The Effect of Antioxidant Supplementation on Fatigue during Exercise: Potential Role for NAD+(H)

This study compared serum pyridine levels (NAD+ /NADH) in trained (n = 6) and untrained (n = 7) subjects after continuous progressive exercise at 50%, 70% then 95% of physical work capacity until fatigue (TTF) after consuming a placebo or antioxidant (AO) cocktail (Lactaway©). An increase of 17% in TTF was observed in AO as compared to placebo (p = 0.032). This was accompanied by a significant increase in serum NAD+ levels (p = 0.037) in the AO supplemented group post exercise. The increases in NAD+ and improved endurance reflect lower oxidative stress-induced suppression of aerobic respiration

Influence of naturally-occurring 5′-pyrophosphate-linked substituents on the binding of adenylic inhibitors to ribonuclease a: An X-ray crystallographic study

Ribonuclease A is the archetype of a functionally diverse superfamily of vertebrate-specific ribonucleases. Inhibitors of its action have potential use in the elucidation of the in vivo roles of these enzymes and in the treatment of pathologies associated therewith. Derivatives of adenosine 5′-pyrophosphate are the most potent nucleotide-based inhibitors known. Here, we use X-ray crystallography to visualize the binding of four naturally-occurring derivatives that contain 5′-pyrophosphate-linked extensions. 5′-ATP binds with the adenine occupying the B2 subsite in the manner of an RNA substrate but with the γ-phosphate at the P1 subsite. Diadenosine triphosphate (Ap3A) binds with the adenine in syn conformation, the β-phosphate as the principal P1 subsite ligand and without order beyond the γ-phosphate. NADPH and NADP+ bind with the adenine stacked against an alternative rotamer of His119, the 2′-phosphate at the P1 subsite, and without order beyond the 5′-α-phosphate. We also present the structure of the complex formed with pyrophosphate ion. The structural data enable existing kinetic data on the binding of these compounds to a variety of ribonucleases to be rationalized and suggest that as the complexity of the 5′-linked extension increases, the need to avoid unfavorable contacts places limitations on the number of possible binding modes. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 995–1008, 2009

Fine Tuning of the Lactate and Diacetyl Production through Promoter Engineering in Lactococcus lactis

Lactococcus lactis is a well-studied bacterium widely used in dairy fermentation and capable of producing metabolites with organoleptic and nutritional characteristics. For fine tuning of the distribution of glycolytic flux at the pyruvate branch from lactate to diacetyl and balancing the production of the two metabolites under aerobic conditions, a constitutive promoter library was constructed by randomizing the promoter sequence of the H2O-forming NADH oxidase gene in L. lactis. The library consisted of 30 promoters covering a wide range of activities from 7,000 to 380,000 relative fluorescence units using a green fluorescent protein as reporter. Eleven typical promoters of the library were selected for the constitutive expression of the H2O-forming NADH oxidase gene in L. lactis, and the NADH oxidase activity increased from 9.43 to 58.17-fold of the wild-type strain in small steps of activity change under aerobic conditions. Meanwhile, the lactate yield decreased from 21.15±0.08 mM to 9.94±0.07 mM, and the corresponding diacetyl production increased from 1.07±0.03 mM to 4.16±0.06 mM with the intracellular NADH/NAD+ ratios varying from 0.711±0.005 to 0.383±0.003. The results indicated that the reduced pyruvate to lactate flux was rerouted to the diacetyl with an almost linear flux variation via altered NADH/NAD+ ratios. Therefore, we provided a novel strategy to precisely control the pyruvate distribution for fine tuning of the lactate and diacetyl production through promoter engineering in L. lactis. Interestingly, the increased H2O-forming NADH oxidase activity led to 76.95% lower H2O2 concentration in the recombinant strain than that of the wild-type strain after 24 h of aerated cultivation. The viable cells were significantly elevated by four orders of magnitude within 28 days of storage at 4°C, suggesting that the increased enzyme activity could eliminate H2O2 accumulation and prolong cell survival

Age Related Changes in NAD+ Metabolism Oxidative Stress and Sirt1 Activity in Wistar Rats

The cofactor nicotinamide adenine dinucleotide (NAD+) has emerged as a key regulator of metabolism, stress resistance and longevity. Apart from its role as an important redox carrier, NAD+ also serves as the sole substrate for NAD-dependent enzymes, including poly(ADP-ribose) polymerase (PARP), an important DNA nick sensor, and NAD-dependent histone deacetylases, Sirtuins which play an important role in a wide variety of processes, including senescence, apoptosis, differentiation, and aging. We examined the effect of aging on intracellular NAD+ metabolism in the whole heart, lung, liver and kidney of female wistar rats. Our results are the first to show a significant decline in intracellular NAD+ levels and NAD∶NADH ratio in all organs by middle age (i.e.12 months) compared to young (i.e. 3 month old) rats. These changes in [NAD(H)] occurred in parallel with an increase in lipid peroxidation and protein carbonyls (o- and m- tyrosine) formation and decline in total antioxidant capacity in these organs. An age dependent increase in DNA damage (phosphorylated H2AX) was also observed in these same organs. Decreased Sirt1 activity and increased acetylated p53 were observed in organ tissues in parallel with the drop in NAD+ and moderate over-expression of Sirt1 protein. Reduced mitochondrial activity of complex I–IV was also observed in aging animals, impacting both redox status and ATP production. The strong positive correlation observed between DNA damage associated NAD+ depletion and Sirt1 activity suggests that adequate NAD+ concentrations may be an important longevity assurance factor

The protonmotive force is required for maintaining ATP homeostasis and viability of hypoxic, nonreplicating Mycobacterium tuberculosis

The persistence of Mycobacterium tuberculosis despite prolonged chemotherapy represents a major obstacle for the control of tuberculosis. The mechanisms used by Mtb to persist in a quiescent state are largely unknown. Chemical genetic and genetic approaches were used here to study the physiology of hypoxic nonreplicating mycobacteria. We found that the intracellular concentration of ATP is five to six times lower in hypoxic nonreplicating Mtb cells compared with aerobic replicating bacteria, making them exquisitely sensitive to any further depletion. We show that de novo ATP synthesis is essential for the viability of hypoxic nonreplicating mycobacteria, requiring the cytoplasmic membrane to be fully energized. In addition, the anaerobic electron transport chain was demonstrated to be necessary for the generation of the protonmotive force. Surprisingly, the alternate ndh-2, but not -1, was shown to be the electron donor to the electron transport chain and to be essential to replenish the [NAD+] pool in hypoxic nonreplicating Mtb. Finally, we describe here the high bactericidal activity of the F0F1 ATP synthase inhibitor R207910 on hypoxic nonreplicating bacteria, supporting the potential of this drug candidate for shortening the time of tuberculosis therapy