Christopher Auger

α-Ketoglutarate Dehydrogenase and Glutamate Dehydrogenase Work in Tandem To Modulate the Antioxidant α-Ketoglutarate during Oxidative Stress in Pseudomonas fluorescens

Alpha-ketoglutarate (KG) is a crucial metabolite in all living organisms, as it participates in a variety of biochemical processes. We have previously shown that this keto acid is an antioxidant and plays a key role in the detoxification of reactive oxygen species (ROS). In an effort to further confirm this intriguing phenomenon, Pseudomonas fluorescens was exposed to menadione-containing media, with various amino acids as the sources of nitrogen. Here, we demonstrate that KG dehydrogenase (KGDH) and NAD-dependent glutamate dehydrogenase (GDH) work in tandem to modulate KG homeostasis. While KGDH was sharply decreased in cells challenged with menadione, GDH was markedly increased in cultures containing arginine (Arg), glutamate (Glu), and proline (Pro). When ammonium (NH(4)) was utilized as the nitrogen source, both KGDH and GDH levels were diminished. These enzymatic profiles were reversed when control cells were incubated in menadione media. (13)C nuclear magnetic resonance and high-performance liquid chromatography studies revealed how KG was utilized to eliminate ROS with the concomitant formation of succinate. The accumulation of KG in the menadione-treated cells was dependent on the redox status of the lipoic acid residue in KGDH. Indeed, the treatment of cellular extracts from the menadione-exposed cells with dithiothreitol, a reducing agent, partially restored the activity of KGDH. Taken together, these data reveal that KG is pivotal to the antioxidative defense strategy of P. fluorescens and also point to the ROS-sensing role for KGDH.

Pseudomonas fluorescens orchestrates a fine metabolic-balancing act to counter aluminium toxicity

Aluminium (Al), an environmental toxin, is known to disrupt cellular functions by perturbing iron (Fe) homeostasis. However, Fe is essential for such metabolic processes as the tricarboxylic acid (TCA) cycle and oxidative phosphorylation, the two pivotal networks that mediate ATP production during aerobiosis. To counter the Fe conundrum induced by Al toxicity, Pseudomonas fluorescens utilizes isocitrate lyase and isocitrate dehydrogenase-NADP dependent to metabolize citrate when confronted with an ineffective aconitase provoked by Al stress. By invoking fumarase C, a hydratase devoid of Fe, this microbe is able to generate essential metabolites. To compensate for the severely diminished enzymes like Complex I, Complex II and Complex IV, the upregulation of a H(2)O-generating NADH oxidase enables the metabolism of citrate, the sole carbon source via a modified TCA cycle. The overexpression of succinyl-CoA synthetase affords an effective route to ATP production by substrate-level phosphorylation in the absence of O(2). This fine metabolic balance enables P. fluorescens to survive the dearth of bioavailable Fe triggered by an Al environment, a feature that may have potential applications in bioremediation technologies.

Dysfunctional mitochondrial bioenergetics and the pathogenesis of hepatic disorders

The liver is involved in a variety of critical biological functions including the homeostasis of glucose, fatty acids, amino acids, and the synthesis of proteins that are secreted in the blood. It is also at the forefront in the detoxification of noxious metabolites that would otherwise upset the functioning of the body. As such, this vital component of the mammalian system is exposed to a notable quantity of toxicants on a regular basis. It therefore comes as no surprise that there are over a hundred disparate hepatic disorders, encompassing such afflictions as fatty liver disease, hepatitis, and liver cancer. Most if not all of liver functions are dependent on energy, an ingredient that is primarily generated by the mitochondrion, the power house of all cells. This organelle is indispensable in providing adenosine triphosphate (ATP), a key effector of most biological processes. Dysfunctional mitochondria lead to a shortage in ATP, the leakage of deleterious reactive oxygen species (ROS), and the excessive storage of fats. Here we examine how incapacitated mitochondrial bioenergetics triggers the pathogenesis of various hepatic diseases. Exposure of liver cells to detrimental environmental hazards such as oxidative stress, metal toxicity, and various xenobiotics results in the inactivation of crucial mitochondrial enzymes and decreased ATP levels. The contribution of the latter to hepatic disorders and potential therapeutic cues to remedy these conditions are elaborated.

Metabolic reengineering invoked by microbial systems to decontaminate aluminum: Implications for bioremediation technologies

As our reliance on aluminum (Al) increases, so too does its presence in the environment and living systems. Although generally recognized as safe, its interactions with most living systems have been nefarious. This review presents an overview of the noxious effects of Al and how a subset of microbes can rework their metabolic pathways in order to survive an Al-contaminated environment. For instance, in order to expulse the metal as an insoluble precipitate, Pseudomonas fluorescens shuttles metabolites toward the production of organic acids and lipids that play key roles in chelating, immobilizing and exuding Al. Further, the reconfiguration of metabolic modules enables the microorganism to combat the dearth of iron (Fe) and the excess of reactive oxygen species (ROS) promoted by Al toxicity. While in Rhizobium spp., exopolysaccharides have been invoked to sequester this metal, an ATPase is known to safeguard Anoxybacillus gonensis against the trivalent metal. Hydroxyl, carboxyl and phosphate moieties have also been exploited by microbes to trap Al. Hence, an understanding of the metabolic networks that are operative in microorganisms residing in polluted environments is critical in devising bioremediation technologies aimed at managing metal wastes. Metabolic engineering is essential in elaborating effective biotechnological processes to decontaminate metal-polluted surroundings.

Histidine is a source of the antioxidant, α‐ketoglutarate, in Pseudomonas fluorescens challenged by oxidative stress

The role of alpha-ketoglutarate (KG) in the detoxification of reactive oxygen species (ROS) has only recently begun to be appreciated. This ketoacid neutralizes ROS in an NADPH-independent manner with the concomitant formation of succinate and CO(2). To further probe this intriguing attribute of KG in living systems, we have evaluated the significance of histidine metabolism in the model organism, Pseudomonas fluorescens, challenged by hydrogen peroxide (H(2)O(2)). Here, we show that this amino acid does contribute to KG homeostasis and appears to be earmarked for the production of KG during oxidative stress. Both the NAD- and the NADP-dependent glutamate dehydrogenases were upregulated in the stressed cells despite the sharp decline in the activities of numerous enzymes mediating the tricarboxylic acid cycle and oxidative phosphorylation. Enzymes such as isocitrate dehydrogenase-NAD dependent, succinate dehydrogenase, alpha-ketoglutarate dehydrogenase, Complex I, and Complex IV were severely affected in the P. fluorescens grown in the presence of H(2)O(2). Studies with fluorocitrate, a potent inhibitor of citrate metabolism, clearly revealed that histidine was preferentially utilized in the production of KG in the H(2)O(2)-challenged cells. Regulation experiments also helped confirm that the metabolic reprogramming, resulting in the enhanced production of KG was induced by H(2)O(2) stress. These data further establish the pivotal role that KG plays in antioxidative defense.

How aluminum, an intracellular ROS generator promotes hepatic and neurological diseases: the metabolic tale

Metal pollutants are a global health risk due to their ability to contribute to a variety of diseases. Aluminum (Al), a ubiquitous environmental contaminant is implicated in anemia, osteomalacia, hepatic disorder, and neurological disorder. In this review, we outline how this intracellular generator of reactive oxygen species (ROS) triggers a metabolic shift towards lipogenesis in astrocytes and hepatocytes. This Al-evoked phenomenon is coupled to diminished mitochondrial activity, anerobiosis, and the channeling of α-ketoacids towards anti-oxidant defense. The resulting metabolic reconfiguration leads to fat accumulation and a reduction in ATP synthesis, characteristics that are common to numerous medical disorders. Hence, the ability of Al toxicity to create an oxidative environment promotes dysfunctional metabolic processes in astrocytes and hepatocytes. These molecular events triggered by Al-induced ROS production are the potential mediators of brain and liver disorders.

Hydrogen peroxide stress provokes a metabolic reprogramming in Pseudomonas fluorescens: Enhanced production of pyruvate

Pseudomonas fluorescens invoked a metabolic reconfiguration that resulted in enhanced production of pyruvate under the challenge of hydrogen peroxide (H₂O₂). Although this stress led to a sharp reduction in the activities of numerous tricarboxylic acid (TCA) cycle enzymes, there was a marked increase in the activities of catalase and various NADPH-generating enzymes to counter the oxidative burden. The upregulation of phosphoenolpyruvate synthase (PEPS) and pyruvate kinase (PK) coupled with the reduction of pyruvate dehydrogenase (PDH) in the H₂O₂-challenged cells appear to be important contributors to the elevated levels of pyruvate found in these bacteria. Increased pyruvate synthesis was evident in the presence of a variety of carbon sources including d-glucose. Intact cells rapidly consumed d-glucose with the concomitant formation of this monocarboxylic acid. At least a 12-fold increase in pyruvate production within 1h was observed in the stressed cells. These findings may be exploited in the development of technologies aimed at the conversion of carbohydrates into pyruvate.

The disruption of L-carnitine metabolism by aluminum toxicity and oxidative stress promotes dyslipidemia in human astrocytic and hepatic cells

L-Carnitine is a critical metabolite indispensable for the metabolism of lipids as it facilitates fatty acid transport into the mitochondrion where β-oxidation occurs. Human astrocytes (CCF-STTG1 cells) and hepatocytes (HepG2 cells) exposed to aluminum (Al) and hydrogen peroxide (H₂O₂), were characterized with lower levels of L-carnitine, diminished β-oxidation, and increased lipid accumulation compared to the controls. γ-Butyrobetainealdehyde dehydrogenase (BADH) and butyrobetaine dioxygenase (BBDOX), two key enzymes mediating the biogenesis of L-carnitine, were sharply reduced during Al and H₂O₂ challenge. Exposure of the Al and H₂O₂-treated cells to α-ketoglutarate (KG), led to the recovery of L-carnitine production with the concomitant reduction in ROS levels. It appears that the channeling of KG to combat oxidative stress results in decreased L-carnitine synthesis, an event that contributes to the dyslipidemia observed during Al and H₂O₂ insults in these mammalian cells. Hence, KG may help alleviate pathological conditions induced by oxidative stress.

The Metabolic Reprogramming Evoked by Nitrosative Stress Triggers the Anaerobic Utilization of Citrate in Pseudomonas fluorescens

Nitrosative stress is an ongoing challenge that most organisms have to contend with. When nitric oxide (NO) that may be generated either exogenously or endogenously encounters reactive oxygen species (ROS), it produces a set of toxic moieties referred to as reactive nitrogen species (RNS). As these RNS can severely damage essential biomolecules, numerous organisms have evolved elaborate detoxification strategies to nullify RNS. However, the contribution of cellular metabolism in fending off nitrosative stress is poorly understood. Using a variety of functional proteomic and metabolomic analyses, we have identified how the soil microbe Pseudomonas fluorescens reprogrammed its metabolic networks to survive in an environment enriched by sodium nitroprusside (SNP), a generator of nitrosative stress. To combat the RNS-induced ineffective aconitase (ACN) and tricarboxylic acid (TCA) cycle, the microbe invoked the participation of citrate lyase (CL), phosphoenolpyruvate carboxylase (PEPC) and pyruvate phosphate dikinase (PPDK) to convert citrate, the sole source of carbon into pyruvate and ATP. These enzymes were not evident in the control conditions. This metabolic shift was coupled to the concomitant increase in the activities of such classical RNS detoxifiers as nitrate reductase (NR), nitrite reductase (NIR) and S-nitrosoglutathione reductase (GSNOR). Hence, metabolism may hold the clues to the survival of organisms subjected to nitrosative stress and may provide therapeutic cues against RNS-resistant microbes.

Glycine metabolism and anti-oxidative defence mechanisms in Pseudomonas fluorescens

The role of metabolism in anti-oxidative defence is only now beginning to emerge. Here, we show that the nutritionally-versatile microbe, Pseudomonas fluorescens, reconfigures its metabolism in an effort to generate NADPH, ATP and glyoxylate in order to fend off oxidative stress. Glyoxylate was produced predominantly via the enhanced activities of glycine dehydrogenase-NADP(+) (GDH), glycine transaminase (GTA) and isocitrate lyase (ICL) in a medium exposed to hydrogen peroxide (H₂O₂). This ketoacid was utilized to produce ATP by substrate-level phosphorylation and to neutralize reactive oxygen species with the concomitant formation of formate. The latter was also a source of NADPH, a process mediated by formate dehydrogenase-NADP(+) (FDH). The increased activities of phosphoenolpyruvate carboxylase (PEPC) and pyruvate orthophosphate dikinase (PPDK) worked in tandem to synthesize ATP in the H₂O₂-challenged cells that had markedly diminished capacity for oxidative phosphorylation. These metabolic networks provide an effective means of combating ROS and reveal therapeutic targets against microbes resistant to oxidative stress.