Nicotinamide adenine dinucleotide (NAD+). Formal potential of the NAD+/NAD· couple and NAD· dimerization rate

The rate constant, k_d, for the dimerization of the free radical (NAD^·), produced on the initial one-electron reduction of NAD⁺, was measured by double potential-step chronoamperometry, fast-scan cyclic voltammetry (cathodic-anodic peak current ratio) and slow-scan cyclic voltammetry (peak potential shift) for a medium in which neither NAD⁺ nor its reduction products are adsorbed at the solution/electrode interface. All three methods give concordant values of k_d (approx. 3·10⁷ M^?1·s^?1), which are in reasonable accord with the values determined by pulse radiolysis but are considerably greater than values previously determined electrochemically. For the NAD⁺/NAD^· couple, the heterogeneous rate constant (k_s,h) exceeds 1 cm·s^?1 at 25°C and the formal potential (E⁰_c) vs. sce is ? 1.155 V at 25°C and ? 1.149 V at 1°C at pH 9.1, with an uncertainty of about ±0.005 V.     

Adriamycin-catalyzed aerobic photooxidation of NAD dimers to NAD+

The photooxidation of the dimers of nicotinamide adenine dinucleotide, (NAD)2, is catalyzed by adriamycin under aerobic conditions. (NAD)2 and O2 react in 1:1 molar ratio to yield 2 mol of NAD+. Experiments carried out by irradiating at 340 and 485 nm, corresponding to the absorption maxima of (NAD)2 and adriamycin, respectively, clearly indicate that the process is primed by photoexcitation of adriamycin. The key step of the process is the redox reaction between (NAD)2 and adriamycin with formation of the semiquinone radical anion, identified by the EPR spectrum. The semiquinone is then oxidized back to adriamycin by oxygen with formation of the superoxide radical.     

NAD kinases use substrate-assisted catalysis for specific recognition of NAD

Here we describe the crystal structures of the NAD kinase (LmNADK1) from Listeria monocytogenes in complex with its substrate NAD, its product NADP, or two synthesized NAD mimics. We identified one of the NAD mimics, di-adenosine diphosphate, as a new substrate for LmNADK1, whereas we showed that the closely related compound di-5'-thioadenosine is a novel non-natural inhibitor for this enzyme. These structures suggest a mechanism involving substrate-assisted catalysis. Indeed, sequence/structure comparison and directed mutagenesis have previously shown that NAD kinases (NADKs) and the distantly related 6-phosphofructokinases share the same catalytically important GGDGT motif. However, in this study we have shown that these enzymes use the central aspartate of this motif differently. Although this acidic residue chelates the catalytic Mg(2+) ion in 6-phosphofructokinases, it activates the phospho-acceptor (NAD) in NADKs. Sequence/structure comparisons suggest that the role of this aspartate would be conserved in NADKs and the related sphingosine and diacylglycerol kinases.     

Reconstructing eukaryotic NAD metabolism

In addition to its well-known role as a coenzyme in oxidation-reduction reactions, the distinct role of NAD as a precursor for molecules involved in cell regulation has been clearly established. The involvement of NAD in these regulatory processes is based on its ability to function as a donor of ADP-ribose; NAD synthesis is therefore required to avoid depletion of the intracellular pool. The rising interest in the biosynthetic routes leading to NAD formation and the highly conserved nature of the enzymes involved prompted us to reconstruct the NAD biosynthetic routes operating in distinct eukaryotic organisms. The evidence obtained from biochemical and computational analysis provides a good example of how complex metabolic pathways may evolve. In particular, it is proposed that the development of several NAD biosynthetic routes during evolution has led to partial functional redundancy, allowing a given pathway to freely acquire novel functions unrelated to NAD biosynthesis.     

NAD+ kinase--a review

NAD+ kinase catalyzes the only (known) biochemical reaction leading to the production of NADP+ from NAD+. Most evidence indicates it is found in the cytoplasm, but reports of its presence in (other) cell bodies can not be discounted. Viewed as a protein, our knowledge of NADK composition and architecture is rudimentary. Though recognized as a large multimeric protein, no agreement is evident for the molecular weight (Mr = approximately 4-65 X 10(4] of the native protein. Is calmodulin an integral subunit of (some, all) NAD+ kinases (analogous to phosphorylase kinase in skeletal muscle)? Or is it an external modulator? Consensus is evident that a subunit of molecular weight 30-35 X 10(3) is a component of the mammalian and yeast kinase. In one case (rabbit liver) two types of subunits are reported to give rise to oligomers differing in molecular weight and catalytic activities. Viewed as an enzyme it is not known why such a complex aggregate is needed for what might otherwise appear to a routine phosphorylation reaction. Rapid equilibrium random (for pigeon liver and C. utilis preparations) and ping-pong (for A. vinelandii kinase) mechanisms have been proposed for the reaction, with multiple reactant binding sites indicated for the random cases. From the perspective of enzyme modulation, the demonstration that green plant and sea urchin egg kinases are targets for calmodulin regulation by intracellular Ca2+ links NADP+ production in these sources to the multi-level discriminatory control functions inherent to this Ca2+-protein complex. Significant questions arise from the results of various investigators considered in this review. These queries offer fertile ground for the selective design of key experiments directed to a better understanding of NAD+ kinase function and pyridine nucleotide biochemistry.     

Probing binding requirements of NAD kinase with modified substrate (NAD) analogues

Synthesis of novel NAD(+) analogues that cannot be phosphorylated by NAD kinase is reported. In these analogues the C2' hydroxyl group of the adenosine moiety was replaced by fluorine in the ribo or arabino configuration (1 and 2, respectively) or was inverted into arabino configuration to give compound 3. Compounds 1 and 2 showed inhibition of human NAD kinase, whereas analogue 3 inhibited both the human and Mycobacterium tuberculosis NAD kinase. An uncharged benzamide adenine dinucleotide (BAD) was found to be the most potent competitive inhibitor (K(i)=90 microM) of the human enzyme reported so far.     

Reactivation of NAD(H) biosynthetic pathway by exogenous NAD+ in Nil cells severely depleted of NAD(H)

The culture of Nil hamster fibroblasts in MEM lacking nicotinamide (NAm-MEM) leads to: (1) the rapid loss of intracellular total nicotinamide adenine dinucleotide (NAD(H)) content in these cells from a level of 150-200 pmoles/10(5) cells to less than 20 pmoles/10(5) cells; (2) the cessation of cell division and inhibition of DNA synthesis; and (3) a reduction of glucose consumption and lactic acid production. In most situations, following nicotinamide starvation, the restoration of intracellular NAD(H) follows rapidly the readdition of NAD+ (oxidized), nicotinamide mononucleotide (NMN), nicotinamide, or nicotinic acid. Resumption of cell division occurs after only a lag of about 24 hours. Nil cells subcultured for three consecutive times in the absence of nicotinamide (3(0) NAm- cells) exhibit different behavior. These severely starved cells are incapable of quickly restoring their intracellular NAD(H) content to normal levels when provided with any pyridine ring compound except NAD+. One-hour exposure of such cells to NAD+ allows utilization of nicotinamide to rapidly restore intracellular NAD(H). This short incubation with NAD+ does not result in any significant restoration of intracellular NAD(H) or lead to the accumulation of an intracellular pool of some precursor. This function of NAD+ as a stimulatory signal to the NAD(H)-biosynthetic pathway in severely starved Nil cells is a previously unreported role of NAD+, and does not require protein synthesis.     

NAD glucohydrolase from bovine retina

The NAD glycohydrolase activity in the retina is very low compared with the one found in the brain. Therefore the retina extracts ahve a sufficiently high NAD level so that they are able to form "in vitro" lactate from glucose in anaerobiosis in presence of only ATP, Mg++ and glucose. The NAD glycohydrolase has been found in the retina in a great extent in the microsomes.     

Evolution of NAD biosynthetic enzymes

Two research groups have solved crystal structures of nicotinic acid phosphoribosyltransferase (PRTase) and made the argument that PRTases in three distinct pathways of nicotinamide adenine dinucleotide (NAD) biosynthesis evolved from a common ancestor (Shin et al., 2005 and Chappie et al., 2005).     

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.     

Manipulation of a nuclear NAD+ salvage pathway delays aging without altering steady-state NAD+ levels

Yeast deprived of nutrients exhibit a marked life span extension that requires the activity of the NAD(+)-dependent histone deacetylase, Sir2p. Here we show that increased dosage of NPT1, encoding a nicotinate phosphoribosyltransferase critical for the NAD(+) salvage pathway, increases Sir2-dependent silencing, stabilizes the rDNA locus, and extends yeast replicative life span by up to 60%. Both NPT1 and SIR2 provide resistance against heat shock, demonstrating that these genes act in a more general manner to promote cell survival. We show that Npt1 and a previously uncharacterized salvage pathway enzyme, Nma2, are both concentrated in the nucleus, indicating that a significant amount of NAD(+) is regenerated in this organelle. Additional copies of the salvage pathway genes, PNC1, NMA1, and NMA2, increase telomeric and rDNA silencing, implying that multiple steps affect the rate of the pathway. Although SIR2-dependent processes are enhanced by additional NPT1, steady-state NAD(+) levels and NAD(+)/NADH ratios remain unaltered. This finding suggests that yeast life span extension may be facilitated by an increase in the availability of NAD(+) to Sir2, although not through a simple increase in steady-state levels. We propose a model in which increased flux through the NAD(+) salvage pathway is responsible for the Sir2-dependent extension of life span.