␣-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.All aerobic organisms have to contend with the dangers associated with reactive oxygen species (ROS), toxic moieties that are routinely generated as a consequence of ATP production via oxidative phosphorylation (34). The transfer of electrons from NADH and reduced flavin adenine dinucleotide to oxygen is mediated by the respiratory complexes, the major sites of intracellular ROS generation (1). These by-products of oxidative phosphorylation are very harmful and have to be nullified if organisms are to survive in an aerobic environment (24). If left unchecked, ROS can damage biological macromolecules, leading to the demise of the cell. Hence, it is not surprising that all aerobic organisms have devised intricate antioxidative defense strategies in an effort to proliferate in the presence of oxygen.Enzymes such as superoxide dismutase and catalase are uniquely bestowed with the task of eliminating superoxide and hydrogen peroxide, two important ROS (11, 12, 21). Glutathione (GSH), a tripeptide, also plays a pivotal role in the detoxification of ROS (22). However, to be effective, all these ROS disposal processes have to be regenerated with the aid of NADPH. This nicotinamide dinucleotide is the main power behind all antioxidative defense strategies, as it provides the reducing fuel necessary to recharge all effectors involved in combating ROS (32). Thus, various enzyme systems and metabolic networks that orchestrate the biogenesis of NADPH have to be activated if an organism is to...