Tricarboxylic acid (TCA) cycle

Tricarboxylic acid cycle also called citrate cycle and Krebs cycle is the third step in aerobic respiration. In eukaryotes, this process takes place in the mitochondrial matrix. It utilises acetyl-CoA from pyruvate oxidation or fatty acid/protein degradation as substrate. In addition, each cycle utilises 2 H2O molecules and generates CO2 and coenzyme-A. This is a cycle of eight enzymes catalysing nine reactions where oxaloacetate, which reacts with acetyl-CoA in the first step, is replenished in the last step. The reverse of this cycle called reverse or reductive Krebs cycle also occurs in some bacteria which utilises CO2 and H2O to synthesise carbon compounds.

 

Genes for all the enzymes of this pathway are present in the Toxoplasma gondii genome and all these enzymes are targeted to mitochondrion (either experimentally or bioinformatics predictions) [1, 2]. There are no evidence or predictions available for two out of three citrate synthase isoenzymes (one with 2.3.3.1 and other with 2.3.3.8 annotations), although the third enzyme (2.3.3.1 annotation) is experimentally verified to be localised to mitochondrion. The only enzyme which is present in T. gondii and absent in Plasmodium falciparum is 2.3.3.8. Although, malate dehydrogenase (1.1.1.37) is localised to mitochondrion in T. gondii, it is localised to cytosol in P. falciparum [3] and malate-quinone oxidoreductase (1.1.99.16), which is shown to be targeted to mitochondrion in Plasmodium yoelii yoelii [4] is annotated to TCA cycle in MPMP.

 

Although P. falciparum has all the enzymes of the TCA cycle, it has been suggested that at least the asexual stages does not require TCA cycle for energy generation [5] and depends on glycolysis and fermentation alone for energy needs [6]. One of the experimental evidence in support of this is the absence of 2-oxoglutarate dehydrogenase activity and the presence of CO2-fixation activities in the erythrocyte stages of P. falciparum [7]. In addition, the isocitrate dehydrogenase uses NADP+ as cofactor [8] and gene identified in the P. falciparum genome is NADP+-dependent isocitrate dehydrogenase. As seen in pyruvate metabolism pathway, the pyruvate dehydrogenase is localised to apicoplast and there is no direct link between glycolysis and Krebs cycle. The enzymes which catalyse oxidation of fatty acids into acetyl-CoA are also absent. Although enzymes of keto-acid dehydrogenase complex, which catalyse the branched chain amino acids degradation, are present (http://sites.huji.ac.il/malaria/maps/LeuIsoValmetpath.html), the downstream enzymes which leads to acetyl-CoA generation are missing. However, the stages in mosquito gut and salivary glands may depend also on Krebs cycle due to reduced glucose availability. There are evidence available for increased expression of genes encoding several TCA cycle enzymes in sporozoites [9]. The proteomics studies also revealed about the upregulation of many enzymes in ookinates compared to asexual stages [10].

 

Although the pyruvate dehydrogenase complex is present in T. gondii apicoplast [11], the presence of complete pathways which catalyse generation of acetyl-CoA from fatty acids (fatty acid recycling and degradation) and branched chain amino acids (leucine, isoleucine and valine metabolism) suggests possible sources of acetyl-coA for Krebs cycle. This also suggests possible role of Krebs cycle in energy generation in at least some life cycle stages of T. gondii. The comparative expression analysis of the TCA cycle enzymes from tachyzoites and early bradyzoites showed similar mRNA levels in both stages [11]. It should be noted that expression levels in sexual stages were not compared. The presence of anaplerotic enzymes which replenish oxaloacetate such as pyruvate carboxylase and PEP carboxykinase (pyruvate metabolism) suggests that Krebs cycle also has a role in biosynthetic pathways. Recent metabolic prfiling studies by MacRae et al showed that glucose is catabolised through a complete functional TCA cycle in intracellular and egressed tachyzoites [12].

 

Protein EC Number Gene id Protein localisation Localisation data source
Malate dehydrogenase 1.1.1.37 TGME49_318430 Mitochondrion Apiloc
Isocitrate dehydrogenase (ICDH2) 1.1.1.42 TGME49_313140 Mitochondrion Apiloc; Previous publication
Malate dehydrogenase (malate-quinone oxidoreductase) 1.1.99.16 (Entry changed to 1.1.5.4) TGME49_288500 Mitochondrion Apiloc; Previous publication
Oxoglutarate dehydrogenase E1 subunit (part of oxoglutarate dehydrogenase complex) 1.2.4.2 TGME49_244200 Mitochondrion Previous publication
Succinate dehydrogenase 1.3.99.1 TGME49_215280 Mitochondrion Previous publication
Succinate dehydrogenase 1.3.99.1 TGME49_215590 Mitochondrion Previous publication
Glutamate dehydrogenase 1.4.1.4 TGME49_293180 Cytosol Previous publication
Dihydrolipoamide dehydrogenase (part of oxoglutarate dehydrogenase complex) 1.8.1.4 TGME49_206470 Mitochondrion Apiloc; Previous publication
Dihydrolipoamide S-succinyl transferase (part of oxoglutarate dehydrogenase complex) 2.3.1.61 TGME49_219550 Mitochondrion Previous publication
Citrate (si)-synthase 2.3.3.1 TGME49_268890 Mitochondrion Apiloc; Previous publication
Citrate (si)-synthase 2.3.3.1 TGME49_203110    
ATP-citrate synthase 2.3.3.8 TGME49_223840    
Fumarate hydratase 4.2.1.2 TGME49_267330 Mitochondrion Previous publication
Aconitate hydratase 4.2.1.3 TGME49_226730 Apicoplast; Mitochondrion Apiloc; Previous publication
Succinate-CoA ligase 6.2.1.4 TGME49_290600 Mitochondrion Apiloc; Previous publication
Succinate-CoA ligase 6.2.1.5 TGME49_309752 Mitochondrion Apiloc; Previous publication
Mitochondrial carrier protein none TGME49_248950 Mitochondrial inner membrane GO annotation
Oxoglutarate/malate translocator none TGME49_274060 Mitochondrial inner membrane Apiloc; GO annotation
Amino acid transporter none TGME49_305470    
Acetyl-CoA transporter none TGME49_215940 Plasma membrane Previous publication

 

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Sources and fates of metabolites

 

Substrate Source pathways Product Fate pathways
CoA Pantothenate and CoA biosynthesis Malate Host
Glutamate Glutamate metabolism Acetyl-CoA Pyruvate metabolism, Tricarboxylic acid (TCA) cycle, Fatty acid elongation in the cytosol, Fatty acid elongation in the ER
2-oxoglutarate Pyruvate metabolism    
Succinate 2-methylcitrate cycle