The tricarboxylic acid (TCA) cycle is an essential metabolic network in all oxidative organisms and provides precursors for anabolic processes and reducing factors (NADH and FADH2) that drive the generation of energy. Here, we show that this metabolic network is also an integral part of the oxidative defence machinery in living organisms and α-ketoglutarate (KG) is a key participant in the detoxification of reactive oxygen species (ROS). Its utilization as an anti-oxidant can effectively diminish ROS and curtail the formation of NADH, a situation that further impedes the release of ROS via oxidative phosphorylation. Thus, the increased production of KG mediated by NADP-dependent isocitrate dehydrogenase (NADP-ICDH) and its decreased utilization via the TCA cycle confer a unique strategy to modulate the cellular redox environment. Activities of α-ketoglutarate dehydrogenase (KGDH), NAD-dependent isocitrate dehydrogenase (NAD-ICDH), and succinate dehydrogenase (SDH) were sharply diminished in the cellular systems exposed to conditions conducive to oxidative stress. These findings uncover an intricate link between TCA cycle and ROS homeostasis and may help explain the ineffective TCA cycle that characterizes various pathological conditions and ageing.

Although aluminum is known to be toxic to most organisms, its precise biochemical interactions are not fully understood. In the present study, we demonstrate that aluminum promotes the inhibition of aconitase (Acn) activity via the perturbation of the Fe-S cluster in Pseudomonas fluorescens. Despite the significant decrease in citrate isomerization activity, cellular survival is assured by the overexpression of isocitrate lyase and isocitrate dehydrogenase (IDH)-NADP ؉ . 13 C NMR spectroscopic studies, Blue Native PAGE, and Western blot analyses indicated that although the decrease in Acn activity is concomitant with the increase of aluminum in the culture, the amount of Acn expressed is not sensitive to the concentration of the trivalent metal. A 6-fold decrease in Acn activity and no discernable change in protein content in aluminum-stressed cultures were observed. The addition of Fe(NH 4 ) 2 (SO 4 ) 2 in a reducing environment led to a significant recovery in Acn activity. This enzymatic activity reverted to normal levels when aluminum-stressed cells were transferred to either a control or an iron-supplemented medium. The overexpression of the two isocitrate-metabolizing enzymes isocitrate lyase and IDH-NADP ؉ appears to mitigate the deficit in Acn activity. The levels of these enzymes are dependent on the aluminum content of the culture and appear to be under transcriptional control. Hence, the regulation of the enzymes involved in the homeostasis of isocitrate constitutes a pivotal component of the global metabolic strategy that ensures the survival of this organism in an aluminum citrate environment.Metabolism is the foundation of all living organisms, and any biological function is the manifestation of the global cellular metabolism. Hence, any cellular behavior is a direct or indirect product of its metabolism. The enzymes/metabolites participating in metabolism provide a precise snapshot of a cellular phenotype (1, 2). As part of our study on molecular adaptation, we have uncovered an interesting model system that allows deciphering the metabolic reconfiguration evoked by metal stress. The metal toxicant was supplied to the microbe Pseudomonas fluorescens, complexed to citrate, the only carbon source. The role of oxalate and phosphatidylethanolamine in the immobilization of aluminum has been demonstrated recently (3, 4). It appears that the cellular metabolism is reconfigured with the aim of providing the metabolic precursors that allow for the survival of the organism in an aluminum environment. Hence, an aluminum-adapted phenotype with an entirely different set of metabolic pathways than in the wild type is promoted.Citrate, the sole carbon source utilized in this system, is known to be cleaved in various organisms, primarily by the enzymes citrate-lyase (CL), 1 ATP-citrate-lyase (ATP-CL), and Acn. Whereas CL mediates the cleavage of citrate to acetate and oxaloacetate, ATP-CL catalyzes the degradation of tricarboxylic acid into acetyl-CoA and oxaloacetate (5, 6). The latter is also referred to as a l...

Iron (Fe) is a critical element in all aerobic organisms as it participates in a variety of metabolic networks. In this study, aluminum (Al) and gallium (Ga), two Fe mimetics, severely impeded the ability of the soil microbe Pseudomonas fluorescens to perform oxidative phosphorylation. This was achieved by disrupting the activity and expression of complexes I, II, and IV. These toxic metals also inactivated aconitase (ACN) and fumarase A (FUM A), two tricarboxylic acid cycle enzymes dependent on Fe for their catalytic activity, while FUM C, an Fe-independent enzyme, displayed an increase in activity and expression under these stressed situations. Furthermore, in the Al-and Ga-exposed cells, the activity and expression of an H 2 O-forming NADH oxidase were markedly increased. The incubation of the Al-and Ga-challenged cells in an Fe-containing medium led to the recovery of the affected enzymatic activities. Taken together, these data provide novel insights into how environmental pollutants such as Al and Ga interfere with cellular Fe metabolism and also illustrate the ability of Pseudomonas fluorescens to modulate metabolic networks to combat this situation.

Gallium (Ga), an iron (Fe) mimetic promoted an oxidative environment and elicited an antioxidative response in Pseudomonas fluorescens. Ga-stressed P. fluorescens was characterized by higher amounts of oxidized lipids and proteins compared to control cells. The oxidative environment provoked by Ga was nullified by increased synthesis of NADPH. The activity and expression glucose 6-phosphate dehydrogenase (G6PDH) and isocitrate dehydrogenase-NADP (ICDH) were stimulated in Ga-cultures. The induction of isoenzymes of these dehydrogenases was also evident in the Ga-stressed cells. Although superoxide dismutase (SOD) activity was significantly enhanced in Ga-stressed cultures, catalase activity experienced a marked diminution. Fe metabolism appeared to be severely impeded by Ga toxicity. This is the first demonstration of the oxidative stress evoked by Ga to be neutralized by a reductive environment generated via the overexpression of NADPH-producing enzymes.

Aluminum (Al) triggered a marked increase in reactive oxygen species (ROS) such as O(2) (-) and H(2)O(2) in Pseudomonas fluorescens. Although the Al-stressed cells were characterized with higher amounts of oxidized lipids and proteins than controls, NADPH production was markedly increased in these cells. Blue native polyacrylamide gel electrophoresis (BN-PAGE) analyses coupled with activity and Coomassie staining revealed that NADP(+) -dependent isocitrate dehydrogenase (ICDH, E.C. 1.1.1.42) and glucose-6-phosphate dehydrogenase (G6PDH, E.C. 1.1.1.49) played a pivotal role in diminishing the oxidative environment promoted by Al. These enzymes were overexpressed in the Al-tolerant microbes and were modulated by the presence of either Al or hydrogen peroxide (H(2)O(2)) or menadione. The activity of superoxide dismutase (SOD, E.C. 1.15.1.1), an enzyme known to combat ROS stress was also increased in the cells cultured in millimolar amounts of Al. Hence, Al-tolerant P. fluorescens invokes an anti-oxidative defense strategy in order to survive.