Glutamate Biosynthesis Research Articles

VGLUTs and Glutamate Synthesis-Focus on DRG Neurons and Pain.

The amino acid glutamate is the principal excitatory transmitter in the nervous system, including in sensory neurons that convey pain sensation from the periphery to the brain. It is now well established that a family of membrane proteins, termed vesicular glutamate transporters (VGLUTs), serve a critical function in these neurons: they incorporate glutamate into synaptic vesicles. VGLUTs have a central role both under normal neurotransmission and pathological conditions, such as neuropathic or inflammatory pain. In the present short review, we will address VGLUTs in the context of primary afferent neurons. We will focus on the role of VGLUTs in pain triggered by noxious stimuli, peripheral nerve injury, and tissue inflammation, as mostly explored in transgenic mice. The possible interplay between glutamate biosynthesis and VGLUT-dependent packaging in synaptic vesicles, and its potential impact in various pain states will be presented.

Reconstruction and analysis of a genome-scale metabolic network of Corynebacterium glutamicum S9114

Corynebacterium glutamicum S9114 is commonly used for industrial glutamate production. Therefore, a comprehensive understanding of the physiological and metabolic characteristics of C. glutamicum is important for developing its potential for industrial production. A genome-scale metabolic model, iJM658, was reconstructed based on genome annotation and literature mining. The model consists of 658 genes, 984 metabolites and 1065 reactions. The model quantitatively predicted C. glutamicum growth on different carbon and nitrogen sources and determined 129 genes to be essential for cell growth. The iJM658 model predicted that C. glutamicum had two glutamate biosynthesis pathways and lacked eight key genes in biotin synthesis. Robustness analysis indicated a relative low oxygen level (1.21mmol/gDW/h) would improve glutamate production rate. Potential metabolic engineering targets for improving γ-aminobutyrate and isoleucine production rate were predicted by in silico deletion or overexpression of some genes. The iJM658 model is a useful tool for understanding and optimizing the metabolism of C. glutamicum and a valuable resource for future metabolic and physiological research.

Dissecting Leishmania infantum Energy Metabolism - A Systems Perspective.

Leishmania infantum, causative agent of visceral leishmaniasis in humans, illustrates a complex lifecycle pertaining to two extreme environments, namely, the gut of the sandfly vector and human macrophages. Leishmania is capable of dynamically adapting and tactically switching between these critically hostile situations. The possible metabolic routes ventured by the parasite to achieve this exceptional adaptation to its varying environments are still poorly understood. In this study, we present an extensively reconstructed energy metabolism network of Leishmania infantum as an attempt to identify certain strategic metabolic routes preferred by the parasite to optimize its survival in such dynamic environments. The reconstructed network consists of 142 genes encoding for enzymes performing 237 reactions distributed across five distinct model compartments. We annotated the subcellular locations of different enzymes and their reactions on the basis of strong literature evidence and sequence-based detection of cellular localization signal within a protein sequence. To explore the diverse features of parasite metabolism the metabolic network was implemented and analyzed as a constraint-based model. Using a systems-based approach, we also put forth an extensive set of lethal reaction knockouts; some of which were validated using published data on Leishmania species. Performing a robustness analysis, the model was rigorously validated and tested for the secretion of overflow metabolites specific to Leishmania under varying extracellular oxygen uptake rate. Further, the fate of important non-essential amino acids in L. infantum metabolism was investigated. Stage-specific scenarios of L. infantum energy metabolism were incorporated in the model and key metabolic differences were outlined. Analysis of the model revealed the essentiality of glucose uptake, succinate fermentation, glutamate biosynthesis and an active TCA cycle as driving forces for parasite energy metabolism and its optimal growth. Finally, through our in silico knockout analysis, we could identify possible therapeutic targets that provide experimentally testable hypotheses.

13C NMR based profiling unveils different α-ketoglutarate pools involved into glutamate and lysine synthesis in the milk yeast Kluyveromyces lactis

BackgroundThe construction of efficient cell factories for the production of metabolites requires the rational improvement/engineering of the metabolism of microorganisms. The subject of this paper is directed towards the quantitative understanding of the respiratory/fermentative Kluyveromyces lactis yeast metabolism and its rag8 casein kinase mutant, taken as a model for all rag gene mutations. Methods13C NMR spectroscopy and [1,2-13C2]glucose were used as metabolic stable-isotope tracer to define the metabolic profiling of a K. lactis yeast and its derivative mutants. ResultsRag8 showed a decrease of all 13C glutamate fractional enrichments, except for [4-13C]glutamate that was higher than wild type ones. A decrease of TCA cycle flux in rag8 mutants and a contribution of a [4-13C]ketoglutarate pool not originating from mitochondria were suggested.13C lysine enrichments confirmed the presence of two compartmentalized α-ketoglutarate (α-KG) pools participating to glutamate and lysine synthesis.Moreover, an increased transaldolase, as compared to transketolase activity, was observed in the rag8 mutant by 13C NMR isotopomer analysis of alanine. Conclusions13C NMR-based isotopomer analysis showed the existence of different α-KG metabolic pools for glutamate and lysine biosynthesis. In the rag8 mutant, 13C labeled pentose phosphate intermediates participated in the synthesis of this compartmentalized α-KG pool. General significanceA compartmentalization of the α-KG pools involved in lysine biosynthesis has been revealed for the first time in K. lactis. Given its great impact in metabolic engineering field, its existence should be validated/compared with other yeasts and/or fungal species.

GLP‐2 reprograms glucose metabolism in intestinal stem cells

Glucagon-like peptide 2 (GLP-2) is an enterotrophic peptide to enhance intestinal crypt cell proliferation. However, cellular mechanisms underlying its tropic action are still unclear. Using the innovative mouse mini-guts models, we wanted to determine if GLP-2 directly modulates metabolic reprogramming (aerobic glycolysis) and cellular function (proliferation) in the intestinal stem cells. Intracellular metabolic fluxes and cell proliferation in the mouse mini-guts were quantified by U-13C-d-glucose tracer and BrdU incorporation. The mouse undifferentiating mini-guts were treated with GLP-2 (100 nM) ± glycolysis inhibitor and labeled with U-13C-d-glucose tracer or BrdU for 90~120 min. We show that the GLP-2 receptor (GLP-2R) was required for the growth of mouse mini-guts; and for GLP-2 to augment cell proliferation in an aerobic glycolysis-dependent manner. Using LC-MS/MS-based metabolomics, moreover, we revealed that GLP-2 shifted 13C metabolic fluxes to aerobic glycolysis and de novo biosynthesis of glutamate in the mini-guts, and this metabolic reprogramming was negated by glycolysis inhibition. These data suggest that via the metabolic reprogramming of glucose, GLP-2 channels glycolytic intermediates for amino acid biosynthesis to support cell proliferation. Furthermore, we identified that GLP-2 acutely induced phosphorylation and nuclear translocation of pyruvate kinase M2 in the mini-guts. We conclude that GLP-2 plays a novel role in metabolic reprogramming of glucose for biosynthesis to support cell proliferation in the crypt stem cells.

Evidence for synergistic control of glutamate biosynthesis by glutamate dehydrogenases and glutamate in Bacillus subtilis.

In the Gram-positive bacterium, Bacillus subtilis glutamate is synthesized by the glutamine synthetase and the glutamate synthase (GOGAT). During growth with carbon sources that exert carbon catabolite repression, the rocG glutamate dehydrogenase (GDH) gene is repressed and the transcription factor GltC activates the expression of the GOGAT encoding gltAB genes. In the presence of amino acids of the glutamate family, the GDH RocG is synthesized and the enzyme prevents GltC from binding to DNA. The dual control of glutamate biosynthesis allows the efficient utilization of the available nutrients. Here we provide genetic and biochemical evidence that, like RocG, also the paralogous GDH GudB can inhibit the transcription factor GltC, thereby controlling glutamate biosynthesis. Contradictory previous observations show that high level of GDH activity does not result in permanent inhibition of GltC. By controlling the intracellular levels of glutamate through feeding with exogenous arginine, we observed that the GDH-dependent control of GltC and thus expression of the gltAB genes inversely correlates with the glutamate pool. These results suggest that the B. subtilis GDHs RocG and GudB in fact act as glutamate sensors. In conclusion, the GDH-mediated control of glutamate biosynthesis seems to depend on the intracellular glutamate concentration.

Ripening of pepper (Capsicum annuum) fruit is characterized by an enhancement of protein tyrosine nitration

Pepper (Capsicum annuum, Solanaceae) fruits are consumed worldwide and are of great economic importance. In most species ripening is characterized by important visual and metabolic changes, the latter including emission of volatile organic compounds associated with respiration, destruction of chlorophylls, synthesis of new pigments (red/yellow carotenoids plus xanthophylls and anthocyanins), formation of pectins and protein synthesis. The involvement of nitric oxide (NO) in fruit ripening has been established, but more work is needed to detail the metabolic networks involving NO and other reactive nitrogen species (RNS) in the process. It has been reported that RNS can mediate post-translational modifications of proteins, which can modulate physiological processes through mechanisms of cellular signalling. This study therefore examined the potential role of NO in nitration of tyrosine during the ripening of California sweet pepper. The NO content of green and red pepper fruit was determined spectrofluorometrically. Fruits at the breaking point between green and red coloration were incubated in the presence of NO for 1 h and then left to ripen for 3 d. Profiles of nitrated proteins were determined using an antibody against nitro-tyrosine (NO2-Tyr), and profiles of nitrosothiols were determined by confocal laser scanning microscopy. Nitrated proteins were identified by 2-D electrophoresis and MALDI-TOF/TOF analysis. Treatment with NO delayed the ripening of fruit. An enhancement of nitrosothiols and nitroproteins was observed in fruit during ripening, and this was reversed by the addition of exogenous NO gas. Six nitrated proteins were identified and were characterized as being involved in redox, protein, carbohydrate and oxidative metabolism, and in glutamate biosynthesis. Catalase was the most abundant nitrated protein found in both green and red fruit. The RNS profile reported here indicates that ripening of pepper fruit is characterized by an enhancement of S-nitrosothiols and protein tyrosine nitration. The nitrated proteins identified have important functions in photosynthesis, generation of NADPH, proteolysis, amino acid biosynthesis and oxidative metabolism. The decrease of catalase in red fruit implies a lower capacity to scavenge H2O2, which would promote lipid peroxidation, as has already been reported in ripe pepper fruit.

Transcript changes in Vibrio cholerae in response to salt stress.

Vibrio cholerae, which is a serious human intestinal pathogen, often resides and thrives in estuaries but requires major self-regulation to overcome intestinal hyperosmotic stress or high salt stress in water and food. In the present study, we selected multiple O1 and O139 group V. cholerae strains that were isolated from different regions and during different years to study their salt tolerance. Based on the mechanisms that other bacteria use to respond to high salt stress, we selected salt stress-response related genes to study the mechanisms which V. cholerae responds to high salt stress.V. cholerae strains showed salt-resistance characteristics that varied in salt concentrations from 4% to 6%. However, group O1 and group O139 showed no significant difference in the degree of salt tolerance. The primary responses of bacteria to salt stress, including Na+ exclusion, K+ uptake and glutamate biosynthesis, were observed in V. cholerae strains. In addition, some sigma factors were up-regulated in V. cholerae strains, suggesting that V. cholerae may recruit common sigma factors to achieve an active salt stress response. However, some changes in gene transcript levels in response to salt stress in V. cholerae were strain-specific. In particular, hierarchical clustering of differentially expressed genes indicated that transcript levels of these genes were correlated with the degree of salt tolerance. Therefore, elevated transcript levels of some genes, including sigma factors and genes involved in peptidoglycan biosynthesis, may be due to the salt tolerance of strains. In addition, high salt-tolerant strains may recruit common as well as additional sigma factors to activate the salt stress response.Electronic supplementary materialThe online version of this article (doi:10.1186/s13099-014-0047-8) contains supplementary material, which is available to authorized users.

Identification of genes potentially involved in solute stress response in Sphingomonas wittichii RW1 by transposon mutant recovery.

The term water stress refers to the effects of low water availability on microbial growth and physiology. Water availability has been proposed as a major constraint for the use of microorganisms in contaminated sites with the purpose of bioremediation. Sphingomonas wittichii RW1 is a bacterium capable of degrading the xenobiotic compounds dibenzofuran and dibenzo-p-dioxin, and has potential to be used for targeted bioremediation. The aim of the current work was to identify genes implicated in water stress in RW1 by means of transposon mutagenesis and mutant growth experiments. Conditions of low water potential were mimicked by adding NaCl to the growth media. Three different mutant selection or separation method were tested which, however recovered different mutants. Recovered transposon mutants with poorer growth under salt-induced water stress carried insertions in genes involved in proline and glutamate biosynthesis, and further in a gene putatively involved in aromatic compound catabolism. Transposon mutants growing poorer on medium with lowered water potential also included ones that had insertions in genes involved in more general functions such as transcriptional regulation, elongation factor, cell division protein, RNA polymerase β or an aconitase.

OmGOGAT-disruption in the ericoid mycorrhizal fungus Oidiodendron maius induces reorganization of the N pathway and reduces tolerance to heavy-metals

Mycorrhizal fungi are key mediators of soil-to-plant movement of mineral nutrients, including essential and non-essential metals. In soil conditions that facilitate mobilization of metal ions, potentially toxic metals can interfere with nitrogen metabolism in both plants and microorganisms. Less is known about possible relationships between nitrogen metabolism and responses to heavy metals. Aim of this study was to investigate this aspect in the ericoid mycorrhizal fungus Oidiodendron maius strain Zn, a metal tolerant ascomycete. Growth of O. maius Zn on zinc and cadmium containing media was significantly affected by the nitrogen source. Screening of a library of O. maius Zn random genetic transformants for sensitivity to heavy metals (zinc and cadmium) and oxidative stress (menadione) yielded a mutant strain that carried a partial deletion of the glutamate synthase (NADH-GOGAT EC 1.4.1.14) gene and its adjacent gene, the APC15 subunit of the anaphase promoting complex. Comparison of WT and OmGOGAT-OmAPC15 mutant strains indicated an impaired N-metabolism and altered stress tolerance, and assays on the OmAPC15-recomplemented strains ascribed the observed phenotypes to the deletion in the OmGOGAT gene. OmGOGAT disruption modified the nitrogen pathway, with a strong reduction of the associated glutamine synthetase (GS, EC 6.3.1.2) activity and an up-regulation of the alternative NADP-glutamate dehydrogenase (NADP-GDH, EC 1.4.1.4) pathway for glutamate biosynthesis. Unless they were supplemented with glutamine, O. maius Zn transformants lacking OmGOGAT were very sensitive to zinc. These results highlight the importance of nitrogen metabolism not only for nitrogen assimilation and transformation, but also for stress tolerance. For mycorrhizal fungi, such as O. maius, this may bear consequences not only to the fungus, but also to the host plant.

Glutamine synthetase 2 is not essential for biosynthesis of compatible solutes in Halobacillus halophilus.

Halobacillus halophilus, a moderately halophilic bacterium isolated from salt marshes, produces various compatible solutes to cope with osmotic stress. Glutamate and glutamine are dominant compatible solutes at mild salinities. Glutamine synthetase activity in cell suspensions of Halobacillus halophilus wild type was shown to be salt dependent and chloride modulated. A possible candidate to catalyze glutamine synthesis is glutamine synthetase A2, whose transcription is stimulated by chloride. To address the role of GlnA2 in the biosynthesis of the osmolytes glutamate and glutamine, a deletion mutant (ΔglnA2) was generated and characterized in detail. We compared the pool of compatible solutes and performed transcriptional analyses of the principal genes controlling the solute production in the wild type strain and the deletion mutant. These measurements did not confirm the hypothesized role of GlnA2 in the osmolyte production. Most likely the presence of another, yet to be identified enzyme has the main contribution in the measured activity in crude extracts and probably determines the total chloride-modulated profile. The role of GlnA2 remains to be elucidated.

Differential contribution of the proline and glutamine pathways to glutamate biosynthesis and nitrogen assimilation in yeast lacking glutamate dehydrogenase

In Saccharomyces cerevisiae, the glutamate dehydrogenase (GDH) enzymes play a pivotal role in glutamate biosynthesis and nitrogen assimilation. It has been proposed that, in GDH-deficient yeast, either the proline utilization (PUT) or the glutamine synthetase–glutamate synthase (GS/GOGAT) pathway serves as the alternative pathway for glutamate production and nitrogen assimilation to the exclusion of the other. Using a gdh-null mutant (gdh1Δ2Δ3Δ), this ambiguity was addressed using a combination of growth studies and pathway-specific enzyme assays on a variety of nitrogen sources (ammonia, glutamine, proline and urea). The GDH-null mutant was viable on all nitrogen sources tested, confirming that alternate pathways for nitrogen assimilation exist in the gdh-null strain. Enzyme assays point to GS/GOGAT as the primary alternative pathway on the preferred nitrogen sources ammonia and glutamine, whereas growth on proline required both the PUT and GS/GOGAT pathways. In contrast, growth on glucose–urea media elicited a decrease in GOGAT activity along with an increase in activity of the PUT pathway specific enzyme Δ1-pyrroline-5-carboxylate dehydrogenase (P5CDH). Together, these results suggest the alternative pathway for nitrogen assimilation in strains lacking the preferred GDH-dependent route is nitrogen source dependent and that neither GS/GOGAT nor PUT serves as the sole compensatory pathway.

Physiology and gene expression profiles of Dekkera bruxellensis in response to carbon and nitrogen availability

The assimilation of nitrate, a nitrogenous compound, was previously described as an important factor favoring Dekkera bruxellensis in the competition with Saccharomyces cerevisiae for the industrial sugarcane substrate. In this substrate, nitrogen sources are limited and diverse, and a recent report showed that amino acids enable D. bruxellensis to grow anaerobically. Thus, understanding the regulation of nitrogen metabolism is one fundamental aspect to comprehend the competiveness of D. bruxellensis in the fermentation environment. In the present study, we evaluated the physiological and transcriptional profiles of D. bruxellensis in response to different carbon and nitrogen supplies to determine their influence on growth, sugar consumption, and ethanol production. Besides, the expression of genes coding for nitrogen permeases and enzymes involved in the biosynthesis of glutamate and energetic metabolism were investigated under these conditions. Our data revealed that genes related to nitrogen uptake in D. bruxellensis are under the control of nitrogen catabolite repression. Moreover, we provide indications that glutamate dehydrogenase and glutamate synthase may switch roles as the major pathway for glutamate biosynthesis in D. bruxellensis. Finally, our data showed that in nonoptimal growth conditions, D. bruxellensis leans toward the respiratory metabolism. The results presented herein show that D. bruxellensis and S. cerevisiae share similar regulation of GDH–GOGAT pathway, while D. bruxellensis converts less glucose to ethanol than S. cerevisiae do when nitrogen is limited. The consequence of this particularity to the industrial process is discussed.

Metabolic evolution of Corynebacterium glutamicum for increased production of L-ornithine

BackgroundL-ornithine is effective in the treatment of liver diseases and helps strengthen the heart. The commercial applications mean that efficient biotechnological production of L-ornithine has become increasingly necessary. Adaptive evolution strategies have been proven a feasible and efficient technique to achieve improved cellular properties without requiring metabolic or regulatory details of the strain. The evolved strains can be further optimised by metabolic engineering. Thus, metabolic evolution strategy was used for engineering Corynebacterium glutamicum to enhance L-ornithine production.ResultsA C. glutamicum strain was engineered by using a combination of gene deletions and adaptive evolution with 70 passages of growth-based selection. The metabolically evolved C. glutamicum strain, named ΔAPE6937R42, produced 24.1 g/L of L-ornithine in a 5-L bioreactor. The mechanism used by C. glutamicum ΔAPE6937R42 to produce L-ornithine was investigated by analysing transcriptional levels of select genes and NADPH contents. The upregulation of the transcription levels of genes involved in the upstream pathway of glutamate biosynthesis and the elevated NADPH concentration caused by the upregulation of the transcriptional level of the ppnK gene promoted L-ornithine production in C. glutamicum ΔAPE6937R42.ConclusionsThe availability of NADPH plays an important role in L-ornithine production in C. glutamicum. Our results demonstrated that the combination of growth-coupled evolution with analysis of transcript abundances provides a strategy to engineer microbial strains for improving production of target compounds.

Astrocytic Control of Biosynthesis and Turnover of the Neurotransmitters Glutamate and GABA

Glutamate and GABA are the quantitatively major neurotransmitters in the brain mediating excitatory and inhibitory signaling, respectively. These amino acids are metabolically interrelated and at the same time they are tightly coupled to the intermediary metabolism including energy homeostasis. Astrocytes play a pivotal role in the maintenance of the neurotransmitter pools of glutamate and GABA since only these cells express pyruvate carboxylase, the enzyme required for de novo synthesis of the two amino acids. Such de novo synthesis is obligatory to compensate for catabolism of glutamate and GABA related to oxidative metabolism when the amino acids are used as energy substrates. This, in turn, is influenced by the extent to which the cycling of the amino acids between neurons and astrocytes may occur. This cycling is brought about by the glutamate/GABA – glutamine cycle the operation of which involves the enzymes glutamine synthetase (GS) and phosphate-activated glutaminase together with the plasma membrane transporters for glutamate, GABA, and glutamine. The distribution of these proteins between neurons and astrocytes determines the efficacy of the cycle and it is of particular importance that GS is exclusively expressed in astrocytes. It should be kept in mind that the operation of the cycle is associated with movement of ammonia nitrogen between the two cell types and different mechanisms which can mediate this have been proposed. This review is intended to delineate the above mentioned processes and to discuss quantitatively their relative importance in the homeostatic mechanisms responsible for the maintenance of optimal conditions for the respective neurotransmission processes to operate.

The Glutamate-Glutamine (GABA) Cycle: Importance of Late Postnatal Development and Potential Reciprocal Interactions between Biosynthesis and Degradation.

The gold standard for studies of glutamate–glutamine (GABA) cycling and its connections to brain biosynthesis from glucose of glutamate and GABA and their subsequent metabolism are the elegant in vivo studies by 13C magnetic resonance spectroscopy (NMR), showing the large fluxes in the cycle. However, simpler experiments in intact brain tissue (e.g., immunohistochemistry), brain slices, cultured brain cells, and mitochondria have also made important contributions to the understanding of details, mechanisms, and functional consequences of glutamate/GABA biosynthesis and degradation. The purpose of this review is to attempt to integrate evidence from different sources regarding (i) the enzyme(s) responsible for the initial conversion of α-ketoglutarate to glutamate; (ii) the possibility that especially glutamate oxidation is essentially confined to astrocytes; and (iii) the ontogenetically very late onset and maturation of glutamine–glutamate (GABA) cycle function. Pathway models based on the functional importance of aspartate for glutamate synthesis suggest the possibility of interacting pathways for biosynthesis and degradation of glutamate and GABA and the use of transamination as the default mechanism for initiation of glutamate oxidation. The late development and maturation are related to the late cortical gliogenesis and convert brain cortical function from being purely neuronal to becoming neuronal-astrocytic. This conversion is associated with huge increases in energy demand and production, and the character of potentially incurred gains of function are discussed. These may include alterations in learning mechanisms, in mice indicated by lack of pairing of odor learning with aversive stimuli in newborn animals but the development of such an association 10–12 days later. The possibility is suggested that analogous maturational changes may contribute to differences in the way learning is accomplished in the newborn human brain and during later development.

American Diabetes Association Research Symposium: Diabetes and the Brain

From 28–30 October 2011, more than 100 people assembled in Alexandria, Virginia, to participate in an American Diabetes Association–sponsored research symposium entitled “Diabetes and the Brain.” The objective of the symposium was to discuss the role of the brain in normal and abnormal metabolism and to consider the impact of diabetes on cerebral structure and function. Symposium participants were particularly interested in understanding how abnormalities in brain metabolism could affect the development of diabetes and obesity and how these diseases could, in turn, affect learning and memory. The growing epidemic of diabetes brought urgency to the meeting because of the ever-increasing number of people placed at risk for the cerebral complications of the disease. In this report, we present meeting highlights in five related areas as follows: 1) metabolism, blood flow, and epigenetics; 2 ) glucose sensing and hypoglycemia counterregulation; 3) insulin resistance and action in the brain; 4) neurocognition and imaging; and 5 ) energy homeostasis. The symposium opened with an outstanding review of cerebral metabolism by Gerald Dienel, PhD, from the University of Arkansas. He emphasized that glucose is the major fuel for the adult brain and that it has multifunctional roles in brain function. Metabolism of glucose via the glycolytic, pentose phosphate shunt, and tricarboxylic acid cycle pathways provides ATP for energy, NADPH for defense against oxidative stress, and carbon for biosynthesis of amino acids and sugars used for synthesis of glycoproteins and glycolipids and for de novo synthesis of tricarboxylic acid cycle-derived neurotransmitters glutamate and γ-aminobutyric acid (GABA) (Fig. 1). Glycogen is stored predominately in astrocytes, and the CO2 fixation reaction required for glutamate biosynthesis occurs in astrocytes. Local rates of blood flow and glucose utilization are closely linked to local activities of brain cells, and metabolic rates increase with increased functional activity and decline when cellular …

The fungal α‐aminoadipate pathway for lysine biosynthesis requires two enzymes of the aconitase family for the isomerization of homocitrate to homoisocitrate

Fungi produce α-aminoadipate, a precursor for penicillin and lysine via the α-aminoadipate pathway. Despite the biotechnological importance of this pathway, the essential isomerization of homocitrate via homoaconitate to homoisocitrate has hardly been studied. Therefore, we analysed the role of homoaconitases and aconitases in this isomerization. Although we confirmed an essential contribution of homoaconitases from Saccharomyces cerevisiae and Aspergillus fumigatus, these enzymes only catalysed the interconversion between homoaconitate and homoisocitrate. In contrast, aconitases from fungi and the thermophilic bacterium Thermus thermophilus converted homocitrate to homoaconitate. Additionally, a single aconitase appears essential for energy metabolism, glutamate and lysine biosynthesis in respirating filamentous fungi, but not in the fermenting yeast S. cerevisiae that possesses two contributing aconitases. While yeast Aco1p is essential for the citric acid cycle and, thus, for glutamate synthesis, Aco2p specifically and exclusively contributes to lysine biosynthesis. In contrast, Aco2p homologues present in filamentous fungi were transcribed, but enzymatically inactive, revealed no altered phenotype when deleted and did not complement yeast aconitase mutants. From these results we conclude that the essential requirement of filamentous fungi for respiration versus the preference of yeasts for fermentation may have directed the evolution of aconitases contributing to energy metabolism and lysine biosynthesis.

Analysis of the rat hypothalamus proteome by data-independent label-free LC-MS/MS.

Studies of neuronal, endocrine, and metabolic disorders would be facilitated by characterization of the hypothalamus proteome. Protein extracts prepared from 16 whole rat hypothalami were measured by data-independent label-free nano LC-MS/MS. Peptide features were detected, aligned, and searched against a rat Swiss-Prot database using ProteinLynx Global Server v.2.5. The final combined dataset comprised 21 455 peptides, corresponding to 622 unique proteins, each identified by a minimum of two distinct peptides. The majority of the proteins (69%) were cytosolic, and 16% were membrane proteins. Important proteins involved in neurological and synaptic function were identified including several members of the Ras-related protein family and proteins involved in glutamate biosynthesis.

Control of glutamate homeostasis in Bacillus subtilis: a complex interplay between ammonium assimilation, glutamate biosynthesis and degradation

Glutamate, the major amino group donor in anabolism, is synthesized by the combined action of the glutamine synthetase (GS) and the glutamate synthase (GOGAT) in Bacillus subtilis. The glutamate dehydrogenase (GDH) exclusively degrades glutamate. GS and GDH are both trigger enzymes, active in nitrogen metabolism and in controlling gene expression. Feedback-inhibited GS (FBI-GS) controls DNA-binding activities of two transcription factors, the repressor GlnR and TnrA, the global regulator of nitrogen metabolism. FBI-GS binds to and activates GlnR. This protein complex inhibits GS formation and thus glutamine synthesis. Moreover, FBI-GS inhibits DNA-binding activity of TnrA. Glutamate biosynthesis, the reaction linking carbon with nitrogen metabolism, is controlled by GDH. Together with glutamate GDH inhibits GltC, the transcription factor that activates expression of the GOGAT genes. Thus, GS and GDH control glutamine and glutamate synthesis, respectively, depending on the nitrogen status of the cell. B. subtilis lacking a functional GDH show a severe growth defect. Interestingly, the growth defect is suppressed by the rapid activation of an inactive GDH. Thus, maintenance of glutamate homeostasis is crucial for cellular vitality. This review covers the recent work on the complex control of glutamine and glutamate metabolism in the Gram-positive model organism B. subtilis.