Metabolic control analysis of the transsulfuration pathway and the compensatory role of the cysteine transport in Trypanosoma cruzi.

Trypanosoma cruzi is the causal agent of American Trypanosomiasis or Chagas Disease in humans. The current drugs for its treatment benznidazole and nifurtimox have inconveniences of toxicity and efficacy; therefore, the search for new therapies continues. Validation through genetic strategies of new drug targets against the parasite metabolism have identified numerous essential genes. Target validation can be further narrowed by applying Metabolic Control Analysis (MCA) to determine the flux control coefficients of the pathway enzymes. That coefficient is a quantitative value that represents the degree in which an enzyme/transporter determines the flux of a metabolic pathway; those with the highest coefficients can be promising drug targets. Previous studies have demonstrated that cysteine (Cys) is a key precursor for the synthesis of trypanothione, the main antioxidant metabolite in the parasite. In this research, MCA was applied in an ex vivo system to the enzymes of the reverse transsulfuration pathway (RTP) for Cys synthesis composed by cystathionine beta synthase (CBS) and cystathionine gamma lyase (CGL). The results indicated that CGL has 90% of the control of the pathway flux. Inhibition of CGL with propargylglycine (PAG) decreased the levels of Cys and trypanothione and depleted those of glutathione in epimastigotes (proliferative stage in the insect vector); these metabolite changes were prevented by supplementing with Cys, suggesting a compensatory role of the Cys transport (CysT). Indeed, Cys supplementation (but not PAG treatment) increased the activity of the CysT in epimastigotes whereas in trypomastigotes (infective stage in mammals) CysT was increased when they were incubated with PAG. Our results suggested that CGL could be a potential drug target given its high control on the RTP flux and its effects on the parasite antioxidant defense. However, the redundant Cys supply pathways in the parasite may require inhibition of the CysT as well. Our findings also suggest differential responses of the Cys supply pathways in different parasite stages.

Phenothiazine-based virtual screening, molecular docking, and molecular dynamics of new trypanothione reductase inhibitors of Trypanosoma cruzi.

Phenothiazine derivatives can unselectively inhibit the trypanothione-dependent antioxidant system enzyme trypanothione reductase (TR). A virtual screening of 2163 phenothiazine derivatives from the ZINC15 and PubChem databases docked on the active site of T. cruzi TR showed that 285 compounds have higher affinity than the natural ligand trypanothione disulfide. 244 compounds showed higher affinity toward the parasite's enzyme than to its human homolog glutathione reductase. Protein-ligand interaction profiling predicted that the main interactions for the top scored compounds were with residues important for trypanothione disulfide binding: Phe396, Pro398, Leu399, His461, Glu466, and Glu467, particularly His461, which participates in catalysis. Two compounds with the desired profiles, ZINC1033681 (Zn_C687) and ZINC10213096 (Zn_C216), decreased parasite growth by 20 % and 50 %, respectively. They behaved as mixed-type inhibitors of recombinant TR, with Ki values of 59 and 47 µM, respectively. This study provides a further understanding of the potential of phenothiazine derivatives as TR inhibitors.

Lactate oxidation is linked to energy conservation and to oxygen detoxification via a putative terminal cytochrome oxidase in Methanosarcina acetivorans.

The marine archaeon Methanosarcina acetivorans contains a putative NAD + -independent d-lactate dehydrogenase (D-iLDH/glycolate oxidase) encoded by the MA4631 gene, belonging to the FAD-oxidase C superfamily. Nucleotide sequences similar to MA4631 gene, were identified in other methanogens and Firmicutes with >90 and 35-40% identity, respectively. Therefore, the lactate metabolism in M. acetivorans is reported here. Cells subjected to intermittent pulses of oxygen (air-adapted; AA-Ma cells) consumed lactate only in combination with acetate, increasing methane production and biomass yield. In AA-Ma cells incubated with d-lactate plus [14C]-l-lactate, the radioactive label was found in methane, CO2 and glycogen, indicating that lactate metabolism fed both methanogenesis and gluconeogenesis. Moreover, d-lactate oxidation was coupled to O2-consumption which was sensitive to HQNO; also, AA-Ma cells showed high transcript levels of gene dld and those encoding subunits A (MA1006) and B (MA1007) of a putative cytochrome bd quinol oxidase, compared to anaerobic control cells. An E. coli mutant deficient in dld complemented with the MA4631 gene, grew with d-lactate as carbon source and showed membrane-bound d-lactate:quinone oxidoreductase activity. The product of the MA4631 gene is a FAD-containing monomer showing activity of iLDH with preference to d-lactate. The results suggested that air adapted M. acetivorans is able to co-metabolize lactate and acetate with associated oxygen consumption by triggering the transcription and synthesis of the D-iLDH and a putative cytochrome bd: methanophenazine (quinol) oxidoreductase. Biomass generation and O2 consumption, suggest a potentially new oxygen detoxification mechanism coupled to energy conservation in this methanogen.

In Vitro and In Silico Analysis of New n-Butyl and Isobutyl Quinoxaline-7-carboxylate 1,4-di-N-oxide Derivatives against Trypanosoma cruzi as Trypanothione Reductase Inhibitors.

American trypanosomiasis is a worldwide health problem that requires attention due to ineffective treatment options. We evaluated n-butyl and isobutyl quinoxaline-7-carboxylate 1,4-di-N-oxide derivatives against trypomastigotes of the Trypanosoma cruzi strains NINOA and INC-5. An in silico analysis of the interactions of 1,4-di-N-oxide on the active site of trypanothione reductase (TR) and an enzyme inhibition study was carried out. The n-butyl series compound identified as T-150 had the best trypanocidal activity against T. cruzi trypomastigotes, with a 13% TR inhibition at 44 µM. The derivative T-147 behaved as a mixed inhibitor with Ki and Ki' inhibition constants of 11.4 and 60.8 µM, respectively. This finding is comparable to the TR inhibitor mepacrine (Ki = 19 µM).

Regulatory role of acetylation on enzyme activity and fluxes of energy metabolism pathways.

BACKGROUND: Most of the enzymes involved in the central carbon metabolism are acetylated in Lys residues. It has been claimed that this covalent modification represents a novel regulatory mechanism by which both enzyme/transporter activities and pathway fluxes can be modulated. METHODS: To establish which enzymes are regulated by acetylation, a systematic experimental analysis of activities and acetylation profile for several energy metabolism enzymes and pathway fluxes was undertaken in cells and mitochondria. RESULTS: The majority of the glycolytic and neighbor enzymes as well as mitochondrial enzymes indeed showed Lys-acetylation, with GLUT1, HPI, CS, ATP synthase displaying comparatively lower acetylation patterns. The incubation of cytosolic and mitochondrial fractions with recombinant Sirt-3 produced lower acetylation signals, whereas incubation with acetyl-CoA promoted protein acetylation. Significant changes in acetylation levels of MDH and IDH-2 from rat liver mitochondria revealed no change in their activities. Similar observations were attained for the cytosolic enzymes from AS-30D and HeLa cells. A minor but significant (23%) increase in the AAT-MDH complex activity induced by acetylation was observed. To examine this question further, AS-30D and HeLa cells were treated with nicotinamide and valproic acid. These compounds promoted changes in the acetylation patterns of glycolytic proteins, although their activities and the glycolytic flux (as well as the OxPhos flux) revealed no clear correlation with acetylation. CONCLUSION: Acetylation seems to play no predominant role in the control of energy metabolism enzyme activities and pathway fluxes. GENERAL SIGNIFICANCE: The physiological function of protein acetylation on energy metabolism pathways remains to be elucidated.

Acetate Promotes a Differential Energy Metabolic Response in Human HCT 116 and COLO 205 Colon Cancer Cells Impacting Cancer Cell Growth and Invasiveness.

Under dysbiosis, a gut metabolic disorder, short-chain carboxylic acids (SCCAs) are secreted to the lumen, affecting colorectal cancer (CRC) development. Butyrate and propionate act as CRC growth inhibitors, but they might also serve as carbon source. In turn, the roles of acetate as metabolic fuel and protein acetylation promoter have not been clearly elucidated. To assess whether acetate favors CRC growth through active mitochondrial catabolism, a systematic study evaluating acetate thiokinase (AcK), energy metabolism, cell proliferation, and invasiveness was performed in two CRC cell lines incubated with physiological SCCAs concentrations. In COLO 205, acetate (+glucose) increased the cell density (50%), mitochondrial protein content (3-10 times), 2-OGDH acetylation, and oxidative phosphorylation (OxPhos) flux (36%), whereas glycolysis remained unchanged vs. glucose-cultured cells; the acetate-induced OxPhos activation correlated with a high AcK activity, content, and acetylation (1.5-6-fold). In contrast, acetate showed no effect on HCT116 cell growth, OxPhos, AcK activity, protein content, and acetylation. However, a substantial increment in the HIF-1α content, HIF-1α-glycolytic protein targets (1-2.3 times), and glycolytic flux (64%) was observed. Butyrate and propionate decreased the growth of both CRC cells by impairing OxPhos flux through mitophagy and mitochondrial fragmentation activation. It is described, for the first time, the role of acetate as metabolic fuel for ATP supply in CRC COLO 205 cells to sustain proliferation, aside from its well-known role as protein epigenetic regulator. The level of AcK determined in COLO 205 cells was similar to that found in human CRC biopsies, showing its potential role as metabolic marker.

Gamma-glutamylcysteine synthetase and tryparedoxin 1 exert high control on the antioxidant system in Trypanosoma cruzi contributing to drug resistance and infectivity.

Trypanothione (T(SH)2) is the main antioxidant metabolite for peroxide reduction in Trypanosoma cruzi; therefore, its metabolism has attracted attention for therapeutic intervention against Chagas disease. To validate drug targets within the T(SH)2 metabolism, the strategies and methods of Metabolic Control Analysis and kinetic modeling of the metabolic pathway were used here, to identify the steps that mainly control the pathway fluxes and which could be appropriate sites for therapeutic intervention. For that purpose, gamma-glutamylcysteine synthetase (γECS), trypanothione synthetase (TryS), trypanothione reductase (TryR) and the tryparedoxin cytosolic isoform 1 (TXN1) were separately overexpressed to different levels in T. cruzi epimastigotes and their degrees of control on the pathway flux as well as their effect on drug resistance and infectivity determined. Both experimental in vivo as well as in silico analyses indicated that γECS and TryS control T(SH)2 synthesis by 60-74% and 15-31%, respectively. γECS overexpression prompted up to a 3.5-fold increase in T(SH)2 concentration, whereas TryS overexpression did not render an increase in T(SH)2 levels as a consequence of high T(SH)2 degradation. The peroxide reduction flux was controlled for 64-73% by TXN1, 17-20% by TXNPx and 11-16% by TryR. TXN1 and TryR overexpression increased H2O2 resistance, whereas TXN1 overexpression increased resistance to the benznidazole plus buthionine sulfoximine combination. γECS overexpression led to an increase in infectivity capacity whereas that of TXN increased trypomastigote bursting. The present data suggested that inhibition of high controlling enzymes such as γECS and TXN1 in the T(SH)2 antioxidant pathway may compromise the parasite's viability and infectivity.

Control and regulation of the pyrophosphate-dependent glucose metabolism in Entamoeba histolytica.

Entamoeba histolytica has neither Krebs cycle nor oxidative phosphorylation activities; therefore, glycolysis is the main pathway for ATP supply and provision of carbon skeleton precursors for the synthesis of macromolecules. Glucose is metabolized through fermentative glycolysis, producing ethanol as its main end-product as well as some acetate. Amoebal glycolysis markedly differs from the typical Embden-Meyerhof-Parnas pathway present in human cells: (i) by the use of inorganic pyrophosphate, instead of ATP, as the high-energy phospho group donor; (ii) with one exception, the pathway enzymes can catalyze reversible reactions under physiological conditions; (iii) there is no allosteric regulation and sigmoidal kinetic behavior of key enzymes; and (iv) the presence of some glycolytic and fermentation enzymes similar to those of anaerobic bacteria. These peculiarities bring about alternative mechanisms of control and regulation of the PPi-dependent fermentative glycolysis in the parasite in comparison to the ATP-dependent and allosterically regulated glycolysis in many other eukaryotic cells. In this review, the current knowledge of the carbohydrate metabolism enzymes in E. histolytica is analyzed. Thermodynamics and stoichiometric analyses indicate 2 to 3.5 ATP yield per glucose metabolized, instead of the often presumed 5 ATP/glucose ratio. PPi derived from anabolism seems insufficient for PPi-glycolysis; hence, alternative ways of PPi supply are also discussed. Furthermore, the underlying mechanisms of control and regulation of the E. histolytica carbohydrate metabolism, analyzed by applying integral and systemic approaches such as Metabolic Control Analysis and kinetic modeling, contribute to unveiling alternative and promising drug targets.

The contribution of two isozymes to the pyruvate kinase activity of Vibrio cholerae: One K+-dependent constitutively active and another K+-independent with essential allosteric activation.

In a previous phylogenetic study of the family of pyruvate kinase EC (2.7.1.40), a cluster with Glu117 and another with Lys117 were found (numbered according to the rabbit muscle enzyme). The sequences with Glu117 have been found to be K+-dependent, whereas those with Lys117 were K+-independent. Interestingly, only γ-proteobacteria exhibit sequences in both branches of the tree. In this context, it was explored whether these phylogenetically distinct pyruvate kinases were both expressed and contribute to the pyruvate kinase activity in Vibrio cholerae. The main findings of this work showed that the isozyme with Glu117 is an active K+-dependent enzyme. At the same substrate concentration, its Vmax in the absence of fructose 1,6 bisphosphate was 80% of that with its effector. This result is in accordance with the non-essential activation described by allosteric ligands for most pyruvate kinases. In contrast, the pyruvate kinase with Lys117 was a K+-independent enzyme displaying an allosteric activation by ribose 5-phosphate. At the same substrate concentration, its activity without the effector was 0.5% of the one obtained in the presence of ribose 5-phosphate, indicating that this sugar monophosphate is a strong activator of this enzyme. This absolute allosteric dependence is a novel feature of pyruvate kinase activity. Interestingly, in the K+-independent enzyme, Mn2+ may "mimic" the allosteric effect of Rib 5-P. Despite their different allosteric behavior, both isozymes display a rapid equilibrium random order kinetic mechanism. The intracellular concentrations of fructose 1,6-bisphosphate and ribose 5-phosphate in Vibrio cholerae have been experimentally verified to be sufficient to induce maximal activation of both enzymes. In addition, Western blot analysis indicated that both enzymes were co-expressed. Therefore, it is concluded that VcIPK and VcIIPK contribute to the activity of pyruvate kinase in this γ-proteobacterium.

The nutritional status of Methanosarcina acetivorans regulates glycogen metabolism and gluconeogenesis and glycolysis fluxes.

Gluconeogenesis is an essential pathway in methanogens because they are unable to use exogenous hexoses as carbon source for cell growth. With the aim of understanding the regulatory mechanisms of central carbon metabolism in Methanosarcina acetivorans, the present study investigated gene expression, the activities and metabolic regulation of key enzymes, metabolite contents and fluxes of gluconeogenesis, as well as glycolysis and glycogen synthesis/degradation pathways. Cells were grown with methanol as a carbon source. Key enzymes were kinetically characterized at physiological pH/temperature. Active consumption of methanol during exponential cell growth correlated with significant methanogenesis, gluconeogenic flux and steady glycogen synthesis. After methanol exhaustion, cells reached the stationary growth phase, which correlated with the rise in glycogen consumption and glycolytic flux, decreased methanogenesis, negligible acetate production and an absence of gluconeogenesis. Elevated activities of carbon monoxide dehydrogenase/acetyl-CoA synthetase complex and pyruvate: ferredoxin oxidoreductase suggested the generation of acetyl-CoA and pyruvate for glycogen synthesis. In the early stationary growth phase, the transcript contents and activities of pyruvate phosphate dikinase, fructose 1,6-bisphosphatase and glycogen synthase decreased, whereas those of glycogen phosphorylase, ADP-phosphofructokinase and pyruvate kinase increased. Therefore, glycogen and gluconeogenic metabolites were synthesized when an external carbon source was provided. Once such a carbon source became depleted, glycolysis and methanogenesis fed by glycogen degradation provided the ATP supply. Weak inhibition of key enzymes by metabolites suggested that the pathways evaluated were mainly transcriptionally regulated. Because glycogen metabolism and glycolysis/gluconeogenesis are not present in all methanogens, the overall data suggest that glycogen storage might represent an environmental advantage for methanosarcinales when carbon sources are scarce. Also, the understanding of the central carbohydrate metabolism in methanosarcinales may help to optimize methane production.

Roles of acetyl-CoA synthetase (ADP-forming) and acetate kinase (PPi-forming) in ATP and PPi supply in Entamoeba histolytica.

BACKGROUND: Acetate is an end-product of the PPi-dependent fermentative glycolysis in Entamoeba histolytica; it is synthesized from acetyl-CoA by ADP-forming acetyl-CoA synthetase (ACS) with net ATP synthesis or from acetyl-phosphate by a unique PPi-forming acetate kinase (AcK). The relevance of these enzymes to the parasite ATP and PPi supply, respectively, are analyzed here. METHODS: The recombinant enzymes were kinetically characterized and their physiological roles were analyzed by transcriptional gene silencing and further metabolic analyses in amoebae. RESULTS: Recombinant ACS showed higher catalytic efficiencies (Vmax/Km) for acetate formation than for acetyl-CoA formation and high acetyl-CoA levels were found in trophozoites. Gradual ACS gene silencing (49-93%) significantly decreased the acetate flux without affecting the levels of glycolytic metabolites and ATP in trophozoites. However, amoebae lacking ACS activity were unable to reestablish the acetyl-CoA/CoA ratio after an oxidative stress challenge. Recombinant AcK showed activity only in the acetate formation direction; however, its substrate acetyl-phosphate was undetected in axenic parasites. AcK gene silencing did not affect acetate production in the parasites but promoted a slight decrease (10-20%) in the hexose phosphates and PPi levels. CONCLUSIONS: These results indicated that the main role of ACS in the parasite energy metabolism is not ATP production but to recycle CoA for glycolysis to proceed under aerobic conditions. AcK does not contribute to acetate production but might be marginally involved in PPi and hexosephosphate homeostasis. SIGNIFICANCE: The previous, long-standing hypothesis that these enzymes importantly contribute to ATP and PPi supply in amoebae can now be ruled out.

Cadmium removal by Euglena gracilis is enhanced under anaerobic growth conditions.

The facultative protist Euglena gracilis, a heavy metal hyper-accumulator, was grown under photo-heterotrophic and extreme conditions (acidic pH, anaerobiosis and with Cd(2+)) and biochemically characterized. High biomass (8.5×10(6)cellsmL(-1)) was reached after 10 days of culture. Under anaerobiosis, photosynthetic activity built up a microaerophilic environment of 0.7% O2, which was sufficient to allow mitochondrial respiratory activity: glutamate and malate were fully consumed, whereas 25-33% of the added glucose was consumed. In anaerobic cells, photosynthesis but not respiration was activated by Cd(2+) which induced higher oxidative stress. Malondialdehyde (MDA) levels were 20 times lower in control cells under anaerobiosis than in aerobiosis, although Cd(2+) induced a higher MDA production. Cd(2+) stress induced increased contents of chelating thiols (cysteine, glutathione and phytochelatins) and polyphosphate. Biosorption (90%) and intracellular accumulation (30%) were the mechanisms by which anaerobic cells removed Cd(2+) from medium, which was 36% higher versus aerobic cells. The present study indicated that E. gracilis has the ability to remove Cd(2+) under anaerobic conditions, which might be advantageous for metal removal in sediments from polluted water bodies or bioreactors, where the O2 concentration is particularly low.

Air-adapted Methanosarcina acetivorans shows high methane production and develops resistance against oxygen stress.

Methanosarcina acetivorans, considered a strict anaerobic archaeon, was cultured in the presence of 0.4-1% O2 (atmospheric) for at least 6 months to generate air-adapted cells; further, the biochemical mechanisms developed to deal with O2 were characterized. Methane production and protein content, as indicators of cell growth, did not change in air-adapted cells respect to cells cultured under anoxia (control cells). In contrast, growth and methane production significantly decreased in control cells exposed for the first time to O2. Production of reactive oxygen species was 50 times lower in air-adapted cells versus control cells, suggesting enhanced anti-oxidant mechanisms that attenuated the O2 toxicity. In this regard, (i) the transcripts and activities of superoxide dismutase, catalase and peroxidase significantly increased; and (ii) the thiol-molecules (cysteine + coenzyme M-SH + sulfide) and polyphosphate contents were respectively 2 and 5 times higher in air-adapted cells versus anaerobic-control cells. Long-term cultures (18 days) of air-adapted cells exposed to 2% O2 exhibited the ability to form biofilms. These data indicate that M. acetivorans develops multiple mechanisms to contend with O2 and the associated oxidative stress, as also suggested by genome analyses for some methanogens.

Maintenance of intracellular hypoxia and adequate heat shock response are essential requirements for pathogenicity and virulence of Entamoeba histolytica.

Adhesion to cells, cytotoxicity and proteolysis are functions required for virulence and pathogenicity of Entamoeba histolytica. However, there was no correlation between these in vitro functions and the early elimination of non-pathogenic E. dispar and non-virulent E. histolytica (nvEh) in experimental amoebic liver abscesses developed in hamsters. Thus, additional functions may be involved in amoebic pathogenicity and virulence. In the present study, an integral experimental assessment, including innovative technologies for analyses of amoebal pathophysiology, cell biology, biochemistry and transcriptomics, was carried out to elucidate whether other cellular processes are involved in amoebal pathogenicity and virulence. In comparison with virulent E. histolytica, the data indicated that the main reasons for the early clearance of nvEh from hamster liver are decreased intracellular H2 O2 detoxification rate and deficient heat shock protein expression, whereas for E. dispar, it is a relatively lower capacity for O2 reduction. Therefore, maintenance of an intracellular hypoxic environment combined with the induction of an adequate parasite response to oxidative stress are essential requirements for Entamoeba survival in the liver, and therefore for pathogenicity.

In vivo identification of the steps that control energy metabolism and survival of Entamoeba histolytica.

The steps that control the Entamoeba histolytica glycolytic flux were here identified by elasticity analysis, an experimental approach of metabolic control analysis. The concentrations of glycolytic metabolites were gradually varied in live trophozoites by (a) feeding with different glucose concentrations and (b) inhibiting the final pathway steps; in parallel, the changes in the pathway flux were determined. From the metabolite concentration-flux relationship, the elasticity coefficients of individual or groups of pathway reactions were determined and used to calculate their respective degrees of control on the glycolytic flux (flux control coefficients). The results indicated that the pathway flux was mainly controlled (72-86%) by the glucose transport/hexokinase/glycogen degradation group of reactions and by bifunctional aldehyde-alcohol dehydrogenase (ADHE; 18%). Further, inhibition of the first pathway reactions with 2-deoxyglucose (2DOG) decreased the glycolytic flux and ATP content by 75% and 50%, respectively. Cell viability was also decreased by 2DOG (25%) and more potently (50%) by 2DOG plus the ADHE inhibitor tetraethylthiuram disulfide (disulfiram). Biosate as an alternative carbon (amino acid) source was unable to replace glucose for ATP supply, which indicated that glucose was the main nutrient for amoebal ATP synthesis and survival. These results indicated that glycolysis in the parasite is mainly controlled by the initial pathway reactions and that their inhibition can decrease the parasite energy load and survival.

Zn-bis-glutathionate is the best co-substrate of the monomeric phytochelatin synthase from the photosynthetic heavy metal-hyperaccumulator Euglena gracilis.

The phytochelatin synthase from photosynthetic Euglena gracilis (EgPCS) was analyzed at the transcriptional, kinetic, functional, and phylogenetic levels. Recombinant EgPCS was a monomeric enzyme able to synthesize, in the presence of Zn(2+) or Cd(2+), phytochelatin2-phytochelatin4 (PC2-PC4) using GSH or S-methyl-GS (S-methyl-glutathione), but not γ-glutamylcysteine or PC2 as a substrate. Kinetic analysis of EgPCS firmly established a two-substrate reaction mechanism for PC2 synthesis with Km values of 14-22 mM for GSH and 1.6-2.5 µM for metal-bis-glutathionate (Me-GS2). EgPCS showed the highest Vmax and catalytic efficiency with Zn-(GS)2, and was inactivated by peroxides. The EgPCS N-terminal domain showed high similarity to that of other PCSases, in which the typical catalytic core (Cys-70, His-179 and Asp-197) was identified. In contrast, the C-terminal domain showed no similarity to other PCSases. An EgPCS mutant comprising only the N-terminal 235 amino acid residues was inactive, suggesting that the C-terminal domain is essential for activity/stability. EgPCS transcription in Euglena cells was not modified by Cd(2+), whereas its heterologous expression in ycf-1 yeast cells provided resistance to Cd(2+) stress. Phylogenetic analysis of the N-terminal domain showed that EgPCS is distant from plants and other photosynthetic organisms, suggesting that it evolved independently. Although EgPCS showed typical features of PCSases (constitutive expression; conserved N-terminal domain; kinetic mechanism), it also exhibited distinct characteristics such as preference for Zn-(GS)2 over Cd-(GS)2 as a co-substrate, a monomeric structure, and ability to solely synthesize short-chain PCs, which may be involved in conferring enhanced heavy-metal resistance.

The bifunctional aldehyde-alcohol dehydrogenase controls ethanol and acetate production in Entamoeba histolytica under aerobic conditions.

By applying metabolic control analysis and inhibitor titration we determined the degree of control (flux control coefficient) of pyruvate:ferredoxin oxidoreductase (PFOR) and bifunctional aldehyde-alcohol dehydrogenase (ADHE) over the fluxes of fermentative glycolysis of Entamoeba histolytica subjected to aerobic conditions. The flux-control coefficients towards ethanol and acetate formation determined for PFOR titrated with diphenyleneiodonium were 0.07 and 0.09, whereas for ADHE titrated with disulfiram were 0.33 and -0.19, respectively. ADHE inhibition induced significant accumulation of glycolytic intermediates and lower ATP content. These results indicate that ADHE exerts significant flux-control on the carbon end-product formation of amoebas subjected to aerobic conditions.

Phosphofructokinase type 1 kinetics, isoform expression, and gene polymorphisms in cancer cells.

Kinetic analysis of PFK-1 from rodent AS-30D, and human HeLa and MCF-7 carcinomas revealed sigmoidal [fructose 6-phosphate, Fru6P]-rate curves with different V(m) values when varying the allosteric activator fructose 2,6 bisphosphate (Fru2,6BP), AMP, Pi, NH(4)(+), or K(+). The rate equation that accurately predicted this behavior was the exclusive ligand binding concerted transition model together with non-essential hyperbolic activation. PFK-1 from rat liver and heart also exhibited the mixed cooperative-hyperbolic kinetic behavior regarding activators. Lowering pH induced decreased affinity for Fru6P, Fru2,6BP, citrate, and ATP (as inhibitor); as well as decreased V(m) and increased content of inactive (T) enzyme forms. High K(+) prompted increased (Fru6P) or decreased (activators) affinities; increased V(m); and increased content of active (R) enzyme forms. mRNA expression analysis and nucleotide sequencing showed that the three PFK-1 isoforms L, M, and C are transcribed in the three carcinomas. However, proteomic analysis indicated the predominant expression of L in liver, of M in heart and MCF-7 cells, of L>M in AS-30D cells, and of C in HeLa cells. PFK-1M showed the highest affinities for F6P and citrate and the lowest for ATP (substrate) and F2,6BP; PFK-1L showed the lowest affinity for F6P and the highest for F2,6BP; and PFK-1C exhibited the highest affinity for ATP (substrate) and the lowest for citrate. Thus, the present work documents the kinetic signature of each PFK-1 isoform, and facilitates the understanding of why this enzyme exerts significant or negligible glycolysis flux-control in normal or cancer cells, respectively, and how it regulates the onset of the Pasteur effect.

Modeling cancer glycolysis.

Most cancer cells exhibit an accelerated glycolysis rate compared to normal cells. This metabolic change is associated with the over-expression of all the pathway enzymes and transporters (as induced by HIF-1α and other oncogenes), and with the expression of hexokinase (HK) and phosphofructokinase type 1 (PFK-1) isoenzymes with different regulatory properties. Hence, a control distribution of tumor glycolysis, modified from that observed in normal cells, can be expected. To define the control distribution and to understand the underlying control mechanisms, kinetic models of glycolysis of rodent AS-30D hepatoma and human cervix HeLa cells were constructed with experimental data obtained here for each pathway step (enzyme kinetics; steady-state pathway metabolite concentrations and fluxes). The models predicted with high accuracy the fluxes and metabolite concentrations found in living cancer cells under physiological O(2) and glucose concentrations as well as under hypoxic and hypoglycemic conditions prevailing during tumor progression. The results indicated that HK≥HPI>GLUT in AS-30D whereas glycogen degradation≥GLUT>HK in HeLa were the main flux- and ATP concentration-control steps. Modeling also revealed that, in order to diminish the glycolytic flux or the ATP concentration by 50%, it was required to decrease GLUT or HK or HPI by 76% (AS-30D), and GLUT or glycogen degradation by 87-99% (HeLa), or decreasing simultaneously the mentioned steps by 47%. Thus, these proteins are proposed to be the foremost therapeutic targets because their simultaneous inhibition will have greater antagonistic effects on tumor energy metabolism than inhibition of all other glycolytic, non-controlling, enzymes.

Pyruvate:ferredoxin oxidoreductase and bifunctional aldehyde-alcohol dehydrogenase are essential for energy metabolism under oxidative stress in Entamoeba histolytica.

The in vitro Entamoeba histolytica pyruvate:ferredoxin oxidoreductase (EhPFOR) kinetic properties and the effect of oxidative stress on glycolytic pathway enzymes and fluxes in live trophozoites were evaluated. EhPFOR showed a strong preference for pyruvate as substrate over other oxoacids. The enzyme was irreversibly inactivated by a long period of saturating O(2) exposure (IC(50) 0.034 mm), whereas short-term exposure (< 30 min) leading to > 90% inhibition allowed for partial restoration by addition of Fe(2+). CoA and acetyl-CoA prevented, whereas pyruvate exacerbated, inactivation induced by short-term saturating O(2) exposure. Superoxide dismutase was more effective than catalase in preventing the inactivation, indicating that reactive oxygen species (ROS) were involved. Hydrogen peroxide caused inactivation in an Fe(2+)-reversible fashion that was not prevented by the coenzymes, suggesting different mechanisms of enzyme inactivation by ROS. Structural analysis on an EhPFOR 3D model suggested that the protection against ROS provided by coenzymes could be attributable to their proximity to the Fe-S clusters. After O(2) exposure, live parasites displayed decreased enzyme activities only for PFOR (90%) and aldehyde dehydrogenase (ALDH; 68%) of the bifunctional aldehyde-alcohol dehydrogenase (EhADH2), whereas acetyl-CoA synthetase remained unchanged, explaining the increased acetate and lowered ethanol fluxes. Remarkably, PFOR and ALDH activities were restored after return of the parasites to normoxic conditions, which correlated with higher ethanol and lower acetate fluxes. These results identified amebal PFOR and ALDH of EhADH2 activities as markers of oxidative stress, and outlined their relevance as significant controlling steps of energy metabolism in parasites subjected to oxidative stress.