scholarly journals Functions of the Membrane-Associated and Cytoplasmic Malate Dehydrogenases in the Citric Acid Cycle of Corynebacterium glutamicum

2000 ◽  
Vol 182 (24) ◽  
pp. 6884-6891 ◽  
Author(s):  
Douwe Molenaar ◽  
Michel E. van der Rest ◽  
André Drysch ◽  
Raif Yücel
Keyword(s):  
Citric Acid ◽  
Corynebacterium Glutamicum ◽  
Malate Dehydrogenase ◽  
Citric Acid Cycle ◽  
Pyridine Nucleotide ◽  
Acid Cycle ◽  
Link Type ◽  
A Site ◽  
Malate Oxidation

ABSTRACT Like many other bacteria, Corynebacterium glutamicumpossesses two types of l-malate dehydrogenase, a membrane-associated malate:quinone oxidoreductase (MQO; EC 1.1.99.16 ) and a cytoplasmic malate dehydrogenase (MDH; EC 1.1.1.37 ) The regulation of MDH and of the three membrane-associated dehydrogenases MQO, succinate dehydrogenase (SDH), and NADH dehydrogenase was investigated. MQO, MDH, and SDH activities are regulated coordinately in response to the carbon and energy source for growth. Compared to growth on glucose, these activities are increased during growth on lactate, pyruvate, or acetate, substrates which require high citric acid cycle activity to sustain growth. The simultaneous presence of high activities of both malate dehydrogenases is puzzling. MQO is the most important malate dehydrogenase in the physiology of C. glutamicum. A mutant with a site-directed deletion in themqo gene does not grow on minimal medium. Growth can be partially restored in this mutant by addition of the vitamin nicotinamide. In contrast, a double mutant lacking MQO and MDH does not grow even in the presence of nicotinamide. Apparently, MDH is able to take over the function of MQO in an mqo mutant, but this requires the presence of nicotinamide in the growth medium. It is shown that addition of nicotinamide leads to a higher intracellular pyridine nucleotide concentration, which probably enables MDH to catalyze malate oxidation. Purified MDH from C. glutamicum catalyzes oxaloacetate reduction much more readily than malate oxidation at physiological pH. In a reconstituted system with isolated membranes and purified MDH, MQO and MDH catalyze the cyclic conversion of malate and oxaloacetate, leading to a net oxidation of NADH. Evidence is presented that this cyclic reaction also takes place in vivo. As yet, no phenotype of an mdh deletion alone was observed, which leaves a physiological function for MDH in C. glutamicumobscure.

scholarly journals Metabolism of [3-13C]pyruvate and [3-13C]propionate in normal and ischaemic rat heart in vivo: 1H- and 13C-NMR studies

1995 ◽  
Vol 312 (1) ◽  
pp. 75-81 ◽  
Author(s):  
B Sumegi ◽  
B Podanyi ◽  
P Forgo ◽  
K E Kover
Keyword(s):  
Citric Acid ◽  
13C Nmr ◽  
Rat Heart ◽  
Citric Acid Cycle ◽  
Nmr Studies ◽  
Acetyl Coa ◽  
1H And 13C Nmr ◽  
Acid Cycle ◽  
Propionyl Carnitine

The oxidation of [3-13C]pyruvate and [3-13C]propionate was studied in vivo in infused rats. The infused [3-13C]pyruvate was quickly converted to [3-13C]lactate in the blood, and the [3-13C]lactate formed was well metabolized in both normoxic and ischaemic hearts. Large differences (200-600%) in the 13C enrichment of alanine (C-3) and acetyl-CoA (C-2) compared with lactate (C-3) were found in both normoxic and ischaemic hearts, suggesting that the extracellular [3-13C]lactate preferentially entered a region of the cytoplasm which specifically transfers the labelled pyruvate (formed from [3-13C]lactate) to the mitochondria. The highly enriched mitochondrial pyruvate gave high enrichment in alanine and acetyl-CoA, which was detected by 1H- and 13C-NMR spectroscopy. Ischaemia increased 13C incorporation into the main cytoplasmic lactate pool and decreased 13C incorporation into citric acid cycle intermediates, mainly decreasing the pyruvate anaplerosis. Isoprenaline-induced ischaemia of the heart caused only a slight decrease in pyruvate oxidation. In contrast to the decreased anaplerosis of pyruvate, the anaplerosis of propionate (and propionyl-carnitine) increased significantly in ischaemic hearts, which may contribute to the protective effect of propionyl-carnitine seen in ischaemia. In addition, we found that [3-13C]propionate preferentially labelled aspartate C-3 in rat heart, suggesting incomplete randomization of label in the succinyl-CoA-malate span of the citric acid cycle. These data show that proton observed 13C edited spectroscopic methods, i.e. heteronuclear spin-echo and the one-dimensional heteronuclear multiple quantum coherence sequence, can be successfully used to study heart metabolism in vivo.

scholarly journals Functions of the Membrane-Associated and Cytoplasmic Malate Dehydrogenases in the Citric Acid Cycle ofEscherichia coli

2000 ◽  
Vol 182 (24) ◽  
pp. 6892-6899 ◽  
Author(s):  
Michel E. van der Rest ◽  
Christian Frank ◽  
Douwe Molenaar
Keyword(s):  
Citric Acid ◽  
Malic Enzyme ◽  
Citric Acid Cycle ◽  
Wild Type ◽  
Batch Cultures ◽  
Acid Cycle ◽  
E Coli ◽  
Link Type ◽  
Phosphoenolpyruvate Synthase ◽  
Rates Of Growth

ABSTRACT Oxidation of malate to oxaloacetate in Escherichia colican be catalyzed by two enzymes: the well-known NAD-dependent malate dehydrogenase (MDH; EC 1.1.1.37 ) and the membrane-associated malate:quinone-oxidoreductase (MQO; EC 1.1.99.16 ), encoded by the genemqo (previously called yojH). Expression of themqo gene and, consequently, MQO activity are regulated by carbon and energy source for growth. In batch cultures, MQO activity was highest during exponential growth and decreased sharply after onset of the stationary phase. Experiments with the β-galactosidase reporter fused to the promoter of the mqo gene indicate that its transcription is regulated by the ArcA-ArcB two-component system. In contrast to earlier reports, MDH did not repressmqo expression. On the contrary, MQO and MDH are active at the same time in E. coli. For Corynebacterium glutamicum, it was found that MQO is the principal enzyme catalyzing the oxidation of malate to oxaloacetate. These observations justified a reinvestigation of the roles of MDH and MQO in the citric acid cycle of E. coli. In this organism, a defined deletion of the mdh gene led to severely decreased rates of growth on several substrates. Deletion of the mqo gene did not produce a distinguishable effect on the growth rate, nor did it affect the fitness of the organism in competition with the wild type. To investigate whether in an mqo mutant the conversion of malate to oxaloacetate could have been taken over by a bypass route via malic enzyme, phosphoenolpyruvate synthase, and phosphenolpyruvate carboxylase, deletion mutants of the malic enzyme genessfcA and b2463 (coding for EC 1.1.1.38 and EC1.1.1.40 , respectively) and of the phosphoenolpyruvate synthase (EC2.7.9.2 ) gene pps were created. They were introduced separately or together with the deletion of mqo. These studies did not reveal a significant role for MQO in malate oxidation in wild-type E. coli. However, comparing growth of themdh single mutant to that of the double mutant containingmdh and mqo deletions did indicate that MQO partly takes over the function of MDH in an mdh mutant.

scholarly journals Quantitative Determination of Metabolic Fluxes during Coutilization of Two Carbon Sources: Comparative Analyses withCorynebacterium glutamicum during Growth on Acetate and/or Glucose

2000 ◽  
Vol 182 (11) ◽  
pp. 3088-3096 ◽  
Author(s):  
Volker F. Wendisch ◽  
Albert A. de Graaf ◽  
Hermann Sahm ◽  
Bernhard J. Eikmanns
Keyword(s):  
Citric Acid ◽  
Citric Acid Cycle ◽  
Glyoxylate Cycle ◽  
Carbon Sources ◽  
Malate Synthase ◽  
Total Carbon ◽  
Acid Cycle ◽  
Consumption Rates ◽  
The Glyoxylate Cycle

ABSTRACT Growth of Corynebacterium glutamicum on mixtures of the carbon sources glucose and acetate is shown to be distinct from growth on either substrate alone. The organism showed nondiauxic growth on media containing acetate-glucose mixtures and simultaneously metabolized these substrates. Compared to those for growth on acetate or glucose alone, the consumption rates of the individual substrates were reduced during acetate-glucose cometabolism, resulting in similar total carbon consumption rates for the three conditions. By13C-labeling experiments with subsequent nuclear magnetic resonance analyses in combination with metabolite balancing, the in vivo activities for pathways or single enzymes in the central metabolism of C. glutamicum were quantified for growth on acetate, on glucose, and on both carbon sources. The activity of the citric acid cycle was high on acetate, intermediate on acetate plus glucose, and low on glucose, corresponding to in vivo activities of citrate synthase of 413, 219, and 111 nmol · (mg of protein)−1 · min−1, respectively. The citric acid cycle was replenished by carboxylation of phosphoenolpyruvate (PEP) and/or pyruvate (30 nmol · [mg of protein]−1 · min−1) during growth on glucose. Although levels of PEP carboxylase and pyruvate carboxylase during growth on acetate were similar to those for growth on glucose, anaplerosis occurred solely by the glyoxylate cycle (99 nmol · [mg of protein]−1 · min−1). Surprisingly, the anaplerotic function was fulfilled completely by the glyoxylate cycle (50 nmol · [mg of protein]−1 · min−1) on glucose plus acetate also. Consistent with the predictions deduced from the metabolic flux analyses, a glyoxylate cycle-deficient mutant ofC. glutamicum, constructed by targeted deletion of the isocitrate lyase and malate synthase genes, exhibited impaired growth on acetate-glucose mixtures.

scholarly journals A photo-switchable yeast isocitrate dehydrogenase to control metabolic flux through the citric acid cycle

2021 ◽  
Author(s):  
Haoqi Chen ◽  
Lianne Mulder ◽  
Hein J. Wijma ◽  
Ronja Wabeke ◽  
Jose Pedro Vila Cha Losa ◽  
...  
Keyword(s):  
Citric Acid ◽  
Isocitrate Dehydrogenase ◽  
Metabolic Flux ◽  
Citric Acid Cycle ◽  
Computational Design ◽  
Metabolic Enzyme ◽  
Turnover Rates ◽  
In Vivo Screening ◽  
Acid Cycle

For various research questions in metabolism, it is highly desirable to have means available, with which the flux through specific pathways can be perturbed dynamically, in a reversible manner, and at a timescale that is consistent with the fast turnover rates of metabolism. Optogenetics, in principle, offers such possibility. Here, we developed an initial version of a photo-switchable isocitrate dehydrogenase (IDH) aimed at controlling the metabolic flux through the citric acid cycle in budding yeast. By inserting a protein-based light switch (LOV2) into computationally identified active/regulatory-coupled sites of IDH and by using in vivo screening in Saccharomyces cerevisiae, we obtained a number of IDH enzymes whose activity can be switched by light. Subsequent in-vivo characterization and optimization resulted in an initial version of photo-switchable (PS) IDH. While further improvements of the enzyme are necessary, our study demonstrates the efficacy of the overall approach from computational design, via in vivo screening and characterization. It also represents one of the first few examples, where optogenetics were used to control the activity of a metabolic enzyme.

Pyruvate and citric acid cycle carbon requirements in isolated skeletal muscle mitochondria

2004 ◽  
Vol 286 (3) ◽  
pp. C565-C572 ◽  
Author(s):  
Jeffrey I. Messer ◽  
Matthew R. Jackman ◽  
Wayne T. Willis
Keyword(s):  
Skeletal Muscle ◽  
Citric Acid ◽  
Citric Acid Cycle ◽  
Thermodynamic Force ◽  
Glycogen Depletion ◽  
Acid Cycle ◽  
Physiological Range ◽  
Skeletal Muscle Mitochondria

Carbohydrate depletion precipitates fatigue in skeletal muscle, but, because pyruvate provides both acetyl-CoA for mainline oxidation and anaplerotic carbon to the citric acid cycle (CAC), the mechanism remains obscure. Thus pyruvate and CAC kinetic parameters were independently quantified in mitochondria isolated from rat mixed skeletal muscle. Mitochondrial oxygen consumption rate ( Jo) was measured polarographically while either pyruvate or malate was added stepwise in the presence of a saturating concentration of the other substrate. These substrate titrations were carried out across a physiological range of fixed extramitochondrial ATP free energy states (ΔGP), established with a creatine kinase energy clamp, and also at saturating [ADP]. The apparent Km,malate for mitochondrial Jo ranged from 21 to 32 μM, and the apparent Km,pyruvate ranged from 12 to 26 μM, with both substrate Km values increasing as ΔGP declined. Vmax for both substrates also increased as ΔGP fell, reflecting thermodynamic control of Jo. Reported in vivo skeletal muscle [malate] are >10-fold greater than the Km,malate determined in this study. In marked contrast, the Km,pyruvate determined is near the [pyruvate] reported in muscle approaching exhaustion associated with glycogen depletion. When data were evaluated in the context of a linear thermodynamic force-flow (ΔGP- Jo) relationship, the ΔGP- Jo slope was essentially insensitive to changes in [malate] in the range observed in vivo but decreased markedly with declining [pyruvate] across the physiological range. Mitochondrial respiration is particularly sensitive to variations in [pyruvate] in the physiological range. In contrast, physiological [malate] exerts very little, if any, influence on mitochondrial pyruvate oxidation measured in vitro.

Quantitative assessment of anaplerosis from propionate in pig heart in vivo

2003 ◽  
Vol 284 (2) ◽  
pp. E351-E356 ◽  
Author(s):  
Wenjun Z. Martini ◽  
William C. Stanley ◽  
Hazel Huang ◽  
Christine Des Rosiers ◽  
Charles L. Hoppel ◽  
...  
Keyword(s):  
Citric Acid ◽  
Citric Acid Cycle ◽  
Contractile Function ◽  
Fatty Acid Uptake ◽  
Cardiac Metabolism ◽  
Acid Uptake ◽  
Acid Cycle ◽  
Isotopomer Distribution ◽  
Mass Isotopomer

Normal cardiac metabolism requires continuous replenishment (anaplerosis) of catalytic intermediates of the citric acid cycle. Little is known about the quantitative aspects of propionate as a substrate of in vivo anaplerosis; therefore, we measured the rate of propionate entry into the citric acid cycle in hearts of anesthetized pigs. [U-13C3]propionate (0.25 mM) was infused in a coronary artery branch for 1 h via an extracorporeal perfusion circuit, and cardiac biopsies were analyzed for the mass isotopomer distribution of citric acid cycle intermediates. Infusion of propionate did not affect myocardial oxygen consumption, heart rate, or contractile function. In the infused territory, propionate infusion did not affect uptake of glucose and lactate but decreased free fatty acid uptake by one-half ( P < 0.05). Propionate extraction and uptake were 57.4 ± 3.3% and 0.078 ± 0.009 μmol · min−1 · g−1. Anaplerosis from propionate, calculated from the mass isotopomer distribution of succinate, accounted for 8.9 ± 1.3% of the citric acid cycle flux. Propioylcarnitine release accounted for only 0.033 ± 0.002% of propionate uptake. Methylcitrate did not accumulate. Thus administration of a low concentration of propionate appears to be a convenient and safe way to boost anaplerosis in the heart.

Sign in / Sign up

Export Citation Format

Share Document