubiquitous redox cofactors, nicotinamide adenine dinucleotide (NAD) and its phosphorylated analog (NADP), collectively contribute to approximately 12% of all biochemical reactions included in the metabolic model of Escherichia coli K-12. A homeostasis of the NAD pool faithfully maintained by the cells results from a dynamic balance in a network of NAD biosynthesis, utilization, decomposition, and recycling pathways that is subject to tight regulation at various levels. A brief overview of NAD utilization processes is provided in this review, including some examples of nonredox utilization. The review focuses mostly on those aspects of NAD biogenesis and utilization in E. coli and Salmonella that emerged within the past 12 years. The first pyridine nucleotide cycle (PNC) originally identified in mammalian systems and termed the Preiss-Handler pathway includes a single-step conversion of niacin (Na) to NaMN by nicotinic acid phosphoribosyltransferase (PncB). In E. coli and many other prokaryotes, this enzyme, together with nicotinamide deamidase (PncA), compose the major pathway for utilization of the pyridine ring in the form of amidated (Nm) or deamidated (Na) precursors. The existence of various regulatory mechanisms and checkpoints that control the NAD biosynthetic machinery reflects the importance of maintaining NAD homeostasis in a variety of growth conditions. Among the most important regulatory mechanisms at the level of individual enzymes are a classic feedback inhibition of NadB, the first enzyme of NAD de novo biosynthesis, by NAD and a metabolic regulation of NadK by reduced cofactors.
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Project description:While interleukin-1? (IL-1?) is a potent pro-inflammatory cytokine essential for host defense, high systemic levels cause life-threatening inflammatory syndromes. ATP, a stimulus of IL-1? maturation, is released from damaged cells along with ?-nicotinamide adenine dinucleotide (?-NAD). Here, we tested the hypothesis that ?-NAD controls ATP-signaling and, hence, IL-1? release. Lipopolysaccharide-primed monocytic U937 cells and primary human mononuclear leukocytes were stimulated with 2'(3')-O-(4-benzoyl-benzoyl)ATP trieethylammonium salt (BzATP), a P2X7 receptor agonist, in the presence or absence of ?-NAD. IL-1? was measured in cell culture supernatants. The roles of P2Y receptors, nicotinic acetylcholine receptors (nAChRs), and Ca2+-independent phospholipase A2 (iPLA2?, PLA2G6) were investigated using specific inhibitors and gene-silencing. Exogenous ?-NAD signaled via P2Y receptors and dose-dependently (IC50 = 15 µM) suppressed the BzATP-induced IL-1? release. Signaling involved iPLA2?, release of a soluble mediator, and nAChR subunit ?9. Patch-clamp experiments revealed that ?-NAD inhibited BzATP-induced ion currents. In conclusion, we describe a novel triple membrane-passing signaling cascade triggered by extracellular ?-NAD that suppresses ATP-induced release of IL-1? by monocytic cells. This cascade links activation of P2Y receptors to non-canonical metabotropic functions of nAChRs that inhibit P2X7 receptor function. The biomedical relevance of this mechanism might be the control of trauma-associated systemic inflammation.
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Project description:AimsAccurate analysis of dinucleotide redox cofactors nicotinamide adenine dinucleotide phosphate reduced (NADPH), nicotinamide adenine dinucleotide phosphate (NADP+), nicotinamide adenine dinucleotide reduced (NADH), and nicotinamide adenine dinucleotide (NAD+) from biological samples is important to understanding cellular redox homeostasis. In this study, we aimed to develop a simple protocol for quenching metabolism and extracting NADPH that avoids interconversion among the reduced forms and the oxidized forms.ResultsWe compared seven different solvents for quenching and extraction of cultured mammalian cells and mouse tissues: a cold aqueous buffer commonly used in enzyme assays with and without detergent, hot aqueous buffer, and cold organic mixtures (80% methanol, buffered 75% acetonitrile, and acidic 40:40:20 acetonitrile:methanol:water with either 0.02 M or 0.1 M formic acid). Extracts were analyzed by liquid chromatography-mass spectrometry (LC-MS). To monitor the metabolite interconversion, cells were grown in 13C6-glucose medium, and unlabeled standards were spiked into the extraction solvents. Interconversion between the oxidized and reduced forms was substantial except for the enzyme assay buffer with detergent, 80% methanol and 40:40:20 acetonitrile:methanol:water, with the 0.1 M formic acid mix giving the least interconversion and best recoveries. Absolute NAD+, NADH, NADP+, and NADPH concentrations in cells and mouse tissues were measured with this approach.InnovationWe found that the interconversion between the reduced and oxidized forms during extraction is a major barrier to accurately measuring NADPH/NADP+ and NADH/NAD+ ratios. Such interconversion can be monitored by isotope labeling cells and spiking NAD(P)(H) standards.ConclusionExtraction with 40:40:20 acetonitrile:methanol:water with 0.1 M formic acid decreases interconversion and, therefore, is suitable for measurement of redox cofactor ratios using LC-MS. This solvent is also useful for general metabolomics. Samples should be neutralized immediately after extraction to avoid acid-catalyzed degradation. When LC-MS is not available and enzyme assays are accordingly used, inclusion of detergent in the aqueous extraction buffer reduces interconversion. Antioxid. Redox Signal. 28, 167-179.
| S-EPMC5737638 | biostudies-literature