Nicotinamide adenine dinucleotide
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Nicotinamide adenine dinucleotide ( NAD) is a coenzyme central to metabolism. Found in all living cells, NAD is called a dinucleotide because it consists of two joined through their phosphate groups. One nucleotide contains an adenine nucleobase and the other, nicotinamide. NAD exists in two forms: an Redox form, abbreviated as NAD and NADH (H for hydrogen), respectively.
In cellular metabolism, NAD is involved in redox reactions, carrying from one reaction to another, so it is found in two forms: NAD is an oxidizing agent, accepting electrons from other molecules and becoming reduced; with H+, this reaction forms NADH, which can be used as a reducing agent to donate electrons. These electron transfer reactions are the main function of NAD. It is also used in other cellular processes, most notably as a substrate of in adding or removing to or from , in posttranslational modifications. Because of the importance of these functions, the enzymes involved in NAD metabolism are targets for drug discovery.
In organisms, NAD can be synthesized from simple building-blocks ( de novo) from either tryptophan or aspartic acid, each a case of an amino acid. Alternatively, more complex components of the coenzymes are taken up from nutritive compounds such as nicotinic acid; similar compounds are produced by reactions that break down the structure of NAD, providing a salvage pathway that recycles them back into their respective active form.
In the name NAD, the superscripted plus sign indicates the positive formal charge on one of its nitrogen atoms. A biological coenzyme that acts as an electron carrier in enzymatic reactions.
Some NAD is converted into the coenzyme nicotinamide adenine dinucleotide phosphate (NADP), whose chemistry largely parallels that of NAD, though its predominant role is as a coenzyme in anabolic metabolism. NADP is a reducing agent in anabolic reactions like the Calvin cycle and lipid and nucleic acid syntheses. NADP exists in two forms: NADP+, the oxidized form, and NADPH, the reduced form. NADP is similar to nicotinamide adenine dinucleotide (NAD), but NADP has a phosphate group at the C-2′ position of the adenosyl.
Physical and chemical properties
The compound accepts or donates the equivalent of H−. Such reactions (summarized in formula below) involve the removal of two hydrogen atoms from a reactant (R), in the form of a hydride (H−), and a proton (H). The proton is released into solution, while the reductant RH is oxidized and NAD reduced to NADH by transfer of the hydride to the nicotinamide ring.
- RH + NAD → NADH + H + R;
From the electron pair of the hydride ion, one electron is attracted to the slightly more electronegative atom of the nicotinamide ring of NAD, becoming part of the nicotinamide moiety. The remaining hydrogen atom is transferred to the carbon atom opposite the N atom. The midpoint potential of the NAD/NADH redox pair is −0.32 , which makes NADH a moderately strong reducing agent. The reaction is easily reversible, when NADH reduces another molecule and is re-oxidized to NAD. This means the coenzyme can continuously cycle between the NAD and NADH forms without being consumed.
In appearance, all forms of this coenzyme are white amorphous solid powders that are hygroscopy and highly water-soluble. The solids are stable if stored dry and in the dark. Solutions of NAD are colorless and stable for about a week at 4 Celsius and neutral pH, but decompose rapidly in acidic or alkaline solutions. Upon decomposition, they form products that are .
Both NAD and NADH strongly absorb ultraviolet light because of the adenine. For example, peak absorption of NAD is at a wavelength of 259 (nm), with an extinction coefficient of 16,900 M−1Centimetre−1. NADH also absorbs at higher wavelengths, with a second peak in UV absorption at 339 nm with an extinction coefficient of 6,220 M−1cm−1. This difference in the ultraviolet absorption spectra between the oxidized and reduced forms of the coenzymes at higher wavelengths makes it simple to measure the conversion of one to another in – by measuring the amount of UV absorption at 340 nm using a spectrophotometer.
NAD and NADH also differ in their fluorescence. Freely diffusing NADH in aqueous solution, when excited at the nicotinamide absorbance of ~335 nm (near-UV), fluoresces at 445–460 nm (violet to blue) with a fluorescence lifetime of 0.4 , while NAD does not fluoresce. The properties of the fluorescence signal changes when NADH binds to , so these changes can be used to measure dissociation constants, which are useful in the study of enzyme kinetics. These changes in fluorescence are also used to measure changes in the redox state of living cells, through fluorescence microscopy.
NADH can be converted to NAD+ in a reaction catalysed by copper, which requires hydrogen peroxide. Thus, the supply of NAD+ in cells requires mitochondrial copper(II).
NAD concentrations are highest in the mitochondria, constituting 40% to 70% of the total cellular NAD. NAD in the cytosol is carried into the mitochondrion by a specific membrane transport protein, since the coenzyme cannot diffusion across membranes. The intracellular half-life of NAD+ was claimed to be between 1–2 hours by one review, whereas another review gave varying estimates based on compartment: intracellular 1–4 hours, cytoplasmic 2 hours, and mitochondrial 4–6 hours.
The balance between the oxidized and reduced forms of nicotinamide adenine dinucleotide is called the NAD/NADH ratio. This ratio is an important component of what is called the redox state of a cell, a measurement that reflects both the metabolic activities and the health of cells. The effects of the NAD/NADH ratio are complex, controlling the activity of several key enzymes, including glyceraldehyde 3-phosphate dehydrogenase and pyruvate dehydrogenase. In healthy mammalian tissues, estimates of the ratio of free NAD to NADH in the cytoplasm typically lie around 700:1; the ratio is thus favorable for oxidative reactions. The ratio of total NAD/NADH is much lower, with estimates ranging from 3–10 in mammals. In contrast, the NADP/NADPH ratio is normally about 0.005, so NADPH is the dominant form of this coenzyme. These different ratios are key to the different metabolic roles of NADH and NADPH.
In a further step, some NAD is converted into NADP by NAD kinase, which phosphorylates NAD. In most organisms, this enzyme uses adenosine triphosphate (ATP) as the source of the phosphate group, although several bacteria such as Mycobacterium tuberculosis and a hyperthermophilic archaeon Pyrococcus, use inorganic polyphosphate as an alternative phosphoryl donor.
Besides assembling NAD de novo from simple amino acid precursors, cells also salvage preformed compounds containing a pyridine base. The three vitamin precursors used in these salvage metabolic pathways are nicotinic acid (NA), nicotinamide (Nam) and nicotinamide riboside (NR). These compounds can be taken up from the diet and are termed vitamin B or niacin. However, these compounds are also produced within cells and by digestion of cellular NAD. Some of the enzymes involved in these salvage pathways appear to be concentrated in the cell nucleus, which may compensate for the high level of reactions that consume NAD in this organelle. There are some reports that mammalian cells can take up extracellular NAD from their surroundings, and both nicotinamide and nicotinamide riboside can be absorbed from the gut.
The salvage pathways used in differ from those of . Some pathogens, such as the yeast Candida glabrata and the bacterium Haemophilus influenzae are NAD – they cannot synthesize NAD – but possess salvage pathways and thus are dependent on external sources of NAD or its precursors. Even more surprising is the intracellular pathogen Chlamydia trachomatis, which lacks recognizable candidates for any genes involved in the biosynthesis or salvage of both NAD and NADP, and must acquire these coenzymes from its host.
There are many different superfamilies of enzymes that bind NAD / NADH. One of the most common superfamilies includes a structural motif known as the Rossmann fold. The motif is named after Michael Rossmann, who was the first scientist to notice how common this structure is within nucleotide-binding proteins.
An example of a NAD-binding bacterial enzyme involved in amino acid metabolism that does not have the Rossmann fold is found in Pseudomonas syringae pv. tomato (; ).
When bound in the active site of an oxidoreductase, the nicotinamide ring of the coenzyme is positioned so that it can accept a hydride from the other substrate. Depending on the enzyme, the hydride donor is positioned either "above" or "below" the plane of the planar C4 carbon, as defined in the figure. Class A oxidoreductases transfer the atom from above; class B enzymes transfer it from below. Since the C4 carbon that accepts the hydrogen is prochiral, this can be exploited in enzyme kinetics to give information about the enzyme's mechanism. This is done by mixing an enzyme with a substrate that has deuterium atoms substituted for the hydrogens, so the enzyme will reduce NAD by transferring deuterium rather than hydrogen. In this case, an enzyme can produce one of two stereoisomerism of NADH.
Despite the similarity in how proteins bind the two coenzymes, enzymes almost always show a high level of specificity for either NAD or NADP. This specificity reflects the distinct metabolic roles of the respective coenzymes, and is the result of distinct sets of amino acid residues in the two types of coenzyme-binding pocket. For instance, in the active site of NADP-dependent enzymes, an ionic bond is formed between a basic amino acid side-chain and the acidic phosphate group of NADP. On the converse, in NAD-dependent enzymes the charge in this pocket is reversed, preventing NADP from binding. However, there are a few exceptions to this general rule, and enzymes such as aldose reductase, glucose-6-phosphate dehydrogenase, and methylenetetrahydrofolate reductase can use both coenzymes in some species.
Since both the oxidized and reduced forms of nicotinamide adenine dinucleotide are used in these linked sets of reactions, the cell maintains significant concentrations of both NAD and NADH, with the high NAD/NADH ratio allowing this coenzyme to act as both an oxidizing and a reducing agent. In contrast, the main function of NADPH is as a reducing agent in anabolism, with this coenzyme being involved in pathways such as fatty acid synthesis and photosynthesis. Since NADPH is needed to drive redox reactions as a strong reducing agent, the NADP/NADPH ratio is kept very low.
Although it is important in catabolism, NADH is also used in anabolic reactions, such as gluconeogenesis. This need for NADH in anabolism poses a problem for prokaryotes growing on nutrients that release only a small amount of energy. For example, nitrification bacteria such as Nitrobacter oxidize nitrite to nitrate, which releases sufficient energy to pump protons and generate ATP, but not enough to produce NADH directly. As NADH is still needed for anabolic reactions, these bacteria use a nitrite oxidoreductase to produce enough chemiosmosis to run part of the electron transport chain in reverse, generating NADH.
Another function of this coenzyme in cell signaling is as a precursor of cyclic ADP-ribose, which is produced from NAD by ADP-ribosyl cyclases, as part of a second messenger system. This molecule acts in calcium signaling by releasing calcium from intracellular stores. It does this by binding to and opening a class of calcium channels called ryanodine receptors, which are located in the membranes of , such as the endoplasmic reticulum, and inducing the activation of the transcription factor NAFC3
NAD is also consumed by different NAD+-consuming enzymes, such as CD38, CD157, PARPs and the NAD-dependent deacetylases (, such as Sir2.). These enzymes act by transferring an acetyl group from their substrate protein to the ADP-ribose moiety of NAD; this cleaves the coenzyme and releases nicotinamide and O-acetyl-ADP-ribose. The sirtuins mainly seem to be involved in regulating transcription through deacetylating histones and altering nucleosome structure. However, non-histone proteins can be deacetylated by sirtuins as well. These activities of sirtuins are particularly interesting because of their importance in the regulation of aging.
Other NAD-dependent enzymes include bacterial , which join two DNA ends by using NAD as a substrate to donate an adenosine monophosphate (AMP) moiety to the 5' phosphate of one DNA end. This intermediate is then attacked by the 3' hydroxyl group of the other DNA end, forming a new phosphodiester bond. This contrasts with eukaryotic DNA ligases, which use ATP to form the DNA-AMP intermediate.
Li et al. have found that NAD directly regulates protein-protein interactions. They also show that one of the causes of age-related decline in DNA repair may be increased binding of the protein DBC1 (Deleted in Breast Cancer 1) to PARP1 (polyADP–ribose polymerase 1) as NAD levels decline during aging. The decline in cellular concentrations of NAD during aging likely contributes to the ageing process and to the pathogenesis of the chronic diseases of aging.Verdin E. NAD⁺ in aging, metabolism, and neurodegeneration. Science. 2015 Dec 4;350(6265):1208-13. doi: 10.1126/science.aac4854. PMID 26785480 Thus, the modulation of NAD may protect against cancer, radiation, and aging.
Because cancer cells utilize increased glycolysis, and because NAD enhances glycolysis, nicotinamide phosphoribosyltransferase (NAD salvage pathway) is often amplified in cancer cells.
It has been studied for its potential use in the therapy of neurodegenerative diseases such as Alzheimer's and Parkinson's disease as well as multiple sclerosis. A placebo-controlled clinical trial of NADH (which excluded NADH precursors) in people with Parkinson's failed to show any effect.
NAD is also a direct drug target of the drug isoniazid, which is used in the treatment of tuberculosis, an infection caused by Mycobacterium tuberculosis. Isoniazid is a prodrug and once it has entered the bacteria, it is activated by a peroxidase enzyme, which oxidizes the compound into a free radical form. This radical then reacts with NADH, to produce adducts that are very potent inhibitors of the enzymes enoyl-acyl carrier protein reductase, and dihydrofolate reductase.
Since many oxidoreductases use NAD and NADH as substrates, and bind them using a highly conserved structural motif, the idea that inhibitors based on NAD could be specific to one enzyme is surprising. However, this can be possible: for example, inhibitors based on the compounds mycophenolic acid and tiazofurin inhibit IMP dehydrogenase at the NAD binding site. Because of the importance of this enzyme in purine metabolism, these compounds may be useful as anti-cancer, anti-viral, or immunosuppressive drugs. Other drugs are not enzyme inhibitors, but instead activate enzymes involved in NAD metabolism. are a particularly interesting target for such drugs, since activation of these NAD-dependent deacetylases extends lifespan in some animal models. Compounds such as resveratrol increase the activity of these enzymes, which may be important in their ability to delay aging in both vertebrate, and invertebrate . In one experiment, mice given NAD for one week had improved nuclear-mitochrondrial communication.
Because of the differences in the metabolic pathways of NAD biosynthesis between organisms, such as between bacteria and humans, this area of metabolism is a promising area for the development of new .
In bacteriology, NAD, sometimes referred to factor V, is used as a supplement to culture media for some fastidious bacteria.
High-cost unlicensed infusions of NAD+ have been claimed in the UK to be "clinically proven" and "effective" treatment for alcoholism and drug abuse. NAD+ is not approved or Medical license in the UK; there are likely breaches of advertising and medicines rules, and no proof that treatments work. Medical experts say "It's complete nonsense" ... "It's untested and unproven. We don't know anything about its efficacy or long-term safety". A November 2024 study, cited 700 times, claiming that NAD+ levels in decreased with age was withdrawn after images were found to have been manipulated, and underlying data was not provided to the publishers on request.
Vitamin precursors of NAD were first identified in 1938, when Conrad Elvehjem showed that liver has an "anti-black tongue" activity in the form of nicotinamide. Then, in 1939, he provided the first strong evidence that nicotinic acid is used to synthesize NAD. In the early 1940s, Arthur Kornberg was the first to detect an enzyme in the biosynthetic pathway. In 1949, the American biochemists Morris Friedkin and Albert L. Lehninger proved that NADH linked metabolic pathways such as the citric acid cycle with the synthesis of ATP in oxidative phosphorylation. In 1958, Jack Preiss and Philip Handler discovered the intermediates and enzymes involved in the biosynthesis of NAD; salvage synthesis from nicotinic acid is termed the Preiss-Handler pathway. In 2004, Charles Brenner and co-workers uncovered the nicotinamide riboside kinase pathway to NAD.
The non-redox roles of NAD(P) were discovered later. The first to be identified was the use of NAD as the ADP-ribose donor in ADP-ribosylation reactions, observed in the early 1960s. Studies in the 1980s and 1990s revealed the activities of NAD and NADP metabolites in cell signaling – such as the action of cyclic ADP-ribose, which was discovered in 1987.
The metabolism of NAD remained an area of intense research into the 21st century, with interest heightened after the discovery of the NAD-dependent protein deacetylases called in 2000, by Shin-ichiro Imai and coworkers in the laboratory of Leonard P. Guarente. In 2009 Imai proposed the "NAD World" hypothesis that key regulators of aging and longevity in mammals are sirtuin 1 and the primary NAD synthesizing enzyme nicotinamide phosphoribosyltransferase (NAMPT). In 2016 Imai expanded his hypothesis to "NAD World 2.0", which postulates that extracellular NAMPT from adipose tissue maintains NAD in the hypothalamus (the control center) in conjunction with from skeletal muscle cells. In 2018, Napa Therapeutics was formed to develop drugs against a novel aging-related target based on the research in NAD metabolism conducted in the lab of Eric Verdin.
Concentration and state in cells
Biosynthesis
De novo production
Salvage pathways
Functions
Oxidoreductase binding of NAD
Role in redox metabolism
Non-redox roles
Extracellular actions of NAD+
Clinical significance
History
See also
Further reading
Function
History
External links
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