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Nicotinamide adenine dinucleotide phosphate
Nicotinamide adenine dinucleotide phosphate
Nicotinamide adenine dinucleotide phosphate
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Nicotinamide adenine dinucleotide phosphate
Identifiers
3D model (JSmol)
ChEBI
ChEMBL
ChemSpider
ECHA InfoCard 100.000.163 Edit this at Wikidata
MeSH NADP
UNII
  • InChI=1S/C21H28N7O17P3/c22-17-12-19(25-7-24-17)28(8-26-12)21-16(44-46(33,34)35)14(30)11(43-21)6-41-48(38,39)45-47(36,37)40-5-10-13(29)15(31)20(42-10)27-3-1-2-9(4-27)18(23)32/h1-4,7-8,10-11,13-16,20-21,29-31H,5-6H2,(H7-,22,23,24,25,32,33,34,35,36,37,38,39)/t10-,11-,13-,14-,15-,16-,20-,21-/m1/s1 checkY
    Key: XJLXINKUBYWONI-NNYOXOHSSA-N checkY
  • InChI=1/C21H28N7O17P3/c22-17-12-19(25-7-24-17)28(8-26-12)21-16(44-46(33,34)35)14(30)11(43-21)6-41-48(38,39)45-47(36,37)40-5-10-13(29)15(31)20(42-10)27-3-1-2-9(4-27)18(23)32/h1-4,7-8,10-11,13-16,20-21,29-31H,5-6H2,(H7-,22,23,24,25,32,33,34,35,36,37,38,39)/t10-,11-,13-,14-,15-,16-,20-,21-/m1/s1
    Key: XJLXINKUBYWONI-NNYOXOHSBN
  • O=C(N)c1ccc[n+](c1)[C@H]2[C@H](O)[C@H](O)[C@H](O2)COP([O-])(=O)OP(=O)(O)OC[C@H]3O[C@@H](n4cnc5c4ncnc5N)[C@@H]([C@@H]3O)OP(=O)(O)O
Properties
C21H28N7O17P3+ (oxidized)
C21H29N7O17P3 (reduced)
Molar mass 744.4 g/mol (oxidized)
745.4 g/mol (reduced)
Except where otherwise noted, data are given for materials in their standard state (at 25 °C [77 °F], 100 kPa).
☒N verify (what is checkY☒N ?)

Nicotinamide adenine dinucleotide phosphate, abbreviated NADP[1][2] or, in older notation, TPN (triphosphopyridine nucleotide), is a cofactor used in anabolic reactions, such as the Calvin cycle and lipid and nucleic acid syntheses, which require NADPH as a reducing agent ('hydrogen source'). NADPH is the reduced form, whereas NADP+ is the oxidized form. NADP+ is used by all forms of cellular life. NADP+ is essential for life because it is needed for cellular respiration.[3]

NADP+ differs from NAD+ by the presence of an additional phosphate group on the 2' position of the ribose ring that carries the adenine moiety. This extra phosphate is added by NAD+ kinase and removed by NADP+ phosphatase.[4]

Biosynthesis

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NADP+

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In general, NADP+ is synthesized before NADPH is. Such a reaction usually starts with NAD+ from either the de-novo or the salvage pathway, with NAD+ kinase adding the extra phosphate group. ADP-ribosyl cyclase allows for synthesis from nicotinamide in the salvage pathway, and NADP+ phosphatase can convert NADPH back to NADH to maintain a balance.[3] Some forms of the NAD+ kinase, notably the one in mitochondria, can also accept NADH to turn it directly into NADPH.[5][6] The prokaryotic pathway is less well understood, but with all the similar proteins the process should work in a similar way.[3]

NADPH

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NADPH is produced from NADP+. The major source of NADPH in animals and other non-photosynthetic organisms is the pentose phosphate pathway, by glucose-6-phosphate dehydrogenase (G6PDH) in the first step. The pentose phosphate pathway also produces pentose, another important part of NAD(P)H, from glucose. Some bacteria also use G6PDH for the Entner–Doudoroff pathway, but NADPH production remains the same.[3]

Ferredoxin–NADP+ reductase, present in all domains of life, is a major source of NADPH in photosynthetic organisms including plants and cyanobacteria. It appears in the last step of the electron chain of the light reactions of photosynthesis. It is used as reducing power for the biosynthetic reactions in the Calvin cycle to assimilate carbon dioxide and help turn the carbon dioxide into glucose. It has functions in accepting electrons in other non-photosynthetic pathways as well: it is needed in the reduction of nitrate into ammonia for plant assimilation in nitrogen cycle and in the production of oils.[3]

There are several other lesser-known mechanisms of generating NADPH, all of which depend on the presence of mitochondria in eukaryotes. The key enzymes in these carbon-metabolism-related processes are NADP-linked isoforms of malic enzyme, isocitrate dehydrogenase (IDH), and glutamate dehydrogenase. In these reactions, NADP+ acts like NAD+ in other enzymes as an oxidizing agent.[7] The isocitrate dehydrogenase mechanism appears to be the major source of NADPH in fat and possibly also liver cells.[8] These processes are also found in bacteria. Bacteria can also use a NADP-dependent glyceraldehyde 3-phosphate dehydrogenase for the same purpose. Like the pentose phosphate pathway, these pathways are related to parts of glycolysis.[3] Another carbon metabolism-related pathway involved in the generation of NADPH is the mitochondrial folate cycle, which uses principally serine as a source of one-carbon units to sustain nucleotide synthesis and redox homeostasis in mitochondria. Mitochondrial folate cycle has been recently suggested as the principal contributor to NADPH generation in mitochondria of cancer cells.[9]

NADPH can also be generated through pathways unrelated to carbon metabolism. The ferredoxin reductase is such an example. Nicotinamide nucleotide transhydrogenase transfers the hydrogen between NAD(P)H and NAD(P)+, and is found in eukaryotic mitochondria and many bacteria. There are versions that depend on a proton gradient to work and ones that do not. Some anaerobic organisms use NADP+-linked hydrogenase, ripping a hydride from hydrogen gas to produce a proton and NADPH.[3]

Like NADH, NADPH is fluorescent. NADPH in aqueous solution excited at the nicotinamide absorbance of ~335 nm (near UV) has a fluorescence emission which peaks at 445-460 nm (violet to blue). NADP+ has no appreciable fluorescence.[10]

Function

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NADPH provides the reducing agents, usually hydrogen atoms, for biosynthetic reactions and the oxidation-reduction involved in protecting against the toxicity of reactive oxygen species (ROS), allowing the regeneration of glutathione (GSH).[11] NADPH is also used for anabolic pathways, such as cholesterol synthesis, steroid synthesis,[12] ascorbic acid synthesis,[12] xylitol synthesis,[12] cytosolic fatty acid synthesis[12] and microsomal fatty acid chain elongation.

The NADPH system is also responsible for generating free radicals in immune cells by NADPH oxidase. These radicals are used to destroy pathogens in a process termed the respiratory burst.[13] It is the source of reducing equivalents for cytochrome P450 hydroxylation of aromatic compounds, steroids, alcohols, and drugs.

Stability

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NADH and NADPH are very stable in basic solutions, but NAD+ and NADP+ are degraded in basic solutions into a fluorescent product that can be used conveniently for quantitation. Conversely, NADPH and NADH are degraded by acidic solutions while NAD+/NADP+ are fairly stable to acid.[14][15]

Enzymes that use NADP(H) as a coenzyme

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Many enzymes that bind NADP share a common super-secondary structure named the "Rossmann fold". The initial beta-alpha-beta (βαβ) fold is the most conserved segment of the Rossmann folds. This segment is in contact with the ADP portion of NADP. Therefore, it is also called an "ADP-binding βαβ fold".[16]

  • Adrenodoxin reductase: This enzyme is present ubiquitously in most organisms.[17] It transfers two electrons from NADPH to FAD. In vertebrates, it serves as the first enzyme in the chain of mitochondrial P450 systems that synthesize steroid hormones.[18]

Enzymes that use NADP(H) as a substrate

[edit]

In 2018 and 2019, the first two reports of enzymes that catalyze the removal of the 2' phosphate of NADP(H) in eukaryotes emerged. First the cytoplasmic protein MESH1 (Q8N4P3),[19] then the mitochondrial protein nocturnin[20] were reported. Of note, the structures and NADPH binding of MESH1 (5VXA) and nocturnin (6NF0) are not related.

Ball-and-stick models of NADP+ and NADPH
  • NADP+
    NADP+
  • NADPH
    NADPH

References

[edit]
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Nicotinamide adenine dinucleotide phosphate

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Nicotinamide adenine dinucleotide phosphate (NADP⁺) is a vital coenzyme found in all living cells, consisting of two nucleotides—one derived from adenine and the other from nicotinamide—joined by a pyrophosphate linkage, with an additional phosphate group esterified to the 2' position of the adenosine ribose, distinguishing it from the related coenzyme nicotinamide adenine dinucleotide (NAD⁺). Its molecular formula is C₂₁H₂₈N₇O₁₇P₃ for the oxidized form, and it cycles between NADP⁺ (oxidized) and NADPH (reduced) states during redox reactions, serving as a carrier of electrons and protons essential for metabolic processes. Unlike NAD⁺/NADH, which primarily participates in catabolic reactions to extract energy from nutrients, NADP⁺/NADPH is predominantly involved in anabolic pathways that build complex molecules, providing reducing power for biosynthesis such as fatty acid and cholesterol synthesis, as well as nucleotide production via the pentose phosphate pathway. NADPH also plays a critical role in maintaining cellular redox homeostasis by donating electrons to regenerate antioxidants like glutathione, thereby protecting against oxidative stress from reactive oxygen species. In plants and photosynthetic bacteria, NADPH is generated during the light-dependent reactions of photosynthesis and consumed in the Calvin-Benson cycle to reduce carbon dioxide into carbohydrates, linking light energy capture to carbon fixation. NADP⁺ is synthesized from NAD⁺ by NAD kinases using ATP, with cellular levels tightly regulated to meet demands for reductive and defense mechanisms. Beyond , NADPH serves as a substrate for enzymes like NADPH oxidases, which produce signaling , and derivatives of NADP⁺, such as NAADP, act as second messengers in pathways. These multifaceted roles underscore NADP⁺/NADPH's indispensability for cellular function, growth, and survival across prokaryotes and eukaryotes.

Overview and Discovery

Chemical Identity and Nomenclature

Nicotinamide adenine dinucleotide phosphate, commonly abbreviated as NADP, is the full chemical name for this coenzyme central to cellular processes. The oxidized form is denoted NADP⁺, while the reduced form is NADPH. NADP serves as a phosphorylated derivative of (NAD), featuring an additional phosphate group attached at the 2′ position of the ring in the adenine moiety. The molecular formula of NADP⁺ is C₂₁H₂₈N₇O₁₇P₃, with a molecular weight of 743.4 g/mol. In its reduced form, NADPH has the formula C₂₁H₃₀N₇O₁₇P₃ and a molecular weight of 745.4 g/mol, reflecting the addition of two hydrogen atoms during reduction. These values represent the neutral zwitterionic forms as commonly referenced in biochemical literature. The systematic IUPAC name for NADP⁺ is [[(2R,3R,4R,5R)-5-(6-aminopurin-9-yl)-3-hydroxy-4-phosphonooxyoxolan-2-yl]methoxy-hydroxyphosphoryl] [(2R,3S,4R,5R)-5-(3-carbamoylpyridin-1-ium-1-yl)-3,4-dihydroxyoxolan-2-yl]methyl phosphate. Historically, NADP was referred to as triphosphopyridine nucleotide (TPN), a term used in early biochemical studies before the adoption of the modern nomenclature. Other synonyms include coenzyme II and codehydrogenase II.

History of Discovery

The discovery of nicotinamide adenine dinucleotide phosphate (NADP) emerged in the context of early 20th-century studies on fermentation, following the identification of (NAD) as a heat-stable coenzyme termed "cozymase" by Arthur Harden and William John Young in 1906. In the early 1930s, Otto and Walter Christian, while investigating oxidation enzymes and fermentation processes in , isolated a distinct coenzyme required for the activity of (G6PD), an catalyzing the oxidation of glucose-6-phosphate. This coenzyme, initially termed cozymase II or codehydrogenase II, was purified from extracts between 1931 and 1934, marking the first recognition of NADP as a separate entity from NAD in reactions. Warburg and Christian's experiments demonstrated its role in transferring hydrogen equivalents, distinguishing it through its specificity for certain dehydrogenase reactions in and animal tissues. By 1936, and Christian elucidated the structural basis for this distinction, identifying an additional group on the moiety of cozymase II compared to NAD (cozymase I), which led to its formal designation as triphosphopyridine (TPN) and later standardized as NADP. This modification was confirmed through and chemical analysis, highlighting NADP's unique participation in biosynthetic and oxidative pathways beyond NAD's primary catabolic functions. A key milestone in understanding NADP's broader physiological significance occurred in the 1950s, when studies on photosynthetic electron transport in chloroplasts revealed its essential role in NADP photoreduction, facilitated by as an intermediate electron carrier. This discovery, advanced by researchers including Daniel Arnon, integrated NADP into the of , where NADPH generation supports carbon fixation.

Molecular Structure

Structure of NADP+

NADP⁺ is a coenzyme composed of a unit linked through a bridge to an unit, with an additional group esterified at the 2' position of the adenosine ribose. This dinucleotide structure features two nucleosides connected by a diphosphate linkage, where the is bound to its via an N-glycosidic bond at the 1' position of the sugar and the 3-position of the ring, and the is similarly attached to its at the N9 position. The bridge consists of two phosphoanhydride bonds, forming a 5'-5' linkage between the ribose moieties. Key structural elements include the positively charged pyridinium ring in the nicotinamide moiety, which acts as the reactive center capable of accepting a hydride ion in redox processes. Both ribose sugars adopt the furanose form, specifically β-D-ribofuranosyl configurations, contributing to the molecule's overall rigidity and orientation in enzymatic binding sites. In total, NADP⁺ possesses three phosphate groups: the two in the pyrophosphate linkage and the single 2'-phosphate on the adenosine ribose. The positively charged pyridinium ring of the nicotinamide moiety contributes +1 charge, while the deprotonated phosphates result in a net negative charge at physiological pH. The systematic IUPAC name is {[(2R,3R,4S,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxyoxolan-2-yl]methoxy}(hydroxy)phosphoryl {[(2R,3R,4S,5R)-5-(3-carbamoyl-1H-pyridin-1-ium-1-yl)-3,4-dihydroxyoxolan-2-yl]methoxy}phosphoric acid, and its molecular formula is C₂₁H₂₈N₇O₁₇P₃⁺. The linear representation of NADP⁺ highlights the sequential connectivity: the nicotinamide-pyridinium ring attached via β-N-glycosidic bond to ribofuranose-5'-, which bridges to the ribose bearing the 2'- and base via another β-N-glycosidic bond. This arrangement ensures the spatial separation of the reactive from the , facilitating specific interactions in protein active sites. In comparison to NAD⁺, NADP⁺ is distinguished only by the additional 2'- group on the ribose, a modification that confers greater specificity by enabling interactions with positively charged residues in NADP⁺-dependent enzymes.

Structure of NADPH

NADPH, the reduced form of nicotinamide adenine dinucleotide phosphate (NADP⁺), features a key structural modification in its nicotinamide moiety compared to the oxidized state. The nicotinamide ring in NADP⁺ is a positively charged pyridinium ion, which undergoes reduction by the addition of a hydride ion (H⁻) to the 4-position of the ring, transforming it into a neutral 1,4-dihydropyridine ring. This reduction process involves the stereospecific transfer of the hydride, distinguishing between the diastereotopic pro-R and pro-S hydrogens at the C4 position, which enzymes exploit for chiral specificity in hydride donation during redox reactions. The overall molecular formula of NADPH is C₂₁H₃₀N₇O₁₇P₃, reflecting the incorporation of the and an additional proton relative to NADP⁺, resulting in a net gain of two electrons and two protons across the molecule. This structural shift renders the dihydropyridine ring non-aromatic, adopting a boat-like conformation that facilitates , while the rest of the structure—including the adenine-ribose-phosphate backbone and the 2'-phosphate group on the moiety—remains unchanged from NADP⁺. The reduction also alters the molecule's spectroscopic properties, shifting the primary maximum from approximately 260 nm in NADP⁺ to 340 nm in NADPH, which is commonly used to monitor its concentration in biochemical assays. In terms of stability, the of NADPH is more susceptible to non-enzymatic oxidation in the presence of air, particularly in aqueous solutions, where it can slowly revert to NADP⁺ through autooxidation, necessitating careful handling to prevent degradation.

Biosynthesis

Synthesis of NADP+ from NAD+

The synthesis of NADP⁺ from NAD⁺ occurs primarily through an ATP-dependent reaction catalyzed by NAD⁺ (NADK, EC 2.7.1.23), which adds a group to the 2'-hydroxyl position of the moiety in NAD⁺. The reaction can be represented as: NAD++ATP→NADP++ADP\text{NAD}^+ + \text{ATP} \rightarrow \text{NADP}^+ + \text{ADP} This process is the primary mechanism for generating the NADP⁺ pool in cells from the existing NAD⁺ pool, with NADK exhibiting specificity for NAD⁺ as the primary substrate over NADH. NADK activity is compartmentalized, occurring in both the cytosol (via cytosolic NADK) and mitochondria (via mitochondrial NADK2 in mammals), allowing localized production of NADP⁺ to support compartment-specific demands. The enzyme relies on NAD⁺ precursors derived from the de novo biosynthesis pathway starting from tryptophan via the kynurenine pathway, the Preiss-Handler salvage pathway from nicotinic acid, or the nicotinamide salvage pathway, which recycles nicotinamide released from NAD⁺-consuming reactions back into NAD⁺ through nicotinamide phosphoribosyltransferase and other enzymes. In prokaryotes, a homologous NADK encoded by the ppnK performs the same phosphorylation, maintaining the NADP⁺/NAD⁺ balance essential for , though prokaryotic variants may utilize as an alternative phosphoryl donor in some species. Eukaryotic NADK is regulated by calcium/calmodulin binding to its N-terminal domain, which activates the in response to intracellular calcium signals, thereby linking NADP⁺ synthesis to cellular signaling events. This regulation is critical for sustaining the cellular NADP⁺ pool, which constitutes approximately 10% of total NAD⁺ levels and supports subsequent metabolic processes.

Generation of NADPH

NADPH is generated through the reduction of NADP⁺ in various metabolic pathways, primarily via the transfer of two electrons and a proton: NADP⁺ + 2e⁻ + H⁺ → NADPH. This process maintains the cellular pool of reducing equivalents essential for biosynthetic and protective reactions. The major routes for NADPH production differ by organism and tissue, balancing the demand for power without overlapping with NAD⁺/NADH systems. The (PPP), particularly its oxidative branch, serves as the primary source of NADPH in most cells. In this pathway, glucose-6-phosphate is oxidized to ribulose-5-phosphate, yielding two molecules of NADPH per molecule of glucose-6-phosphate through sequential dehydrogenations. Tissues with high biosynthetic demands, such as the liver, favor the PPP for NADPH generation to support processes like synthesis. The pathway's flux can adjust based on cellular needs, with approximately 5-10% of glucose metabolism directed through its oxidative branch in hepatocytes under fed conditions. Additional cytosolic and mitochondrial routes contribute to NADPH production, including the NADP⁺-dependent s and malic enzyme. Cytosolic 1 (IDH1) and mitochondrial 2 (IDH2) each produce one NADPH per isocitrate molecule converted to α-ketoglutarate, often utilizing citrate exported from mitochondria. Similarly, cytosolic malic enzyme 1 (ME1) and mitochondrial malic enzyme 3 (ME3) generate one NADPH per malate decarboxylated to pyruvate, linking to tricarboxylic acid cycle intermediates for flexible supply. In photosynthetic organisms, NADPH is produced in chloroplasts during the light-dependent reactions via ferredoxin-NADP⁺ reductase. This enzyme transfers electrons from reduced ferredoxin—generated by photosystem I—to NADP⁺, yielding NADPH to fuel the Calvin cycle. The process occurs primarily in the stroma and thylakoid membranes, adjusting the ATP:NADPH ratio through linear and cyclic electron flows. To sustain NADPH generation, the reduced form is reoxidized in anabolic reductions or antioxidant defenses, regenerating NADP⁺ for continuous cycling in these pathways. This dynamic balance ensures efficient utilization of NADP⁺ derived from NAD⁺ phosphorylation.

Biological Functions

Role in Redox Reactions and Anabolism

NADPH serves as a key carrier in cellular , providing reducing power through (H⁻) transfer in anabolic processes. Its standard (E°') is approximately -0.324 V, enabling it to donate electrons for the reduction of substrates in biosynthetic pathways. In contrast, the NAD⁺/NADH couple has a similar E°' of -0.320 V but is primarily utilized in catabolic reactions for production. This functional distinction arises largely from their compartmentalization within the cell, despite the comparable . The high NADPH/NADP⁺ ratio in the (often around 100:1) supports reductive by maintaining a reduced pool available for anabolic enzymes, whereas the NAD⁺/NADH ratio is more oxidized in mitochondria to favor . This spatial separation ensures that NADPH drives synthetic reactions in the and , while NADH facilitates breakdown processes in mitochondria. The general mechanism involves NADPH transferring a to an oxidized substrate, as depicted in the following equation: Substrateox+NADPH+H+→Substratered+NADP+\text{Substrate}_{\text{ox}} + \text{NADPH} + \text{H}^+ \rightarrow \text{Substrate}_{\text{red}} + \text{NADP}^+ This reaction powers diverse anabolic pathways, including the synthesis of lipids and nucleotides. In fatty acid synthesis, NADPH provides the reducing equivalents required by fatty acid synthase to elongate acyl chains, with 14 molecules of NADPH consumed per palmitate produced in the cytosol. Similarly, cholesterol biosynthesis relies on NADPH for multiple reductions, notably in the HMG-CoA reductase step, which converts 3-hydroxy-3-methylglutaryl-CoA to mevalonate using two equivalents of NADPH; overall, up to 21 NADPH molecules are needed per cholesterol molecule. In steroid hormone production, NADPH supports cytochrome P450-mediated hydroxylations and reductions in the adrenal cortex and gonads, where it is generated locally to fuel the conversion of cholesterol to active hormones like cortisol and testosterone. These roles underscore NADPH's essential contribution to building complex biomolecules essential for cellular growth and hormone regulation.

Involvement in Cellular Protection and Signaling

NADPH plays a crucial role in cellular defense by serving as the for the reduction of oxidized (GSSG) to its reduced form (GSH) through the enzyme . This reaction, represented as: GSSG + NADPH + H+→2GSH + NADP+\text{GSSG + NADPH + H}^+ \rightarrow 2\text{GSH + NADP}^+ maintains a high GSH/GSSG ratio, which is essential for detoxifying (ROS) such as via glutathione peroxidase.30301-1) In mammalian cells, this pathway supports the neutralization of oxidative damage, preventing and protein oxidation during metabolic stress. Similarly, NADPH fuels to regenerate reduced , which reduces disulfide bonds in oxidized proteins and peroxiredoxins, thereby protecting cellular components from ROS-induced harm. The system, dependent on NADPH, is particularly vital in maintaining in the and nucleus, influencing and regulation. Beyond defense, NADPH contributes to ROS generation through (NOX) enzymes, which produce as a signaling molecule and agent in . In neutrophils and macrophages, assembles upon pathogen recognition to catalyze the transfer of electrons from NADPH to oxygen, forming that aids in microbial killing during the respiratory burst. This controlled ROS production also facilitates intracellular signaling, such as activation of transcription factors for inflammatory responses, highlighting NADPH's dual role in protection and immunity. Dysregulation of NOX activity can lead to excessive ROS, but in balanced contexts, it supports defense without overwhelming systems. NADPH participates in cellular signaling pathways, including the generation of lipid second messengers via phospholipase activation. , stimulated by various signals, produces , which directly activates components, linking lipid metabolism to ROS-mediated in immune cells. Additionally, diacylglycerol from cooperates with to promote oxidase assembly, amplifying downstream effects like cytoskeletal reorganization. In , NAD kinase (NADK) is regulated by in response to Ca²⁺ elevations, phosphorylating NAD⁺ to NADP⁺ and thereby boosting NADPH availability for signaling cascades. This modulation ensures rapid NADPH supply during Ca²⁺-dependent events, such as or neuronal activity. Imbalances in NADPH levels, often due to deficiencies in generating enzymes like , heighten in erythrocytes, leading to membrane damage and . In such cases, insufficient NADPH impairs glutathione regeneration, allowing ROS accumulation that oxidizes and cytoskeletal proteins, reducing deformability and lifespan. This vulnerability is evident in conditions like G6PD deficiency, where erythrocytes exhibit increased susceptibility to oxidant-induced injury.

Enzymes Interacting with NADP(H)

Enzymes Using NADP(H) as Coenzymes

Nicotinamide adenine dinucleotide phosphate (NADP(H)) serves as a coenzyme in numerous enzymatic reactions, primarily facilitating reversible transfer to support processes without being consumed. In these reactions, NADPH donates a stereospecifically from the A-side (pro-R position) of its ring to the substrate or , while NADP⁺ accepts a hydride in the reverse direction, enabling efficient shuttling in anabolic and protective pathways. A prominent example is (G6PD), the rate-limiting enzyme of the oxidative (PPP), which catalyzes the conversion of glucose-6-phosphate to 6-phosphogluconolactone while reducing NADP⁺ to NADPH. This reaction provides essential NADPH for biosynthetic processes and antioxidant defense in the . Similarly, 6-phosphogluconate dehydrogenase (6PGD), the second enzyme in the PPP, oxidizes 6-phosphogluconate to ribulose-5-phosphate, generating a second molecule of NADPH per glucose-6-phosphate molecule processed, thereby amplifying NADPH production for reductive such as and . In the , 1 (IDH1) catalyzes the reversible NADP+-dependent of isocitrate to α-ketoglutarate (producing NADPH in the oxidative direction) and contributes to NADPH by supporting reductive carboxylation under hypoxia or nutrient limitation, linking the tricarboxylic acid cycle to reductive processes. Cytosolic malic enzyme 1 (ME1) similarly employs NADP⁺ to decarboxylate malate to pyruvate, yielding NADPH that supports and tumor growth by fueling . These enzymes highlight NADP(H)'s role in compartmentalized anabolic reactions. In photosynthesis, ferredoxin-NADP⁺ reductase (FNR) catalyzes the transfer of electrons from reduced to NADP⁺, producing NADPH during the of , which is crucial for the Calvin-Benson cycle and CO₂ fixation in chloroplasts. Other notable enzymes include 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), which uses two molecules of NADPH to reduce to mevalonate in the committed step of biosynthesis, and NADPH-dependent methemoglobin reductase (also known as cytochrome b5 reductase), which reduces to in erythrocytes using NADPH as the via flavin intermediates. Evolutionarily, anabolic enzymes preferentially utilize NADPH over NADH due to the separation of distinct cellular pools, allowing specialized for catabolic (NAD(H)) and anabolic (NADP(H)) .

Enzymes Using NADP(H) as Substrates

Enzymes that utilize NADP(H) as substrates typically catalyze irreversible modifications or degradation, thereby consuming the cofactor and influencing cellular and metabolic balance. A key class includes that remove the 2'-phosphate group from NADP(H), converting it to NAD(H) plus inorganic . The enzyme MESH1 (also known as HDDC3), identified in 2020, functions as a cytosolic NADPH requiring as a cofactor; it hydrolyzes NADPH to NADH, thereby depleting cytosolic NADPH pools and promoting under conditions. Similarly, nocturnin (NOCT), discovered in 2019 to possess NADP(H) activity, catalyzes the of both NADP⁺ to NAD⁺ and NADPH to NADH, with a slight preference for the ; NOCT exhibits circadian , peaking in activity during the early dark phase, and localizes primarily to mitochondria but also the , where it modulates local NADP(H) concentrations. Beyond phosphatases, certain bacterial toxins act as glycohydrolases that irreversibly degrade NADP(H), analogous to their action on NAD⁺. For instance, Tse6, a type VI secretion system effector from identified in 2015, hydrolyzes both NAD⁺ and NADP⁺ into and ADP-ribose phosphate, leading to cofactor depletion in target bacterial cells and interbacterial antagonism; this activity requires binding to Tu for delivery into the cytoplasm.01189-7) In eukaryotic contexts, poly(ADP-ribose) polymerases (PARPs) primarily consume NAD⁺ for in and signaling, but NADP⁺ binds competitively to their catalytic sites without serving as a substrate due to the obstructing 2'-phosphate, instead acting as an endogenous inhibitor that fine-tunes PARP activity. These substrate-consuming enzymes play critical roles in regulating the NADP/NAD , which is essential for maintaining and preventing metabolic imbalances. For example, MESH1 depletion preserves NADPH levels, enhancing cellular resistance to and oxidative damage, while NOCT's mitochondrial localization links its activity to organelle-specific and circadian control of . Dysregulation of such enzymes has been implicated in mitochondrial dysfunction, as seen in cancer and metabolic disorders where altered NADP(H) consumption disrupts energy production and stress responses.

Stability and Regulation

Chemical Stability

Nicotinamide adenine dinucleotide phosphate (NADP⁺) and its reduced form (NADPH) exhibit distinct -dependent stability profiles. NADP⁺ remains stable at acidic values below 7, but undergoes base-catalyzed at higher , leading to cleavage of the linkage or the nicotinamide-ribose . In contrast, NADPH is stable under basic conditions but degrades rapidly in acidic environments ( < 6), primarily through oxidation or proton-catalyzed breakdown of the dihydronicotinamide ring. These differences arise from the structural features of the oxidized and reduced forms, where the positively charged ring in NADP⁺ is susceptible to nucleophilic attack in , while the reduced form's dihydropyridine is prone to and auto-oxidation in acid. Thermal stability of NADP(H) is limited in aqueous solutions, with half-lives on the order of hours at physiological s. For NADPH in neutral buffer ( 7-8), the half-life is approximately 1-2 hours at 37°C, accelerating with increasing due to enhanced rates of and oxidation pathways. NADP⁺ shows greater thermal resilience than NADPH under neutral conditions but degrades faster above 8, with significant loss (up to 50%) observed after 30 minutes at 60°C in alkaline media. Degradation products include , ADP-ribose, and phosphorylated fragments, monitored via UV absorbance changes at 260 nm or 340 nm. NADP(H) is sensitive to light exposure, and protection from light is recommended during storage to maintain coenzyme activity. This instability is exacerbated in solution, where NADPH undergoes auto-oxidation at a rate of about 1-2% per hour in aerated neutral buffer at room temperature, converting to NADP⁺ and generating . To mitigate these effects, storage as lyophilized powder at -20°C (or preferably -80°C) in the dark is recommended, maintaining stability for months to years; aqueous solutions should be prepared fresh and used within hours. The primary non-enzymatic chemical reaction affecting NADP⁺ stability is the of the bond under basic conditions, yielding and . For NADPH, degradation involves multiple routes, including acid-catalyzed ring opening and oxygen-dependent oxidation, but lacks a dominant single pathway outside of extremes.

Cellular Regulation of Levels

Cells maintain NADP(H) homeostasis through dynamic of synthesis and consumption to support balance and biosynthetic demands. NAD (NADK) plays a central role by phosphorylating NAD⁺ to NADP⁺, with its activity upregulated under to replenish the NADP(H) pool and enhance cellular defense against . Additionally, the NADPH/NADP⁺ ratio provides feedback control on flux through the (PPP), where high NADPH levels inhibit (G6PD), the rate-limiting enzyme, thereby preventing overproduction and maintaining equilibrium. NADP(H) levels are further regulated by compartmentalization, ensuring localized availability for specific functions. In the and (ER), NADPH concentrations are elevated to support and protein synthesis, primarily generated by cytosolic NADK and malic enzyme 1 (ME1). In mitochondria, 2 (IDH2) provides a dedicated NADPH supply for defense and biosynthesis, independent of cytosolic pools. Physiological shifts in NADP(H) levels occur in response to hormonal and age-related signals. Insulin stimulates NADPH production via upregulation of ME1 activity in adipocytes, facilitating during nutrient abundance. With aging, competition for the NAD⁺ pool by deacetylases contributes to reduced NADP(H) availability, as declining NAD⁺ levels limit NADK-mediated conversion to NADP⁺. NADP(H) ratios are monitored in cells using , exploiting NADPH's excitation at 340 nm and emission at ~460 nm, which allows distinction from NADH. In the cytosol, the typical NADPH/NADP⁺ ratio is highly reduced, approximately 100:1, reflecting the compartment's anabolic role.

Clinical and Research Significance

Medical Implications

(G6PD) deficiency, an X-linked , represents a prominent medical implication of NADP(H) dysregulation, affecting an estimated 443 million individuals worldwide as of 2021. This condition impairs the first step of the , reducing NADPH production essential for maintaining in its to counteract in erythrocytes. Under triggers such as infection, certain drugs like , or ingestion of fava beans, affected individuals experience acute due to heightened oxidative damage and destruction. Defects in , a key enzyme complex utilizing NADPH to generate (ROS) for microbial killing, underlie (CGD), a rare . Mutations in genes encoding NADPH oxidase subunits, such as CYBB, lead to impaired ROS production in , resulting in recurrent severe bacterial and fungal infections, formation, and inflammatory complications. This NADPH-dependent dysfunction compromises innate immunity, highlighting the coenzyme's critical role in host defense. In metabolic disorders, elevated NADPH levels contribute to cancer progression by fueling reductive biosynthesis pathways, particularly synthesis required for rapid and formation. Cancer cells often upregulate NADPH production via the to support de novo , enhancing tumor growth and survival under metabolic stress. Similarly, mutations in NADP+-dependent 1 (IDH1), common in gliomas, convert α-ketoglutarate to the oncometabolite 2-hydroxyglutarate (2-HG), which disrupts epigenetic regulation and promotes tumorigenesis while altering NADPH . These mutant enzymes produce 2-HG at the expense of normal NADPH generation, fostering a pro-oncogenic metabolic environment. Therapeutically, supplementation, as a precursor to NAD+, has been shown to elevate cellular NADP pools, potentially mitigating in conditions like non-alcoholic by enhancing NADPH availability. In , where induces oxidative damage, targeting the aim to bolster NADPH-mediated regeneration, thereby protecting against complications such as nephropathy. This approach underscores NADP(H)'s role in preserving cellular balance amid pathological stress.

Recent Developments in Research

Recent studies from 2021 to 2024 have elucidated the role of mitochondrial enzymes like MESH1 (also known as HDDC3) in regulating NADP(H) , which impacts in cancer cells. MESH1 functions as a Mn²⁺-dependent that converts NADPH to NADH in the under stress conditions, thereby modulating balance essential for processes such as the folate cycle and serine synthesis. In cancer contexts, MESH1 overexpression depletes NADPH levels, elevating (ROS) and sensitizing cells to while promoting tumor progression; conversely, MESH1 knockdown induces proliferative arrest via TAZ degradation and enhances anti-tumor effects by restoring NADPH for one-carbon pathways supporting synthesis and . These findings highlight MESH1 as a potential therapeutic target, as mitochondrial NADPH generated via enzymes like MTHFD2 supports integrity in the one-carbon cycle, crucial for serine-derived carbon units in proliferating cancer cells. In , post-2020 advances have focused on engineering NADP-specific enzymes to optimize NADPH regeneration for production. A 2023 study demonstrated the relocation of NADP-specific to mitochondria, enhancing NADPH availability and boosting 3-hydroxypropionate yields—a key precursor—by integrating it into the TCA cycle for efficient cofactor . Similarly, dynamic regulation strategies for NADP(H) in biocatalysis, including immobilization and coenzyme systems using NADP-dependent oxidoreductases, have improved NADPH turnover rates in microbial hosts, enabling scalable production of like from without cofactor depletion. These engineered pathways prioritize NADP specificity to minimize crosstalk with NAD(H) pools, achieving improved productivity in fermentative processes compared to native systems. Research from 2022 onward has linked NADP decline in aging and neurodegeneration to impaired NAD kinase (NADK) activity, particularly in (AD). A 2022 analysis revealed that posttranslational modifications, such as , dysregulate NADK in aging brains, contributing to NADPH shortages that impair defenses like regeneration; this decline correlates with amyloid-β accumulation and cognitive deficits in AD patients. Recent 2024 studies further indicate that mitochondrial NADK2-dependent NADPH controls aggregation in AD models, suggesting a role in neuronal pathology. Emerging evidence suggests NADP boosters, including NADK activators or NADPH precursors, could mitigate these effects by restoring , with preclinical trials showing improved mitochondrial function and reduced in AD mouse models. Between 2020 and 2022, investigations into COVID-19 highlighted the role of NADPH oxidase (NOX)-derived ROS in driving inflammation, with NADP(H) modulation emerging as a therapeutic avenue. NOX enzymes, fueled by NADPH, generate superoxide in immune cells, amplifying cytokine storms and endothelial damage in severe cases; studies showed elevated NOX2 and NOX5 expression in COVID-19 lung tissues, correlating with ROS-mediated thrombosis and multiorgan failure. Inhibition of NOX activity reduced ROS bursts in patient-derived neutrophils, alleviating hyperinflammation without compromising antiviral responses. Furthermore, NADP(H) regeneration strategies, such as enhancing pentose phosphate pathway flux, were explored in antiviral contexts to counterbalance NOX consumption, with preliminary data indicating that NADPH supplementation mitigates oxidative lung injury in SARS-CoV-2 models.

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