Investigation of the TCA cycle and the glyoxylate shunt inEscherichia coli BL21 and JM109 using13C-NMR/MS

2000 ◽  
Vol 68 (3) ◽  
pp. 316-327 ◽  
Author(s):  
S. B. Noronha ◽  
H. J. C. Yeh ◽  
T. F. Spande ◽  
J. Shiloach
Keyword(s):  
Tca Cycle ◽  
Glyoxylate Shunt ◽  
Inescherichia Coli ◽  
The Tca Cycle

scholarly journals Metabolic flux partitioning between the TCA cycle and glyoxylate shunt combined with a reversible methyl citrate cycle provide nutritional flexibility for Mycobacterium tuberculosis

2021 ◽  
Author(s):  
Khushboo Borah ◽  
Tom A. Mendum ◽  
Nathaniel D. Hawkins ◽  
Jane L. Ward ◽  
Michael H. Beale ◽  
...  
Keyword(s):  
Metabolic Network ◽  
Metabolic Flux ◽  
Carbon Sources ◽  
Tca Cycle ◽  
Flux Analysis ◽  
Glyoxylate Shunt ◽  
Citrate Cycle ◽  
Metabolic Fluxes ◽  
The Tca Cycle ◽  
Carbon Substrates

AbstractThe utilisation of multiple host-derived carbon substrates is required by Mycobacterium tuberculosis (Mtb) to successfully sustain a tuberculosis infection thereby identifying the Mtb specific metabolic pathways and enzymes required for carbon co-metabolism as potential drug targets. Metabolic flux represents the final integrative outcome of many different levels of cellular regulation that contribute to the flow of metabolites through the metabolic network. It is therefore critical that we have an in-depth understanding of the rewiring of metabolic fluxes in different conditions. Here, we employed 13C-metabolic flux analysis using stable isotope tracers (13C and 2H) and lipid fingerprinting to investigate the metabolic network of Mtb growing slowly on physiologically relevant carbon sources in a steady state chemostat. We demonstrate that Mtb is able to efficiently co-metabolise combinations of either cholesterol or glycerol along with C2 generating carbon substrates. The uniform assimilation of the carbon sources by Mtb throughout the network indicated no compartmentalization of metabolism in these conditions however there were substrate specific differences in metabolic fluxes. This work identified that partitioning of flux between the TCA cycle and the glyoxylate shunt combined with a reversible methyl citrate cycle as the critical metabolic nodes which underlie the nutritional flexibility of Mtb. These findings provide new insights into the metabolic architecture that affords adaptability of Mtb to divergent carbon substrates.ImportanceEach year more than 1 million people die of tuberculosis (TB). Many more are infected but successfully diagnosed and treated with antibiotics, however antibiotic-resistant TB isolates are becoming ever more prevalent and so novel therapies are urgently needed that can effectively kill the causative agent. Mtb specific metabolic pathways have been identified as an important drug target in TB. However the apparent metabolic plasticity of this pathogen presents a major obstacle to efficient targeting of Mtb specific vulnerabilities and therefore it is critical to define the metabolic fluxes that Mtb utilises in different conditions. Here, we used 13C-metabolic flux analysis to measure the metabolic fluxes that Mtb uses whilst growing on potential in vivo nutrients. Our analysis identified selective use of the metabolic network that included the TCA cycle, glyoxylate shunt and methyl citrate cycle. The metabolic flux phenotypes determined in this study improves our understanding about the co-metabolism of multiple carbon substrates by Mtb identifying a reversible methyl citrate cycle and the glyoxylate shunt as the critical metabolic nodes which underlie the nutritional flexibility of Mtb.

The Glyoxylate Shunt, 60 Years On

2018 ◽  
Vol 72 (1) ◽  
pp. 309-330 ◽  
Author(s):  
Stephen K. Dolan ◽  
Martin Welch
Keyword(s):  
Citric Acid ◽  
Citric Acid Cycle ◽  
Tca Cycle ◽  
Oxidative Decarboxylation ◽  
Glyoxylate Shunt ◽  
Cellular Functions ◽  
Acid Cycle ◽  
E Coli ◽  
80Th Anniversary ◽  
The Tca Cycle

2017 marks the 60th anniversary of Krebs’ seminal paper on the glyoxylate shunt (and coincidentally, also the 80th anniversary of his discovery of the citric acid cycle). Sixty years on, we have witnessed substantial developments in our understanding of how flux is partitioned between the glyoxylate shunt and the oxidative decarboxylation steps of the citric acid cycle. The last decade has shown us that the beautifully elegant textbook mechanism that regulates carbon flux through the shunt in E. coli is an oversimplification of the situation in many other bacteria. The aim of this review is to assess how this new knowledge is impacting our understanding of flux control at the TCA cycle/glyoxylate shunt branch point in a wider range of genera, and to summarize recent findings implicating a role for the glyoxylate shunt in cellular functions other than metabolism.

scholarly journals Yeast Aconitase in Two Locations and Two Metabolic Pathways: Seeing Small Amounts Is Believing

2005 ◽  
Vol 16 (9) ◽  
pp. 4163-4171 ◽  
Author(s):  
Neta Regev-Rudzki ◽  
Sharon Karniely ◽  
Nitzan Natani Ben-Haim ◽  
Ophry Pines
Keyword(s):  
Metabolic Pathways ◽  
Tca Cycle ◽  
Tricarboxylic Acid ◽  
Glyoxylate Shunt ◽  
Mitochondrial Matrix ◽  
Hybrid Gene ◽  
Subcellular Compartments ◽  
The Tca Cycle ◽  
Dual Distribution ◽  
Fundamental Process

The distribution of identical enzymatic activities between different subcellular compartments is a fundamental process of living cells. At present, the Saccharomyces cerevisiae aconitase enzyme has been detected only in mitochondria, where it functions in the tricarboxylic acid (TCA) cycle and is considered a mitochondrial matrix marker. We developed two strategies for physical and functional detection of aconitase in the yeast cytosol: 1) we fused the α peptide of the β-galactosidase enzyme to aconitase and observed α complementation in the cytosol; and 2) we created an ACO1-URA3 hybrid gene, which allowed isolation of strains in which the hybrid protein is exclusively targeted to mitochondria. These strains display a specific phenotype consistent with glyoxylate shunt elimination. Together, our data indicate that yeast aconitase isoenzymes distribute between two distinct subcellular compartments and participate in two separate metabolic pathways; the glyoxylate shunt in the cytosol and the TCA cycle in mitochondria. We maintain that such dual distribution phenomena have a wider occurrence than recorded currently, the reason being that in certain cases there is a small fraction of one of the isoenzymes, in one of the locations, making its detection very difficult. We term this phenomenon of highly uneven isoenzyme distribution “eclipsed distribution.”

scholarly journals Three enzymes and one substrate; regulation of flux through the glyoxylate shunt in the opportunistic pathogen, Pseudomonas aeruginosa

2018 ◽  
Author(s):  
Audrey Crousilles ◽  
Stephen K. Dolan ◽  
Paul Brear ◽  
Dimitri Y. Chirgadze ◽  
Martin Welch
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Tca Cycle ◽  
Opportunistic Pathogen ◽  
Oxidative Decarboxylation ◽  
Glyoxylate Shunt ◽  
Reciprocal Regulation ◽  
Cycle Enzyme ◽  
The Tca Cycle ◽  
Flux Partitioning ◽  
Opportunistic Human Pathogen

AbstractThe glyoxylate shunt bypasses the oxidative decarboxylation steps of the tricarboxylic acid (TCA) cycle, thereby conserving carbon skeletons for biosynthesis. The branchpoint between the TCA cycle and the glyoxylate shunt is therefore widely considered to be one of the most important junctions in the whole of microbial metabolism. In Escherichia coli, AceK-mediated phosphorylation and inactivation of the TCA cycle enzyme, isocitrate dehydrogenase (ICD), is necessary to redirect flux through the first enzyme of the glyoxylate shunt, isocitrate lyase (ICL). In contrast, Mycobacterial species lack AceK and employ a phosphorylation-insensitive isocitrate dehydrogenase (IDH) at the branchpoint. Flux partitioning here is controlled “rheostatically” through cross-activation of IDH by the product of ICL activity, glyoxylate. However, the opportunistic human pathogen, Pseudomonas aeruginosa, expresses IDH, ICD, ICL and AceK. Here, we present the structure, kinetics and regulation of each branchpoint enzyme. We show that flux partitioning is coordinated through reciprocal regulation of the enzymes involved, beautifully linking carbon flux with the availability of key gluconeogenic precursors in a way that cannot be extrapolated from an understanding of the branchpoint enzymes in other organisms.

Liquid-liquid extraction of succinate using a dicopper cryptate

2020 ◽  
Author(s):  
Riccardo Mobili ◽  
Sonia La Cognata ◽  
Francesca Merlo ◽  
Andrea Speltini ◽  
Massimo Boiocchi ◽  
...  
Keyword(s):  
Aqueous Phase ◽  
Visible Spectrum ◽  
Tca Cycle ◽  
Residual Concentration ◽  
Waste Products ◽  
Liquid Liquid Extraction ◽  
The Tca Cycle ◽  
Quantitative Extraction ◽  
Uv Visible

<div> <p>The extraction of the succinate dianion from a neutral aqueous solution into dichloromethane is obtained using a lipophilic cage-like dicopper(II) complex as the extractant. The quantitative extraction exploits the high affinity of the succinate anion for the cavity of the azacryptate. The anion is effectively transferred from the aqueous phase, buffered at pH 7 with HEPES, into dichloromethane. A 1:1 extractant:anion adduct is obtained. Extraction can be easily monitored by following changes in the UV-visible spectrum of the dicopper complex in dichloromethane, and by measuring the residual concentration of succinate in the aqueous phase by HPLC−UV. Considering i) the relevance of polycarboxylates in biochemistry, as e.g. normal intermediates of the TCA cycle, ii) the relevance of dicarboxylates in the environmental field, as e.g. waste products of industrial processes, and iii) the recently discovered role of succinate and other dicarboxylates in pathophysiological processes including cancer, our results open new perspectives for research in all contexts where selective recognition, trapping and extraction of polycarboxylates is required. </p> </div>

scholarly journals Mitochondrial Mistranslation in Brain Provokes a Metabolic Response Which Mitigates the Age-Associated Decline in Mitochondrial Gene Expression

2021 ◽  
Vol 22 (5) ◽  
pp. 2746
Author(s):  
Dimitri Shcherbakov ◽  
Reda Juskeviciene ◽  
Adrián Cortés Sanchón ◽  
Margarita Brilkova ◽  
Hubert Rehrauer ◽  
...  
Keyword(s):  
Gene Expression ◽  
Metabolic Response ◽  
Mitochondrial Gene ◽  
Tca Cycle ◽  
Brain Mitochondria ◽  
Mitochondrial Gene Expression ◽  
Rna Seq ◽  
The Tca Cycle ◽  
Neurological Phenotype

Mitochondrial misreading, conferred by mutation V338Y in mitoribosomal protein Mrps5, in-vivo is associated with a subtle neurological phenotype. Brain mitochondria of homozygous knock-in mutant Mrps5V338Y/V338Y mice show decreased oxygen consumption and reduced ATP levels. Using a combination of unbiased RNA-Seq with untargeted metabolomics, we here demonstrate a concerted response, which alleviates the impaired functionality of OXPHOS complexes in Mrps5 mutant mice. This concerted response mitigates the age-associated decline in mitochondrial gene expression and compensates for impaired respiration by transcriptional upregulation of OXPHOS components together with anaplerotic replenishment of the TCA cycle (pyruvate, 2-ketoglutarate).

scholarly journals Metabolomics of aging in primary fibroblasts from small and large breed dogs

GeroScience ◽  
2021 ◽  
Author(s):  
Paul S. Brookes ◽  
Ana Gabriela Jimenez
Keyword(s):  
Aging Process ◽  
Tca Cycle ◽  
Therapeutic Strategies ◽  
Targeted Metabolomics ◽  
The Tca Cycle ◽  
Age Dependent ◽  
Primary Fibroblasts ◽  
Aging Rates ◽  
Underlying Mechanisms ◽  
Coenzyme A Biosynthesis

AbstractAmong several animal groups (eutherian mammals, birds, reptiles), lifespan positively correlates with body mass over several orders of magnitude. Contradicting this pattern are domesticated dogs, with small dog breeds exhibiting significantly longer lifespans than large dog breeds. The underlying mechanisms of differing aging rates across body masses are unclear, but it is generally agreed that metabolism is a significant regulator of the aging process. Herein, we performed a targeted metabolomics analysis on primary fibroblasts isolated from small and large breed young and old dogs. Regardless of size, older dogs exhibited lower glutathione and ATP, consistent with a role for oxidative stress and bioenergetic decline in aging. Furthermore, several size-specific metabolic patterns were observed with aging, including the following: (i) An apparent defect in the lower half of glycolysis in large old dogs at the level of pyruvate kinase. (ii) Increased glutamine anaplerosis into the TCA cycle in large old dogs. (iii) A potential defect in coenzyme A biosynthesis in large old dogs. (iv) Low nucleotide levels in small young dogs that corrected with age. (v) An age-dependent increase in carnitine in small dogs that was absent in large dogs. Overall, these data support the hypothesis that alterations in metabolism may underlie the different lifespans of small vs. large breed dogs, and further work in this area may afford potential therapeutic strategies to improve the lifespan of large dogs.

scholarly journals The Metabolic Fates of Pyruvate in Normal and Neoplastic Cells

Cells ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 762
Author(s):  
Edward V. Prochownik ◽  
Huabo Wang
Keyword(s):  
Metabolic Pathways ◽  
Redox State ◽  
Pyruvate Dehydrogenase Complex ◽  
Tca Cycle ◽  
Vascular Supply ◽  
Extrinsic Factors ◽  
The Tca Cycle ◽  
Initial Substrate ◽  
Numerous Cell ◽  
Bio Synthesis

Pyruvate occupies a central metabolic node by virtue of its position at the crossroads of glycolysis and the tricarboxylic acid (TCA) cycle and its production and fate being governed by numerous cell-intrinsic and extrinsic factors. The former includes the cell’s type, redox state, ATP content, metabolic requirements and the activities of other metabolic pathways. The latter include the extracellular oxygen concentration, pH and nutrient levels, which are in turn governed by the vascular supply. Within this context, we discuss the six pathways that influence pyruvate content and utilization: 1. The lactate dehydrogenase pathway that either converts excess pyruvate to lactate or that regenerates pyruvate from lactate for use as a fuel or biosynthetic substrate; 2. The alanine pathway that generates alanine and other amino acids; 3. The pyruvate dehydrogenase complex pathway that provides acetyl-CoA, the TCA cycle’s initial substrate; 4. The pyruvate carboxylase reaction that anaplerotically supplies oxaloacetate; 5. The malic enzyme pathway that also links glycolysis and the TCA cycle and generates NADPH to support lipid bio-synthesis; and 6. The acetate bio-synthetic pathway that converts pyruvate directly to acetate. The review discusses the mechanisms controlling these pathways, how they cross-talk and how they cooperate and are regulated to maximize growth and achieve metabolic and energetic harmony.

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