Tricarboxylic Acid Cycle Enzymes of the Ectomycorrhizal Basidiomycete, Suillus bovinus

2001 ◽  
Vol 56 (5-6) ◽  
pp. 334-342 ◽  
Author(s):  
Norbert Grotjohann ◽  
Yi Huangb ◽  
Wolfgang Kowallik
Keyword(s):  
Succinate Dehydrogenase ◽  
Malate Dehydrogenase ◽  
Isocitrate Dehydrogenase ◽  
Citrate Synthase ◽  
Tricarboxylic Acid Cycle ◽  
Tricarboxylic Acid ◽  
Technical Problems ◽  
Acid Cycle ◽  
Suillus Bovinus ◽  
Cell Extracts

In crude cell extracts of the ectomycorrhizal fungus, Suillus bovinus, activities of citrate synthase, aconitase, isocitrate dehydrogenase, succinate dehydrogenase, fumarase, and malate dehydrogenase have been proved and analyzed. Citrate synthase exhibited high affinities for both its substrates: oxaloacetate (Km = 0.018 mᴍ) and acetyl-CoA (Km = 0.014 mᴍ) . Aconitase showed better affinity for isocitrate (Km = 0.62 mᴍ) than for citrate (Km = 3.20 mᴍ) . Analysis of isocitrate dehydrogenase revealed only small maximum activity (60 nmol x mg protein-1 x min -1), the enzyme being exclusively NADP+-dependent. Using the artificial electron acceptor dichlorophenol indophenol, activity and substrate affinity of succinate dehydrogenase were rather poor. Fumarase proved Fe2+-independent. Its affinity for malate was found higher ( Km = 1.19 mᴍ) than that for fumarate ( Km = 2.09 mᴍ) . High total activity of malate dehydrogenase could be separated by native PAGE into a slowly running species of (mainly) cytosolic (about 80%) and a faster running species of (mainly) mitochondrial origin. Affinities for oxaloacetate of the two enzyme species were found identical within limits of significance (Km = 0.24 mᴍ and 0.22 mᴍ) . The assumed cytosolic enzyme exhibited affinity for malate (Km = 5.77 mᴍ) more than one order of magnitude lower than that for oxaloacetate. FPLC on superose 12 revealed only one activity band at a molecular mass of 100 ± 15 kDa. Activities of 2-oxoglutarate dehydrogenase and of succinyl-CoA synthetase could not be found. Technical problems in their detection, but also existence of an incomplete tricarboxylic acid cycle are considered. Metabolite affinities, maximum activities and pʜ-dependences of fumarase and of malate dehydrogenase allow the assumption of a reductive instead of oxidative function of these enzymes in vivo.

scholarly journals Tricarboxylic acid cycle and proton gradient in Pandoravirus massiliensis: Is it still a virus?

2020 ◽  
Author(s):  
Sarah Aherfi ◽  
Djamal Brahim Belhaouari ◽  
Lucile Pinault ◽  
Jean-Pierre Baudoin ◽  
Philippe Decloquement ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Isocitrate Dehydrogenase ◽  
Energy Production ◽  
Citrate Synthase ◽  
Tricarboxylic Acid Cycle ◽  
Tricarboxylic Acid ◽  
Proton Gradient ◽  
Giant Virus ◽  
Acid Cycle ◽  
Key Enzyme

ABSTRACTSince the discovery of Acanthamoeba polyphaga Mimivirus, the first giant virus of amoeba, the historical hallmarks defining a virus have been challenged. Giant virion sizes can reach up to 2.3 µm, making them visible by optical microscopy. They have large genomes of up to 2.5 Mb that encode proteins involved in the translation apparatus. Herein, we investigated possible energy production in Pandoravirus massiliensis, the largest of our giant virus collection. MitoTracker and TMRM mitochondrial membrane markers allowed for the detection of a membrane potential in virions that could be abolished by the use of the depolarizing agent CCCP. An attempt to identify enzymes involved in energy metabolism revealed that 8 predicted proteins of P. massiliensis exhibited low sequence identities with defined proteins involved in the universal tricarboxylic acid cycle (acetyl Co-A synthase; citrate synthase; aconitase; isocitrate dehydrogenase; α-ketoglutarate decarboxylase; succinate dehydrogenase; fumarase). All 8 viral predicted ORFs were transcribed together during viral replication, mainly at the end of the replication cycle. Two of these proteins were detected in mature viral particles by proteomics. The product of the ORF132, a predicted protein of P. massiliensis, cloned and expressed in Escherichia coli, provided a functional isocitrate dehydrogenase, a key enzyme of the tricarboxylic acid cycle, which converts isocitrate to α-ketoglutarate. We observed that membrane potential was enhanced by low concentrations of Acetyl-CoA, a regulator of the tricarboxylic acid cycle. Our findings show for the first time that energy production can occur in viruses, namely, pandoraviruses, and the involved enzymes are related to tricarboxylic acid cycle enzymes. The presence of a proton gradient in P. massiliensis coupled with the observation of genes of the tricarboxylic acid cycle make this virus a form a life for which it is legitimate to question ‘what is a virus?’.

scholarly journals [13C]propionate oxidation in wild-type and citrate synthase mutant Escherichia coli: evidence for multiple pathways of propionate utilization

1993 ◽  
Vol 291 (3) ◽  
pp. 927-932 ◽  
Author(s):  
C T Evans ◽  
B Sumegi ◽  
P A Srere ◽  
A D Sherry ◽  
C R Malloy
Keyword(s):  
Escherichia Coli ◽  
Citrate Synthase ◽  
Tricarboxylic Acid Cycle ◽  
Tricarboxylic Acid ◽  
Spin Coupling ◽  
Wild Type ◽  
Acid Cycle ◽  
E Coli ◽  
Cell Extracts ◽  
In Cells

The metabolism of propionate was examined in wild-type Escherichia coli and cells lacking citrate synthase by high-resolution 13C n.m.r. Spectra of cell extracts from wild-type E. coli show that glutamate becomes highly enriched in 13C when 13C-enriched propionate is the sole carbon source. No glutamate labelling was detected when the tricarboxylic acid cycle was blocked either by deletion of citrate synthase or by inhibition of succinate dehydrogenase by malonate. The 13C fractional enrichment in glutamate C-2, C-3 and C-4 in wild-type cells was quantitatively and qualitatively different when [2-13C]propionate as opposed to [3-13C]propionate was supplied. Approximately equal labelling occurred in the C-2, C-3 and C-4 positions of glutamate when [3-13C]propionate was available, and multiplets due to carbon-carbon spin-spin coupling were observed. However, in cells supplied with [2-13C]propionate, very little 13C appeared in the glutamate C-4 position, and the remaining glutamate resonances all appeared as singlets. The unequal and non-identical labelling of glutamate in cells supplied with [2-13C]- as opposed to [3-13C]propionate is consistent with the utilization of propionate by E. coli via two pathways, oxidation of propionate to pyruvate and carboxylation of propionate to succinate. These intermediates are further metabolized to glutamate by the action of the tricarboxylic acid cycle. The existence of an organized tricarboxylic acid cycle is discussed as a consequence of the ability to block utilization of propionate in tricarboxylic acid-cycle-defective E. coli.

scholarly journals Tricarboxylic acid-cycle and related enzymes in restricted facultative methylotrophs

1975 ◽  
Vol 148 (3) ◽  
pp. 505-511 ◽  
Author(s):  
J Colby ◽  
L J Zatman
Keyword(s):  
Succinate Dehydrogenase ◽  
Dehydrogenase Activity ◽  
Isocitrate Dehydrogenase ◽  
Tricarboxylic Acid Cycle ◽  
Tricarboxylic Acid ◽  
Methylotrophic Bacteria ◽  
Acid Cycle ◽  
Oxoglutarate Dehydrogenase ◽  
Specific Activities ◽  
Succinyl Coa Synthetase

The isolation is described of pure cultures of three non-methane-utilizing methylotrophic bacteria which, together with the previously described Bacillus PM6, have a very limited range of growth substrates; these organisms are designated “restricted facultative’ methylotrophs. Two of these isolates, W6A and W3A1, grow only on glucose out of 50 non-C1 compounds tested, whereas the third isolate S2A1 and Bacillus PM6 grow on betaine, glucose, gluconate, alanine, glutamate, citrate and nutrient agar, but not on any of a further 56 non-C1 compounds. Crude sonic extracts of trimethylamine-grown and glucose-grown W6A and W3A1 isolates, and of trimethylamine-grown C2A1 (an obligate methylotroph) contain (i) no detectable 2-oxogltarate dehydrogenase activity, (ii) very low or zero specific activities of succinate dehydrogenase and succinyl-CoA synthetase and (iii) NAD+-dependent isocitrate dehydrogenase activity. Extracts of trimethylamine-grown PM6 and S2A1 methylotrophs have (i) very low 2-oxoglutarate dehydrogenase specific activities, (ii) comparatively high specific activities of succinate dehydrogenase, malate dehydrogenase and succinyl-CoA synthetase and (iii) NADP+-dependent isocitrate dehydrogenase activity but no NAD+-dependent isocitrate dehydrogenase activity. The activities of most of these enzymes are increased during growth on glucose, alanine, glutamate or citrate, but only very low 2-oxoglutarate dehydrogenase activities are present under all growth conditions. The restricted facultative methylotrophs grow on certain non-C1 compounds in the absence of 2-oxoglutarate dehydrogenase and, in some cases, of other enzymes of the tricarboxylic acid cycle; these lesions cannot therefore be the sole cause of obligate methylotrophy.

Tricarboxylic acid cycle enzymes of a pseudophyllid cestode Penetrocephalus ganapatii

1990 ◽  
Vol 64 (1) ◽  
pp. 51-53
Author(s):  
S. Dhandayuthapani ◽  
K. Nellaiappan
Keyword(s):  
Isocitrate Dehydrogenase ◽  
Citrate Synthase ◽  
Tricarboxylic Acid Cycle ◽  
Tca Cycle ◽  
Tricarboxylic Acid ◽  
Reverse Direction ◽  
Acid Cycle ◽  
Tca Cycle Enzymes ◽  
The Tca Cycle ◽  
Atp Generation

ABSTRACTStudies on the tricarboxylic acid cycle (TCA cycle) enzymes of Penetrocephalus ganapatii reveal that the TCA cycle is only partially operative, as some of the enzymes at the start of the cycle viz. citrate synthase, aconitase and isocitrate dehydrogenase are found to be low in their activities. The high activities of malate dehydrogenase and fumarase, showing affinity towards a reverse direction, indicate that the TCA cycle operates in the reverse direction resulting in the formation of fumarate. The low succinate dehydrogenase/fumarate reductase ratio suggests that ATP generation may occur at site I of the respiratory chain during the reduction of fumarate into succinate.

Complex formation between malate dehydrogenase and isocitrate dehydrogenase fromBacillus subtilisis regulated by tricarboxylic acid cycle metabolites

FEBS Journal ◽  
2014 ◽  
Vol 281 (4) ◽  
pp. 1132-1143 ◽  
Author(s):  
Maike Bartholomae ◽  
Frederik M. Meyer ◽  
Fabian M. Commichau ◽  
Andreas Burkovski ◽  
Wolfgang Hillen ◽  
...  
Keyword(s):  
Complex Formation ◽  
Malate Dehydrogenase ◽  
Isocitrate Dehydrogenase ◽  
Tricarboxylic Acid Cycle ◽  
Tricarboxylic Acid ◽  
Acid Cycle

THE ENZYMES OF THE TRICARBOXYLIC ACID CYCLE OF PSEUDOMONAS AERUGINOSA

1956 ◽  
Vol 2 (4) ◽  
pp. 433-440 ◽  
Author(s):  
Jack J. R. Campbell ◽  
Roberts A. Smith
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Malic Acid ◽  
Tricarboxylic Acid Cycle ◽  
Tricarboxylic Acid ◽  
The Other ◽  
Acid Cycle ◽  
High Ph ◽  
Optimum Activity ◽  
Fresh Cell ◽  
Cell Extracts

It was demonstrated that Pseudomonas aeruginosa possesses all the enzymes necessary for the oxidation of pyruvate to CO2 and water without passing through the conventional intermediates oxalosuccinate and α-ketoglutarate. These intermediates are bypassed by the action of the enzyme isocitratase which splits d-isocitrate to succinate plus glyoxylate. This reaction was shown to be readily reversible. The malic acid dehydrogenase content was low and in addition this enzyme required a high pH for optimum activity. In fresh cell extracts at pH 7.4 its activity was only 10% that of the other enzymes of the cycle. The malic and isocitric dehydrogenases were TPN specific. The organism was also shown to possess all the enzymes necessary for the operation of the conventional tricarboxylic acid cycle.

Saccharomyces cerevisiae contains two functional citrate synthase genes

1986 ◽  
Vol 6 (6) ◽  
pp. 1936-1942
Author(s):  
K S Kim ◽  
M S Rosenkrantz ◽  
L Guarente
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Carbon Source ◽  
Citrate Synthase ◽  
Tricarboxylic Acid Cycle ◽  
Nuclear Gene ◽  
Tricarboxylic Acid ◽  
Yeast Genome ◽  
Gene Encoding ◽  
Acid Cycle ◽  
Nonfermentable Carbon Source

The tricarboxylic acid cycle occurs within the mitochondria of the yeast Saccharomyces cerevisiae. A nuclear gene encoding the tricarboxylic acid cycle enzyme citrate synthase has previously been isolated (M. Suissa, K. Suda, and G. Schatz, EMBO J. 3:1773-1781, 1984) and is referred to here as CIT1. We report here the isolation, by an immunological method, of a second nuclear gene encoding citrate synthase (CIT2). Disruption of both genes in the yeast genome was necessary to produce classical citrate synthase-deficient phenotypes: glutamate auxotrophy and poor growth on rich medium containing lactate, a nonfermentable carbon source. Therefore, the citrate synthase produced from either gene was sufficient for these metabolic roles. Transcription of both genes was maximally repressed in medium containing both glucose and glutamate. However, transcription of CIT1 but not of CIT2 was derepressed in medium containing a nonfermentable carbon source. The significance of the presence of two genes encoding citrate synthase in S. cerevisiae is discussed.

scholarly journals Association of the malate dehydrogenase-citrate synthase metabolon is modulated by intermediates of the Krebs tricarboxylic acid cycle

2021 ◽  
Author(s):  
Joy Omini ◽  
Izabela Wojciechowska ◽  
Aleksandra Skirycz ◽  
Hideaki Moriyama ◽  
Toshihiro Obata
Keyword(s):  
Malate Dehydrogenase ◽  
Metabolic Flux ◽  
Citrate Synthase ◽  
Tricarboxylic Acid Cycle ◽  
Feedback Regulation ◽  
Dynamic Structure ◽  
Tca Cycle ◽  
Tricarboxylic Acid ◽  
Enzyme Complex ◽  
Acetyl Coa

Mitochondrial malate dehydrogenase (MDH)-citrate synthase (CS) multi-enzyme complex is a part of the Krebs tricarboxylic acid (TCA) cycle 'metabolon' which is enzyme machinery catalyzing sequential reactions without diffusion of reaction intermediates into a bulk matrix. This complex is assumed to be a dynamic structure involved in the regulation of the cycle by enhancing metabolic flux. Microscale Thermophoresis analysis of the porcine heart MDH-CS complex revealed that substrates of the MDH and CS reactions, NAD+ and acetyl-CoA, enhance complex association while products of the reactions, NADH and citrate, weaken the affinity of the complex. Oxaloacetate enhanced the interaction only when it was presented together with acetyl-CoA. Structural modeling using published CS structures suggested that the binding of these substrates can stabilize the closed format of CS which favors the MDH-CS association. Two other TCA cycle intermediates, ATP, and low pH also enhanced the association of the complex. These results suggest that dynamic formation of the MDH-CS multi-enzyme complex is modulated by metabolic factors responding to respiratory metabolism, and it may function in the feedback regulation of the cycle and adjacent metabolic pathways.

Tricarboxylic acid cycle dehydrogenases inhibition by naringenin: experimental and molecular modelling evidence

2020 ◽  
Vol 123 (10) ◽  
pp. 1117-1126
Author(s):  
Pauline Maciel August ◽  
Mateus Grings ◽  
Marcelo Sartori Grunwald ◽  
Geancarlo Zanatta ◽  
Vinícius Stone ◽  
...  
Keyword(s):  
Citrate Synthase ◽  
Tricarboxylic Acid Cycle ◽  
Cell Metabolism ◽  
Tricarboxylic Acid ◽  
Metabolic Effects ◽  
Similar Reduction ◽  
Acid Cycle ◽  
Docking Simulations ◽  
Female Wistar Rats

AbstractThe study of polyphenols’ effects on health has been gaining attention lately. In addition to reacting with important enzymes, altering the cell metabolism, these substances can present either positive or negative metabolic alterations depending on their consumption levels. Naringenin, a citrus flavonoid, already presents diverse metabolic effects. The objective of this work was to evaluate the effect of maternal naringenin supplementation during pregnancy on the tricarboxylic acid cycle activity in offspring’s cerebellum. Adult female Wistar rats were divided into two groups: (1) vehicle (1 ml/kg by oral administration (p.o.)) or (2) naringenin (50 mg/kg p.o.). The offspring were euthanised at 7th day of life, and the cerebellum was dissected to analyse citrate synthase, isocitrate dehydrogenase (IDH), α-ketoglutarate dehydrogenase (α-KGDH) and malate dehydrogenase (MDH) activities. Molecular docking used SwissDock web server and FORECASTER Suite, and the proposed binding pose image was created on UCSF Chimera. Data were analysed by Student’s t test. Naringenin supplementation during pregnancy significantly inhibited IDH, α-KGDH and MDH activities in offspring’s cerebellum. A similar reduction was observed in vitro, using purified α-KGDH and MDH, subjected to pre-incubation with naringenin. Docking simulations demonstrated that naringenin possibly interacts with dehydrogenases in the substrate and cofactor binding sites, inhibiting their function. Naringenin administration during pregnancy may affect cerebellar development and must be evaluated with caution by pregnant women and their physicians.

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