Oxidative Stress Evokes a Metabolic Adaptation That Favors Increased NADPH Synthesis and Decreased NADH Production in Pseudomonas fluorescens

Abstract

The fate of all aerobic organisms is dependent on the varying intracellular concentrations of NADH and NADPH. The former is the primary ingredient that fuels ATP production via oxidative phosphorylation, while the latter helps maintain the reductive environment necessary for this process and other cellular activities. In this study we demonstrate a metabolic network promoting NADPH production and limiting NADH synthesis as a consequence of an oxidative insult. The activity and expression of glucose-6-phosphate dehydrogenase, malic enzyme, and NADP ؉ -isocitrate dehydrogenase, the main generators of NADPH, were markedly increased during oxidative challenge. On the other hand, numerous tricarboxylic acid cycle enzymes that supply the bulk of intracellular NADH were significantly downregulated. These metabolic pathways were further modulated by NAD ؉ kinase (NADK) and NADP ؉ phosphatase (NADPase), enzymes known to regulate the levels of NAD ؉ and NADP ؉ . While in menadione-challenged cells, the former enzyme was upregulated, the phosphatase activity was markedly increased in control cells. Thus, NADK and NADPase play a pivotal role in controlling the cross talk between metabolic networks that produce NADH and NADPH and are integral components of the mechanism involved in fending off oxidative stress.If an aerobic organism is to survive, it is essential that an adequate supply of NADPH is available. This nicotinamide nucleotide provides a reductive environment that enables the oxidative cell to nullify the reactive oxygen species (ROS) generated as a consequence of oxidative phosphorylation, a process key to the generation of ATP (9,18,22). All organisms that utilize oxygen as the terminal e Ϫ acceptor have evolved intricate molecular strategies that allow them to combat the inherent dangers associated with living in an aerobic environment (11,26). Catalase, superoxide dismutase (SOD), and glutathione peroxidase are some of the enzymes that help decrease oxidative tension during aerobic respiration (4). However, the effectiveness of these proteins as the scavengers of ROS depends on the availability of NADPH. This nucleotide supplies the reductive power necessary to quell the oxidative potential of ROS. Hence, the production of this reducing agent is an integral part of the oxidative energy-generating machinery of all aerobic organisms. Production of ATP via oxidative phosphorylation cannot proceed effectively in the absence of a continual supply of NADPH (14, 31).Glucose-6-phosphate dehydrogenase (G6PDH), NADP ϩ -isocitrate dehydrogenase (ICDH-NADP ϩ ), malic enzyme (ME), 6-phosphogluconate dehydrogenase (6PGDH), and glutamate dehydrogenase-NADP ϩ (GDH-NADP ϩ ) are some of the important enzymes that enable aerobic cells to fulfill their requirement for NADPH (34). NADH, which is generated essentially during the catabolism of acetyl-coenzyme A via the tricarboxylic acid (TCA) cycle, is a potent prooxidant as its downstream metabolism mediated by complexes I, III, and IV produces the majority of the ROS generated in...

Role of Glyoxylate Shunt in Oxidative Stress Response

“…2A). The level of NADH in PQ-treated WT cells dropped markedly; a similar observation has been made after antibiotic treatment (29). Our data suggested that WT cells experienced increased oxidative stress when treated with PQ, whereas mutants did not.…”

“…The NADH/NAD ϩ ratio under PQ conditions was lower in the WT, likely due to the fact that the glyoxylate shunt bypasses two NADH-generating steps. Cells show an increased NADH/NAD ϩ ratio when NADH oxidation is slowed by limitation of electron acceptors (29). The addition of PQ did not alter the NADH/NAD ϩ ratio in the aceA mutants, indicating that respiration in these cells was maintained, perhaps through a combination of pathways; lower oxygen consumption and higher denitrification-related events were observed (Fig.…”

H<sub>2</sub>O<sub>2</sub> modulates the energetic metabolism of the cloud microbiome

“…Second, H 2 O 2 could impact different metabolic pathways which are interconnected, including glutathione metabolism, glycolysis, the TCA cycle, and the DNA repair system. The functioning of the enzymes in these pathways and also the activity of the ATP synthase are dependent on the redox potential of the cells (NAD + /NADH, NADP + /NADPH ratios), and as a consequence the ATP concentration is regulated by this redox potential (Haddock and Jones, 1977;Singh et al, 2007;Oka et al, 2012). If for instance NAD + is depleted when the repair system is activated to avoid potential DNA damages induced by H 2 O 2 , then ATP is depleted, and finally all the metabolic pathways involving these compounds are impacted and a complete change in the metabolome can be expected.…”

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