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

α-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 (NH₄) 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. ¹³C 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 acquire ATP via the reduction of oxygen (23). We have recently shown the crucial role played by NADK, an enzyme that mediates the formation of NADP, a key ingredient known to tilt cellular metabolism toward the synthesis of NADPH and away from the formation of NADH, a prooxidant (28). Hence, aerobic respiration, ROS production, and antioxidative defense strategies have to be intricately modulated.Although the elimination of ROS is critical to the survival of all organisms, it is also important to appreciate the role that adaptative mechanisms play to lower the production of ROS. Our laboratory has recently identified an intriguing role of the tricarboxylic acid (TCA) cycle in this regard (15, 27). By modulating the production of NADH and NADPH, this metabolic network appears to be instrumental in striking the proper balance between the generation of ROS and the aerobic formation of ATP. As part of our study to delineate the molecular mechanisms that allow cellular systems to adapt to oxidative stress, we have identified how α-ketoglutarate dehydrogenase (KGDH) and glutamate dehydrogenase (GDH) play a critical role in modulating α-ketoglutarate (KG) homeostasis in Pseudomonas fluorescens challenged with menadione. This keto acid can readily nullify these ROS with the concomitant formation of succinate, a moiety that may signal anaerobic metabolism. The roles of KGDH in sensing ROS and limiting NADH production are also discussed.     

Distribution of ω-Amino Acid: Pyruvate Transaminase and Aminobutyrate: α-Ketoglutarate Transaminase in Microorganisms

The distribution of ω-amino acid transaminases in microorganisms was investigated, ω-Amino acid: pyruvate transaminase (ω-APT) was found in bacteria and yeasts, but not in actinomycetes and fungi. On the contrary, aminobutyrate: α-ketoglutarate transaminase (GABA-T) was shown in most of the microorganisms from bacteria to fungi. β-Alanine is a preferred amino donor for the co-APT reaction. Although bacterial and yeast GABA-T are inactive for β-alanine, fungal and actinomycete enzymes react with this compound and γ-aminobutyrate. In comparing these results with those of plant and mammalian enzymes, two different pathways of co-amino acid metabolism are suggested for bacteria, yeast and plants, i.e. one for β-alanine and the other for γ-aminobutyrate, catalyzed by ω-APT and GABA-T, respectively. In actinomycetes, fungi, and mammals GABA-T may be involved in the metabolism of both ω-amino acids. In addition, evolutionary changes of ω-amino acid transaminases are discussed.     

Irreversible Inactivation of l-Ornithine : α-Ketoglutarate δ-Aminotransferase by Gabaculine

The action of endo-1,4-β-d-galactanase from Penicillium citrinum on o-nitrophenyl-β-d-galactopyranoside (ONPG) was investigated.

In reaction mixtures with various concentrations of ONPG, liberation of o-nitrophenol (ONP) was observed after a lag phase and then oligosaccharides with and without ONP-group were found to accumulate. The products were separated by activated carbon column chromatography and paper chromatography, and found to be a series of β-1,4-linked galactooligosaccharides and their ONP-substituted derivatives.

Liberation of ONP from the ONP-substituted oligosaccharides by the enzyme occured without a lag phase. Furthermore, the lag phase of ONP liberation from ONPG was eliminated by the addition of β-1,4-galactotriose and -tetraose to the reaction mixture.

The formation of ONP-substituted oligosaccharides before the liberation of ONP is assumed to be the cause of the observed lag.     

Uptake of α-Ketoglutarate by Citrate Transporter CitP Drives Transamination in Lactococcus lactis

Transamination is the first step in the conversion of amino acids into aroma compounds by lactic acid bacteria (LAB) used in food fermentations. The process is limited by the availability of α-ketoglutarate, which is the best α-keto donor for transaminases in LAB. Here, uptake of α-ketoglutarate by the citrate transporter CitP is reported. Cells of Lactococcus lactis IL1403 expressing CitP showed significant levels of transamination activity in the presence of α-ketoglutarate and one of the amino acids Ile, Leu, Val, Phe, or Met, while the same cells lacking CitP showed transamination activity only after permeabilization of the cell membrane. Moreover, the transamination activity of the cells followed the levels of CitP in a controlled expression system. The involvement of CitP in the uptake of the α-keto donor was further demonstrated by the increased consumption rate in the presence of l-lactate, which drives CitP in the fast exchange mode of transport. Transamination is the only active pathway for the conversion of α-ketoglutarate in IL1403; a stoichiometric conversion to glutamate and the corresponding α-keto acid from the amino acids was observed. The transamination activity by both the cells and the cytoplasmic fraction showed a remarkably flat pH profile over the range from pH 5 to pH 8, especially with the branched-chain amino acids. Further metabolism of the produced α-keto acids into α-hydroxy acids and other flavor compounds required the coupling of transamination to glycolysis. The results suggest a much broader role of the citrate transporter CitP in LAB than citrate uptake in the citrate fermentation pathway alone.     

Artefacts in cell culture: α-Ketoglutarate can scavenge hydrogen peroxide generated by ascorbate and epigallocatechin gallate in cell culture media

Ascorbate and several phenolic compounds readily oxidise in cell culture media to generate hydrogen peroxide. However, addition of α-ketoglutarate, which is known to be released by several cell types, decreased the levels of H₂O₂, and the α-ketoglutarate was depleted and converted to succinate. These observations could account for previous reports of the protective effects of α-ketoglutarate in promoting the growth of cells in culture, and may contribute to explaining some of the variability in the literature in reported rates of H₂O₂ production from autoxidisable compounds in cell culture systems.     

Presence and Regulation of α-Ketoglutarate Dehydrogenase Complex in a Glutamate-Producing Bacterium,Brevibacterium flavum

The presence of α-ketoglutarate (α-KG) dehydrogenase complex in the glutamate-producing bacteria was demonstrated for the first time with Brevibacterium flavum. The partially purified enzyme, which was specific to KG and NAD⁺ with the usual requirements for other co-factors, was labile and stabilized by glycerol, Mg²⁺, and thiamine pyrophosphate. The enzyme showed an optimum pH of 7.6 and K_ms of 80, 86, and 61 μm for KG, NAD⁺, and CoA, respectively, cis-Aconitate, succinyl-CoA, NADPH, NADH, pyruvate, and oxalacetate strongly inhibited the activity, while it was activated by acetyl-CoA, but not by AMP. Various inorganic and organic salts also inhibited the activity. When cells were cultured in glucose and acetate media, the specific activity of the cell extracts increased markedly and reached to a maximum at the late-logarithmic phase. Then, it decreased to the basal level. The addition of glutamate stimulated the synthesis of the enzyme.     

The Krebs Cycle Enzyme α-Ketoglutarate Decarboxylase Is an Essential Glycosomal Protein in Bloodstream African Trypanosomes

α-Ketoglutarate decarboxylase (α-KDE1) is a Krebs cycle enzyme found in the mitochondrion of the procyclic form (PF) of Trypanosoma brucei. The bloodstream form (BF) of T. brucei lacks a functional Krebs cycle and relies exclusively on glycolysis for ATP production. Despite the lack of a functional Krebs cycle, α-KDE1 was expressed in BF T. brucei and RNA interference knockdown of α-KDE1 mRNA resulted in rapid growth arrest and killing. Cell death was preceded by progressive swelling of the flagellar pocket as a consequence of recruitment of both flagellar and plasma membranes into the pocket. BF T. brucei expressing an epitope-tagged copy of α-KDE1 showed localization to glycosomes and not the mitochondrion. We used a cell line transfected with a reporter construct containing the N-terminal sequence of α-KDE1 fused to green fluorescent protein to examine the requirements for glycosome targeting. We found that the N-terminal 18 amino acids of α-KDE1 contain overlapping mitochondrion- and peroxisome-targeting sequences and are sufficient to direct localization to the glycosome in BF T. brucei. These results suggest that α-KDE1 has a novel moonlighting function outside the mitochondrion in BF T. brucei.     

Dual Functions of α-Ketoglutarate Dehydrogenase E2 in the Krebs Cycle and Mitochondrial DNA Inheritance in Trypanosoma brucei

The dihydrolipoyl succinyltransferase (E2) of the multisubunit α-ketoglutarate dehydrogenase complex (α-KD) is an essential Krebs cycle enzyme commonly found in the matrices of mitochondria. African trypanosomes developmentally regulate mitochondrial carbohydrate metabolism and lack a functional Krebs cycle in the bloodstream of mammals. We found that despite the absence of a functional α-KD, bloodstream form (BF) trypanosomes express α-KDE2, which localized to the mitochondrial matrix and inner membrane. Furthermore, α-KDE2 fractionated with the mitochondrial genome, the kinetoplast DNA (kDNA), in a complex with the flagellum. A role for α-KDE2 in kDNA maintenance was revealed in α-KDE2 RNA interference (RNAi) knockdowns. Following RNAi induction, bloodstream trypanosomes showed pronounced growth reduction and often failed to equally distribute kDNA to daughter cells, resulting in accumulation of cells devoid of kDNA (dyskinetoplastic) or containing two kinetoplasts. Dyskinetoplastic trypanosomes lacked mitochondrial membrane potential and contained mitochondria of substantially reduced volume. These results indicate that α-KDE2 is bifunctional, both as a metabolic enzyme and as a mitochondrial inheritance factor necessary for the distribution of kDNA networks to daughter cells at cytokinesis.     

« Biology/medicine 2.0 »: an overview

In the last decade, the applications known as "web 2.0" (Facebook, Youtube, Twitter…) have changed our daily life and gradually influence the field of research. This article aims at proposing a critical overview of these new services, and emphasizes the changes induced for researchers (practice of scientific publication, sharing and mutualization of research data and discussion between researchers…) especially in the field of biology/medicine. A focus is done on the limitations that prevent most of scientists from using these services in their common practice (lack of knowledge about these tools, time-consuming, fear of sharing data and ideas). Despite these restrictions, some mutations affecting researcher's information uses are unavoidable, and these new tools may rapidly contribute to scientific advances.     

Short-Term Regulation of the α-Ketoglutarate Dehydrogenase Complex by Energy-Linked and Some Other Effectors

The question of regulation of α-ketoglutarate dehydrogenase complex (KGDHC) has been considered in the biochemical literature very rarely. Moreover, such information is not usually accurate, especially in biochemical textbooks. From the mini-review of research works published during the last 25 years, the following basic view is clear: a) animal KGDHC is very sensitive to ADP, P_i, and Ca²⁺; b) these positive effectors increase manifold the affinity of KGDHC to α-ketoglutarate; c) KGDHC is inhibited by ATP, NADH, and succinyl-CoA; d) the ATP effect is realized in several ways, probably mainly via opposition versus ADP activation; e) NADH, besides inhibiting dihydrolipoamide dehydrogenase component competitively versus NAD⁺, decreases the affinity of α-ketoglutarate dehydrogenase to substrate and inactivates it; f) thioredoxin protects KGDHC from self-inactivation during catalysis; g) bacterial and plant KGDHC is activated by AMP instead of ADP. These main effects form the basis of short-term regulation of KGDHC.__________Translated from Biokhimiya, Vol. 70, No. 7, 2005, pp. 880–884.Original Russian Text Copyright © 2005 by Strumilo.     

α-Ketoglutarate dehydrogenase: a mitochondrial redox sensor

α-Ketoglutarate dehydrogenase (KGDH), a key regulatory enzyme within the Krebs cycle, is sensitive to mitochondrial redox status. Treatment of mitochondria with H?O? results in reversible inhibition of KGDH due to glutathionylation of the cofactor, lipoic acid. Upon consumption of H?O?, glutathione is removed by glutaredoxin restoring KGDH activity. Glutathionylation appears to be enzymatically catalysed or require a unique microenvironment. This may represent an antioxidant response, diminishing the flow of electrons to the respiratory chain and protecting sulphydryl residues from oxidative damage. KGDH is, however, also susceptible to oxidative damage. 4-Hydroxy-2-nonenal (HNE), a lipid peroxidation product, reacts with lipoic acid resulting in enzyme inactivation. Evidence indicates that HNE modified lipoic acid is cleaved from KGDH, potentially the first step of a repair process. KGDH is therefore a likely redox sensor, reversibly altering metabolism to reduce oxidative damage and, under severe oxidative stress, acting as a sentinel of mitochondrial viability.     

Introduction: Special Issue in Honor of Eva Syková

Neurochemical Research -     

Functional Elements in Molecular Chaperone α-Crystallin: Identification of Binding Sites in αB-Crystallin

α-Crystallin, the predominant eye lens protein with sequence homology to small heat shock proteins, acts like a molecular chaperone by suppressing the aggregation of damaged crystallins and proteins. To gain an insight into the amino acid sequences in α-crystallin involved in chaperone-like function, we used a cleavable, fluorescent, photoactive, crosslinking agent, sulfosuccinimidyl-2(7-azido-4-methylcoumarin-3-acetamido)-ethyl-1,3′ dithiopropionate (SAED), to derivatize yeast alcohol dehydrogenase (ADH) and allowed it to complex with bovine α-crystallin at 48°C. The complex was photolyzed and reduced with DTT and the subunits of α-crystallin, αA- and αB-, were separated. Fluorescence analysis showed that both αA- and αB-crystallins interacted with ADH during chaperone-like function. Tryptic digestion, amino acid sequencing, and mass spectral analysis of αB-crystallin revealed that APSWIDTGLSEMR (57-69) and VLGDVIEVHGKHEER (93-107) sequences were involved in binding with ADH.