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

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.

Download Full-text

Malate : Quinone Oxidoreductase and Malic Enzyme are required for the Plant Pathogen Pseudomonas syringae pv. tomato DC3000 to Utilize Malate

American Journal of Undergraduate Research ◽

10.33697/ajur.2016.016 ◽

2016 ◽

Vol 13 (2) ◽

Author(s):

Zabrina Ebert ◽

Preston Jacob ◽

Katrina Jose ◽

Lina Fouad ◽

Katherine Vercellino ◽

...

Keyword(s):

Arabidopsis Thaliana ◽

Citric Acid ◽

Pseudomonas Syringae ◽

Plant Pathogen ◽

Malic Enzyme ◽

Citric Acid Cycle ◽

Wild Type ◽

In Planta ◽

Quinone Oxidoreductase ◽

Acid Cycle

Pseudomonas syringae pv. tomato strain DC3000 (DC3000) is a gram-negative bacterial plant pathogen that causes disease on tomato and the model plant Arabidopsis thaliana. Interestingly, previous studies showed that malate:quinone oxidoreductase (Mqo), an enzyme in the citric acid cycle, is required for DC3000 to cause disease on these plants. In addition, growth of DC3000 lacking the mqo gene in minimal medium with malate was significantly delayed, but eventually reached wild-type levels of growth, which is similar to growth in planta. This suggests that malate may be an important carbon source for DC3000. One reason the mqo::KO bacteria may be able to reach wild-type levels of growth in culture and plants is that the DC3000 malic enzyme may be used to complete the citric acid cycle. Our research shows that a mutant strain lacking a functional mqo gene and malic enzyme gene (mqo::KO;ME::pJP) fails to grow in minimal media cultures with malate and has reduced growth in media with citrate, indicating that both Mqo and ME are required for normal growth when utilizing these carbon sources. Future studies looking at growth of this double mutant in plants will identify how important the activities of both of these genes are for DC3000 to cause disease in plants. KEY WORDS: Malate:quinone Oxidoreductase; Malic Enzyme; MQO; Pseudomonas syringae; Arabidopsis thaliana; Malate; Citrate; DC3000

Download Full-text

The regulation of glucose and pyruvate formation from glutamine and citric-acid-cycle intermediates in the kidney cortex of rats, dogs, rabbits and guinea pigs

Biochemical Journal ◽

10.1042/bj1880741 ◽

1980 ◽

Vol 188 (3) ◽

pp. 741-748 ◽

Cited By ~ 21

Author(s):

M Watford ◽

P Vinay ◽

G Lemieux ◽

A Gougoux

Keyword(s):

Citric Acid ◽

Guinea Pig ◽

Guinea Pigs ◽

Malic Enzyme ◽

Citric Acid Cycle ◽

Alternative Pathway ◽

Lactate Production ◽

Kidney Cortex ◽

Acid Cycle ◽

Pig Kidney

The suppression by 3-mercaptopicolinate of gluconeogenesis from glutamine or 2-oxoglutarate in rat or dog kidney tubules did not affect the amount of these substrates undergoing complete oxidation. Furthermore, 3-mercaptopicolinate caused an accumulation of lactate in dog tubules. 3-Mercaptopicolinate abolished both gluconeogenesis and substrate oxidation in tubules from rabbit and guinea-pig kidney. These results imply the presence of an alternative pathway to phosphoenolpyruvate carboxykinase/pyruvate kinase for the production of pyruvate from citric-acid-cycle intermediates in the kidney cortex of rats and dogs but not in that of rabbits or guinea pigs. Oxaloacetate decarboxylase (present in the kidney cortex of all four species) or ‘malic’ enzyme (present in rat and dog but absent in rabbit and guinea-pig kidney cortex) could function in this role. Our observations indicate that ‘malic’ enzyme is probably implicated in this phenomenon. The lactate production observed in dog tubules in the presence of 3-mercaptopicolinate can be suppressed when aspartate formation is inhibited by 2-amino-4-methoxy-trans-but-3-enoic acid. This suggests that the provision of cytosolic NADH from citric-acid-cycle intermediates is facilitated by accumulation of aspartate acting as a ‘sink’ for cytosolic oxaloacetate.

Download Full-text

The ms2io6A37 Modification of tRNA inSalmonella typhimurium Regulates Growth on Citric Acid Cycle Intermediates

Journal of Bacteriology ◽

10.1128/jb.180.12.3144-3151.1998 ◽

1998 ◽

Vol 180 (12) ◽

pp. 3144-3151 ◽

Cited By ~ 18

Author(s):

Britt C. Persson ◽

Ólafur Ólafsson ◽

Hans K. Lundgren ◽

Lars Hederstedt ◽

Glenn R. Björk

Keyword(s):

Escherichia Coli ◽

Citric Acid ◽

Salmonella Typhimurium ◽

Gene Product ◽

Respiratory Chain ◽

Citric Acid Cycle ◽

Dicarboxylic Acids ◽

Acid Cycle ◽

E Coli ◽

Isopentenyl Adenosine

ABSTRACT The modified nucleoside 2-methylthio-N-6-isopentenyl adenosine (ms2i6A) is present in position 37 (adjacent to and 3′ of the anticodon) of tRNAs that read codons beginning with U except tRNA I,V Ser inEscherichia coli. In Salmonella typhimurium, 2-methylthio-N-6-(cis-hydroxy)isopentenyl adenosine (ms2io6A; also referred to as 2-methylthio cis-ribozeatin) is found in tRNA, most likely in the species that have ms2i6A in E. coli. Mutants (miaE) of S. typhimurium in which ms2i6A hydroxylation is blocked are unable to grow aerobically on the dicarboxylic acids of the citric acid cycle. Such mutants have normal uptake of dicarboxylic acids and functional enzymes of the citric acid cycle and the aerobic respiratory chain. The ability of S. typhimurium to grow on succinate, fumarate, and malate is dependent on the state of modification in position 37 of those tRNAs normally having ms2io6A37 and is not due to a second cellular function of tRNA (ms2io6A37)hydroxylase, themiaE gene product. We suggest that S. typhimurium senses the hydroxylation status of the isopentenyl group of the tRNA and will grow on succinate, fumarate, or malate only if the isopentenyl group is hydroxylated.

Download Full-text

Differences between mouse and rat pancreatic islets: succinate responsiveness, malic enzyme, and anaplerosis

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00041.2002 ◽

2002 ◽

Vol 283 (2) ◽

pp. E302-E310 ◽

Cited By ~ 44

Author(s):

Michael J. MacDonald

Keyword(s):

Citric Acid ◽

Pancreatic Islets ◽

Insulin Release ◽

Malic Enzyme ◽

Citric Acid Cycle ◽

Acetyl Coa ◽

Acid Cycle ◽

Rat Islets ◽

Rat Pancreatic Islets ◽

Mouse Islets

Succinic acid methyl esters are potent insulin secretagogues in rat pancreatic islets, but they do not stimulate insulin release in mouse islets. Unlike rat and human islets, mouse islets lack malic enzyme and, therefore, are unable to form pyruvate from succinate-derived malate for net synthesis of acetyl-CoA. Dimethyl-[2,3-14C]succinate is metabolized in the citric acid cycle in mouse islets to the same extent as in rat islets, indicating that endogenous acetyl-CoA condenses with oxaloacetate derived from succinate. However, without malic enzyme, the net synthesis from succinate of the citric acid cycle intermediates citrate, isocitrate, and α-ketoglutarate cannot occur. Glucose and other nutrients that augment α-ketoglutarate formation are secretagogues in mouse islets with potencies similar to those in rat islets. All cycle intermediates can be net-synthesized from α-ketoglutarate. Rotenone, an inhibitor of site I of the electron transport chain, inhibits methyl succinate-induced insulin release in rat islets even though succinate oxidation forms ATP at sites II and III of the respiratory chain. Thus generating ATP, NADH, and anaplerosis of succinyl-CoA plus the four-carbon dicarboxylic acids of the cycle and its metabolism in the citric acid cycle is insufficient for a fuel to be insulinotropic; it must additionally promote anaplerosis of α-ketoglutarate or two intermediates interconvertible with α-ketoglutarate, citrate, and isocitrate.

Download Full-text

The Glyoxylate Shunt, 60 Years On

Annual Review of Microbiology ◽

10.1146/annurev-micro-090817-062257 ◽

2018 ◽

Vol 72 (1) ◽

pp. 309-330 ◽

Cited By ~ 34

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.

Download Full-text

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

Journal of Bacteriology ◽

10.1128/jb.182.24.6884-6891.2000 ◽

2000 ◽

Vol 182 (24) ◽

pp. 6884-6891 ◽

Cited By ~ 80

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.

Download Full-text

Abstract 15454: Inhibition of Mechanistic Target of Rapamycin by Heterozygous Deletion of Raptor Ameliorates Pressure-overload Induced Heart Failure and the Associated Proteomics Remodeling

Circulation ◽

10.1161/circ.130.suppl_2.15454 ◽

2014 ◽

Vol 130 (suppl_2) ◽

Author(s):

Dao Fu Dai

Keyword(s):

Heart Failure ◽

Citric Acid ◽

Citric Acid Cycle ◽

Heterozygous Deletion ◽

Wild Type ◽

Proteomics Analysis ◽

Mtor Inhibition ◽

Acid Cycle ◽

Mechanistic Target Of Rapamycin ◽

Mtor Complex 1

Background: We reported that inhibition of mechanistic target of rapamycin (mTOR) by short-term rapamycin or caloric restriction ameliorates age-dependent cardiac hypertrophy and diastolic dysfunction. Although inhibition of mTOR signaling is well known to regulate metabolism and suppress protein synthesis, the mechanisms of beneficial effect of mTOR inhibition in cardiac hypertrophy and failure are not fully understood. Method: To investigate the mechanisms underlying beneficial effect of mTOR inhibition, we used the transverse aortic constriction (TAC)-induced heart failure model and examined the effect of heterozygous deletion of Raptor (Raptor het), a component of mTOR complex 1, and transgenic overexpression of cardiac specific wild type or mutant 4EBP1, one of the main downstream target of mTOR complex 1. Global proteomics analysis was performed using an improved label-free quantitative shotgun approach, followed by Ingenuity Pathway analysis. Results: In wild-type mice with TAC-induced heart failure, global proteomics analysis revealed decreased abundance of proteins involved in mitochondrial function, electron transport chain, citric acid cycle and fatty acid metabolism and increased abundance of proteins involved in several signaling pathways (RhoA, actin, integrin) as well as oxidative stress response and protein ubiquitin pathways. Raptor het attenuate TAC induced heart failure, accompanied by better preservation of proteomics remodeling, especially the proteins involved in mitochondrial function, citric acid cycle and protein ubiquitination pathways. In contrast, either transgenic overexpression of wild type 4EBP1 or mutant 4EBP1 abolish the adaptive hypertrophy in response to TAC by suppressing protein translation, and thereby aggravate heart failure, in parallel with adverse remodeling of left ventricular proteomes. Neonatal cardiomyocyte experiments reveal that PGC1-α and Sirt3 are among the candidate signaling mechanisms linking the mTOR inhibition and mitochondrial metabolism. Conclusion: mTOR inhibition by Raptor heterozygous deletion, but not overexpression of 4EBP1, ameliorates TAC-induced heart failure and associated with better preservation of mitochondrial proteome.

Download Full-text

EFFECT OF SERUM GONADOTROPHIN ON DEHYDROGENASES OF THE CITRIC ACID CYCLE IN THE OVARY OF THE RAT

Acta Endocrinologica ◽

10.1530/acta.0.0420480 ◽

1963 ◽

Vol 42 (4) ◽

pp. 480-484 ◽

Cited By ~ 2

Author(s):

B. Eckstein ◽

R. Landsberg

Keyword(s):

Citric Acid ◽

Citric Acid Cycle ◽

Age Groups ◽

Succinic Dehydrogenase ◽

Immature Rat ◽

Acid Cycle ◽

Immature Rats ◽

Rat Ovary

ABSTRACT The succinic, malic and isocitric dehydrogenases in the ovary of immature and mature, normal and serum gonadotrophin injected rats were examined. The Qo2 of these enzymes were markedly enhanced in the gonadotrophin injected rats of both age groups, except in the case of succinic dehydrogenase in the ovary of the immature rats, where a slight non-significant decrease was noted. It is concluded that in the mature rat ovary, gonadotrophin administration stimulates the activity of all the examined dehydrogenases of the citric acid cycle, whereas in the immature rat ovary, at least the isocitric- and malic dehydrogenases are thus stimulated.

Download Full-text