Oxidation of excited-state NADH and NAD dimer in aqueous medium involvement of O2− as a mediator in the presence of oxygen

It has been shown that direct excitation of NADH (or NADPH) in aqueous medium at 254 nm, or at wavelengths longer than 320 nm (where only the reduced nicotinamide moiety absorbs), leads to generation of NAD⁺ (or NADP⁺). The reaction proceeds both in the presence and absence of oxygen. Under aerobic conditions the reaction is accompanied by formation of H₂O₂ at a level equimolar with that of the NADH present in solution. On irradiation at wavelengths longer than 320 nm, conversion of NADH to enzymatically active NAD⁺ is about 75%. Under analogous irradiation conditions, the dimers (NAD)₂ and (NADP)₂ undergo disproportionation to NAD⁺ and NADP⁺, respectively, to the extent of 90%. Both physicochemical and enzymatic criteria were employed to formulate mechanisms for the photooxidation of NADH and the photodisproportionation of the dimer (NAD)₂.     

Photocatalyzed oxidation of NAD dimers

A mixture of dimers of nicotinamide adenine dinucleotide, largely 4,4?-linked, obtained by electrochemical reduction of NAD⁺, can be photooxidized back to NAD⁺ in the presence of oxygen. Oxygen is consumed during the photooxidation process with the production of hydrogen peroxide. The oxidation is almost pH independent and is stimulated by light whose wavelength exceeds 300 nm. Lactate dehydrogenase and alcohol dehydrogenase added to the solutions under irradiation increased the oxygen uptake by the NAD dimers in a concentration-dependent way. These observations suggest that light induces the homolytic cleavage of NAD dimers to NAD radicals which in turn are oxidized to NAD⁺ by oxygen.     

Adaptive changes in NAD+ metabolism in ultraviolet light-irradiated murine lymphoma cells

We have determined the ability of UV_254nm-irradiated murine lymphoma cells to adapt their NAD⁺ metabolism to the increased NAD⁺ consumption for the poly ADP-ribosylation of chromatin proteins. Two murine lymphoma sublines with differential UV-sensitivity and poly(ADP-ribose) turnover were used as a model system. The first subline, designated LY-R is UV_254nm-sensitive and tumorigenic in DBA/2 mice. The second subline, LY-S is UV_254nm-resistant and nontumorigenic. Following treatment of these cells with 2 mM benzamide, an inhibitor of the NAD⁺-utilizing enzyme poly(ADP-ribose) polymerase, NAD⁺ levels slowly increased up to about 160% of control levels after 3 hours. When benzamide was added to these cultures 20 min after UV_254nm irradiation, a dramatic transient increase of NAD⁺ levels was observed within 4 min in LY-R cells and more moderately in LY-S cells. At later times after UV_254nm irradiation, the NAD⁺ levels increased in both sublines reaching up to 200% of the concentrations prior to benzamide treatment. These results demonstrate an adaptative response of NAD⁺ metabolism to UV_254nm irradiation. In parallel, we observed a differential repartitioning of ADP-ribosyl residues between the NAD⁺ and poly(ADP-ribose) pools of LY-R and LY-S cells that correlates with the differential UV sensitivity of these cells.     

Photochemical and enzymatic synthesis of methanol from formaldehyde with alcohol dehydrogenase from Saccharomyces cerevisiae and water-soluble zinc porphyrin

We evaluated the photochemical and enzymatic synthesis of methanol from formaldehyde with alcohol dehydrogenase (ADH) from Saccharomyces cerevisiae and NAD⁺ photoreduction by the visible-light sensitization of zinc tetraphenylporphyrin tetrasulfonate (ZnTPPS) in the presence of methylviologen (MV²⁺), diaphorase, and triethanolamine (TEOA). When the sample solution containing ZnTPPS, MV²⁺, NAD⁺, diaphorase, and TEOA in potassium phosphate buffer solution was irradiated, the NADH produced increased with the irradiation time. After irradiation for 180 min, the conversion yield of NAD⁺ to NADH was about 60% under 0.1 mM NAD⁺ condition. The methanol production also depended on the conversion yield of NAD⁺ to NADH. After irradiation for 180 min, 0.38 μM of methanol was produced from formaldehyde (16 μM). The conversion ratio of formaldehyde to methanol was about 2.3%. This result indicates that a system for the photochemical synthesis of methanol from formaldehyde was developed with ADH and the NADH produced by the photosensitization of ZnTPPS in water media.     

Subcellular Distribution of NAD+ between Cytosol and Mitochondria Determines the Metabolic Profile of Human Cells

The mitochondrial NAD pool is particularly important for the maintenance of vital cellular functions. Although at least in some fungi and plants, mitochondrial NAD is imported from the cytosol by carrier proteins, in mammals, the mechanism of how this organellar pool is generated has remained obscure. A transporter mediating NAD import into mammalian mitochondria has not been identified. In contrast, human recombinant NMNAT3 localizes to the mitochondrial matrix and is able to catalyze NAD⁺ biosynthesis in vitro. However, whether the endogenous NMNAT3 protein is functionally effective at generating NAD⁺ in mitochondria of intact human cells still remains to be demonstrated. To modulate mitochondrial NAD⁺ content, we have expressed plant and yeast mitochondrial NAD⁺ carriers in human cells and observed a profound increase in mitochondrial NAD⁺. None of the closest human homologs of these carriers had any detectable effect on mitochondrial NAD⁺ content. Surprisingly, constitutive redistribution of NAD⁺ from the cytosol to the mitochondria by stable expression of the Arabidopsis thaliana mitochondrial NAD⁺ transporter NDT2 in HEK293 cells resulted in dramatic growth retardation and a metabolic shift from oxidative phosphorylation to glycolysis, despite the elevated mitochondrial NAD⁺ levels. These results suggest that a mitochondrial NAD⁺ transporter, similar to the known one from A. thaliana, is likely absent and could even be harmful in human cells. We provide further support for the alternative possibility, namely intramitochondrial NAD⁺ synthesis, by demonstrating the presence of endogenous NMNAT3 in the mitochondria of human cells.     

Transport of NAD in Percoll-Purified Potato Tuber Mitochondria: Inhibition of NAD Influx and Efflux by N-4-Azido-2-nitrophenyl-4-aminobutyryl-3'-NAD

A mechanism by which intact potato (Solanum tuberosum) mitochondria may regulate the matrix NAD content was studied in vitro. If mitochondria were incubated with NAD⁺ at 25°C in 0.3 molar mannitol, 10 millimolar phosphate buffer (pH 7.4), 5 millimolar MgCl₂, and 5 millimolar α-ketoglutarate, the NAD pool size increased with time. In the presence of uncouplers, net uptake was not only inhibited, but NAD⁺ efflux was observed instead. Furthermore, the rate of NAD⁺ accumulation in the matrix space was strongly inhibited by the analog N-4-azido-2-nitrophenyl-4-aminobutyryl-3′-NAD⁺. When suspended in a medium that avoided rupture of the outer membrane, intact purified mitochondria progressively lost their NAD⁺ content. This led to a slow decrease of NAD⁺-linked substrates oxidation by isolated mitochondria The rate of NAD⁺ efflux from the matrix space was strongly temperature dependent and was inhibited by the analog inhibitor of NAD⁺ transport indicating that a carrier was required for net flux in either direction. It is proposed that uptake and efflux operate to regulate the total matrix NAD pool size.     

Kinetic properties of N-(2-carboxyethyl)-NAD(H) and poly(ethylene glycol)-bound NAD(H) for alcohol, lactate, malate and glyceraldehyde-3-phosphate dehydrogenase from different organisms

The steady-state kinetics of alcohol dehydrogenases (alcohol:NAD⁺ oxidoreductase, EC 1.1.1.1 and alcohol:NADP⁺ oxidoreductase, EC 1.1.1.2), lactate dehydrogenases (l-lactate:NAD⁺ oxidoreductase, EC 1.1.1.27 and d-lactate:NAD⁺ oxidoreductase, EC 1.1.1.28), malate dehydrogenase (l-malate:NAD⁺ oxidoreductase, EC 1.1.1.37), and glyceraldehyde-3-phosphate dehydrogenases [d-glyceraldehyde-3-phosphate:NAD⁺ oxidoreductase (phosphorylating), EC 1.2.1.12] from different sources (prokaryote and eukaryote, mesophilic and thermophilic organisms) have been studied using NAD(H), N⁶-(2-carboxyethyl)-NAD(H), and poly(ethylene glycol)-bound NAD(H) as coenzymes. The kinetic constants for NAD(H) were changed by carboxyethylation of the 6-amino group of the adenine ring and by conversion to macromolecular form. Enzymes from thermophilic bacteria showed especially high activities for the derivatives. The relative values of the maximum velocity (NAD = 1) of Thermus thermophilus malate dehydrogenase for N⁶-(2-carboxyethyl)-NAD and poly(ethylene glycol)-bound NAD were 5.7 and 1.9, respectively, and that of Bacillus stearothermophilus glyceraldehyde-3-phosphate dehydrogenase for poly(ethylene glycol)-bound NAD was 1.9.     

Analysis of calorimetric data for isodesmic self-associating systems.

ATP and respiration (NADH)-driven NAD(P)⁺ transhydrogenase (EC 1.6.1.1) activities are low in membranes from Escherichia coli cultured on yeast extract medium (17 and 21 nmol/min × mg) but high on glucose (82 and 142 nmol/min × mg). The ATPase and respiratory activities in both cases appeared comparable. Growth of the bacteria in yeast extract medium followed by washing and replacement into a glucose medium showed that after 3 h the energy-linked and energy-independent NAD(P)⁺ transhydrogenase (reduction of acetylpyridine NAD⁺ by NADPH) activities had appeared simultaneously. Incorporation of chloramphenicol or omission of glucose in the induction medium resulted in no increase in these activities indicating that de novo protein synthesis is required for the induction of energy-linked and -independent NAD(P)⁺ transhydrogenase. It was found that the K_m values for acetylpyridine NAD⁺ and NADPH for the energy-independent reaction in membranes from glucose grown cells (143 and 62 μm) were similar to those in membranes from cells grown on glucose-yeast extract (135 and 45 μm), respectively, but the maximum velocity at infinite acetyl pyridine NAD⁺ and NADPH increased from 353 to 2175 nmol/min × mg. Furthermore, the membrane-bound NAD(P)⁺ transhydrogenase in glucose-yeast extract grown cells showed substrate inhibition at high NADPH and low acetyl pyridine NAD⁺ levels. Further kinetic data demonstrate that the mechanism of the energy-independent NAD(P)⁺ transhydrogenase in E. coli is similar to that of the mitochondrial enzyme and exhibits similar responses to competitive inhibitors at the NAD⁺ and NADPH sites.     

Effects of overexpression of NAPRTase,NAMNAT, and NAD synthetase in the NAD(H) biosynthetic pathways on the NAD(H) pool,NADH/NAD+ ratio,and succinic acid production with different carbon sources by metabolically engineered Escherichia coli

Escherichia coli BA002, the ldhA and pflB deletion strain, cannot utilize glucose anaerobically due to the inability to regenerate NAD⁺. To regulate NAD(H) pool size and NADH/NAD⁺ ratio, overexpression of the enzymes in the NAD(H) biosynthetic pathways in BA002 was investigated. The results clearly demonstrate that the increased NAD(H) pool size and the decreased NADH/NAD⁺ ratio improved the glucose consumption and cell growth, which improved succinic acid production. When the pncB and the nadD genes were co-overexpressed in CA102, the ratio of NADH/NAD⁺ was decreased from 0.60 to 0.12, and the concentration of NAD(H) was the highest among that of all the strains. Moreover, the dry cell weight (DCW), glucose consumption, and the concentration of succinic acid in CA102 were also the highest. Based on the sufficient NAD⁺ supply after gene modification in the NAD(H) biosynthetic pathways, reductive carbon sources with different amounts of NADH can further change the distribution of metabolites. When sorbitol was used as a carbon source in CA102, the byproducts were lower than those of glucose fermentation, and the yield of succinic acid was increased.     

Sex-related differences in human plasma NAD+/NADH levels depend on age

Nicotinamide adenine dinucleotide (NAD) is a coenzyme in metabolic reactions and cosubstrate in signaling pathways of cells. While the intracellular function of NAD is well described, much less is known about its importance as an extracellular molecule. Moreover, there is only little information about the concentration of extracellular NAD and the ratio between its oxidized (NAD⁺) and reduced (NADH) form in humans. Therefore, our study aimed at the analysis of total NAD and NAD⁺/NADH ratio in human plasma depending on sex and age. First, an enzymatic assay was established for detecting NAD⁺ and NADH in human plasma samples. Then, plasma NAD was analyzed in 205 probands without severe diseases (91 men, 114 women) being 18–83 years old. The total plasma NAD concentration was determined with median 1.34 µM (0.44–2.88 µM) without difference between men and women. Although the amounts of NAD⁺ and NADH were nearly balanced, women had higher plasma NAD⁺/NADH ratios than men (median 1.33 vs. 1.09, P<0.001). The sex-related difference in the plasma NAD⁺/NADH ratio reduces with increasing age, an effect that was more obvious for two parameters of the biological age (skin autofluorescence, brachial-femoral pulse wave velocity (PWV)) than for the chronological age. However, plasma values for total NAD and NAD⁺/NADH ratio did not generally alter with increasing age. In conclusion, human plasma contains low micromolar concentrations of total NAD with higher NAD⁺/NADH redox ratios in adult but not older women compared with same-aged men.     

Energetics of the one-electron steps in the NAD+/NADH redox couple

The one-electron reduction potential of NAD⁺ has been determined using pulse radiolysis to study electron-transfer equilibria between it and a low potential bipyridylium compound. The determined value E¹₇ (NAD⁺/NAD^.) = ?922 ± 8 mV (NHE scale) is used to calculate E²₇ (NAD^./NADH) = +282 mV. E¹₇ for 1-methylnicotinamide, E¹₇ (MeN⁺/MeN^.) = ?918 ± 7 mV.     

Improving the production of NAD+ via multi-strategy metabolic engineering in Escherichia coli

Nicotinamide adenine dinucleotide (NAD⁺) is an essential coenzyme involved in numerous physiological processes. As an attractive product in the industrial field, NAD⁺ also plays an important role in oxidoreductase-catalyzed reactions, drug synthesis, and the treatment of diseases, such as dementia, diabetes, and vascular dysfunction. Currently, although the biotechnology to construct NAD⁺-overproducing strains has been developed, limited regulation and low productivity still hamper its use on large scales. Here, we describe multi-strategy metabolic engineering to address the NAD⁺-production bottleneck in E. coli. First, blocking the degradation pathway of NAD(H) increased the accumulation of NAD⁺ by 39%. Second, key enzymes involved in the Preiss-Handler pathway of NAD⁺ synthesis were overexpressed and led to a 221% increase in the NAD⁺ concentration. Third, the PRPP synthesis module and Preiss-Handler pathway were combined to strengthen the precursors supply, which resulted in enhancement of NAD⁺ content by 520%. Fourth, increasing the ATP content led to an increase in the concentration of NAD⁺ by 170%. Finally, with the combination of all above strategies, a strain with a high yield of NAD⁺ was constructed, with the intracellular NAD⁺ concentration reaching 26.9 μmol/g DCW, which was 834% that of the parent strain. This study presents an efficient design of an NAD⁺-producing strain through global regulation metabolic engineering.     

Detection of early unfolding events in a dimeric protein by amide proton exchange and native electrospray mass spectrometry

Oligomeric proteins generally undergo unfolding through a dissociation/denaturation mechanism wherein the subunits first dissociate and then unfold. This mechanism can be detected by the fact that the proteins exhibit a concentration dependence of the denaturation curve. However, the concentration dependence does not answer the question of whether there are thermally induced conformational changes that facilitate subunit dissociation. To fully probe these mechanisms it is desirable to have an analytical approach that is capable of measuring both subunit dissociation and protein denaturation in a highly sensitive manner. In this article, we demonstrate that the combined use of native mass spectrometry to detect subunit mixing, and amide hydrogen/deuterium exchange to detect transient unfolding events can provide a very unique insight into the pre‐melting transitions in a protein oligomer. Both methods keep an isotopic record of each transformation event, without the dependence on equilibrium of the unfolding reaction. Here, we use a combined form of H/D exchange/mass spectrometry and isotopic labeling/native electrospray mass spectrometry to study the pre‐unfolding events of Bacillus subtilis NAD⁺ synthetase, a symmetrical dimer protein, which plays a vital role in the lifecycle of the bacteria. In the experimental outcome provided, we were able to clearly illustrate that at elevated temperatures, the NAD synthetase dimer undergoes reversible dissociation without monomer unfolding, while at temperatures where monomer unfolding is observed to take place, the rate of dimer dissociation still yet exceeds the rate of unfolding. Information provided by combining these two mass spectrometric methods was found to be very robust, and allowed us to establish an NAD synthetase unfolding model, where primary dissociation occurs prior to the complete unfolding of the NAD⁺ synthetase.     

Dual emission fluorescent silver nanoclusters for sensitive detection of the biological coenzyme NAD+/NADH

Fluorescent silver nanoclusters (Ag NCs) displaying dual-excitation and dual-emission properties have been developed for the specific detection of NAD⁺ (nicotinamide adenine dinucleotide, oxidized form). With the increase of NAD⁺ concentrations, the longer wavelength emission (with the peak at 550 nm) was gradually quenched due to the strong interactions between the NAD⁺ and Ag NCs, whereas the shorter wavelength emission (peaking at 395 nm) was linearly enhanced. More important, the dual-emission intensity ratio (I₃₉₅/I₅₅₀), fitting by a single-exponential decay function, can efficiently detect various NAD⁺ levels from 100 to 4000 μM, as well as label NAD⁺/NADH (reduced form of NAD) ratios in the range of 1–50.     

Activity of liver mitochondrial Krebs cycle NAD<Superscript>+</Superscript>-dependent dehydrogenases in rats with hepatitis induced by acetaminophen under conditions of alimentary protein deficiency

Activity of isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, malate dehydrogenase, and the NAD⁺/NADН ratio were studied in the liver mitochondrial fraction of rats with toxic hepatitis induced by acetaminophen under conditions of alimentary protein deficiency. Acetaminophen-induced hepatitis was characterized by a decrease of isocitrate dehydrogenase, α-ketoglutarate dehydrogenase and malate dehydrogenase activities, while the mitochondrial NAD⁺/NADН ratio remained at the control level. Modeling of acetaminophen-induced hepatitis in rats with alimentary protein deficiency caused a more pronounced decrease in the activity of studied Krebs cycle NAD⁺-dependent dehydrogenases and a 2.2-fold increase of the mitochondrial NAD⁺/NADН ratio.     

Soluble and chloroplast malate dehydrogenase isoenzymes of Triticum aestivum

Three isoenzymes of malate dehydrogenase have been isolated from 9-day-old wheat shoots. The microbody (peroxisome) and chloroplast MDH are similar in their electrophoretic behaviour. The mitochondrial MDH, soluble MDH and chloroplast MDH differ in K_m values for malate and NAD. The activity of MDH isoenzymes with NAD⁺-analogues as substrate was in the order 3-AP-NAD⁺ > 3-AP-deam NAD⁺ > NAD⁺ > TN-NAD⁺ and deam NAD⁺. The thermal stabilities of the isoenzymes were significantly different: C-MDH > m-MDH > S-MDH.     

Kinetic properties of NAD malic enzyme from cauliflower

The kinetic characteristics of NAD malic enzyme purified to homogeneity from cauliflower florets have been examined. Free NAD⁺ is the active form of this coenzyme. Double-reciprocal plots of data obtained by varying NAD⁺ and malate^2? at a saturating concentration of Mg²⁺ or by varying Mg²⁺ and NAD⁺ at a saturating level of malate^2? are of intersecting type. This indicates that NAD malic enzyme obeys a sequential mechanism. Analysis of these sets of data suggests that each of these substrate pairs binds randomly to the enzyme. However, each substrate binds tighter when others are already present on the enzyme. NAD malic enzyme cannot decarboxylate malate^2? in the absence of either Mg²⁺ or NAD⁺. Arrhenius plots of the NAD-linked reaction are concave downward, indicating the existence of two rate-determining steps with activation energies of 26.5 and 14.2 kcal/mol, respectively. In addition to Mg²⁺, the enzyme can also use Mn²⁺ and Co²⁺. Using Co²⁺ in place of Mg²⁺ does not change V_max or K_m,malate^2? but the K_m for metal and NAD⁺ are greatly decreased. At pH 7.0 and above, Mn²⁺ isotherms and malate^2? curves with Mn²⁺ are nonlinear and appear to be composed of two separate saturation curves. NAD malic enzyme is completely and irreversibly inactivated by N-ethylmaleimide. The enzyme is also irreversibly inactivated approximately 50% by KCNO.     

Genesis of Drosophila ADH: the shaping of the enzymatic activity from a SDR ancestor

Drosophila alcohol dehydrogenase (ADH) is an NAD(H)-dependent oxidoreductase that catalyzes the oxidation of alcohols and aldehydes. Structurally and biochemically distinct from all the reported ADHs (typically, the mammalian medium-chain dehydrogenase/reductase–ethanol-metabolizing enzyme), it stands as the only small-alcohol transforming system that has originated from a short-chain dehydrogenase/reductase (SDR) ancestor. The crystal structures of the apo, binary (E·NAD⁺) and three ternary (E·NAD⁺·acetone, E·NAD⁺·3-pentanone and E·NAD⁺·cyclohexanone) forms of Drosophila lebanonensis ADH have allowed us to infer the structural and kinetic features accounting for the generation of the ADH activity within the SDR lineage.