Investigating the Role of ADP-forming Acetyl-CoA Synthetase from the Protozoan Parasite Entamoeba histolytica

ADP-forming acetyl-CoA synthetase (ACD; EC 6.2.1.13) catalyzes the reversible conversion of acetyl-CoA to acetate coupled to the production of ATP. This enzyme is present only in certain acetate-producing archaea and a limited number of bacteria and eukaryotes. ACD belongs to the same NDP-forming acyl-CoA synthetase enzyme superfamily as succinyl-CoA synthetase (SCS; EC 6.2.1.4) from the citric acid cycle, and a similar three-step mechanism involving a phosphoenzyme intermediate was originally proposed for this enzyme. ACD has been postulated to be a major acetate-producing enzyme in the protozoan parasite Entamoeba histolytica and may contribute to ATP production. Biochemical and kinetic characterization of recombinant E. histolytica ACD (EhACD) revealed that this enzyme may function in the direction of acetate production for generation of ATP and CoA during growth in the high glucose environment of the small intestine, and in acetate assimilation to acetyl-CoA in the high acetate environment of the lower intestine during colonization. EhACD utilizes multiple substrates including propionate and propionyl-CoA supporting an additional proposed role in amino acid degradation. EhACD activity is regulated by both ATP and PPi, important energy molecules in E. histolytica. The ACD mechanism has been controversial, as a required second phosphorylation step was proposed for the Pyrococcus furiosus enzyme. Investigation of the catalytic role of the two proposed phosphorylation sites in EhACD revealed that His252, the site of phosphorylation in the original three-step mechanism, is essential for activity and His533, the proposed second phosphorylation site, is important but not essential. Likewise, Glu213, proposed to play a role in phosphorylation/ dephosphorylation of His252, is also required but Asp674 thought to stabilize the phosphohistidine is not. These results suggest that EhACD follows a three-step mechanism with a single phosphoenzyme intermediate. Additional conserved active site residues were examined for their role in catalysis. Asp314 was shown to be essential for activity, possibly in both a catalytic role and a structural role. Alteration at this position resulted in complete loss of activity, and computational modeling based on the Candidatus Korarchaeum cryptofilum ACD-I structure suggests that this residue may be critical for dimerization. Future directions for understanding the complex mechanism of ACD and its physiological role are presented

Cell Death after Photodynamic Therapy Treatment in Unicellular Protozoan Parasite <em>Tritrichomonas foetus</em>

Programmed cell death in T. foetus does not seem to make sense at first sight; however, different mechanisms of cellular death in this unicellular organism have been observed. This review summarizes the available data related to programmed cell death already published for the cattle parasite T. foetus and attempts to clarify some crucial points to understand this mechanism found in non-mitochondriates parasites, as well as assist in future research. Important results with different treatments showed that the T. foetus can choose among different pathways how to initiate cell death. Thus, a major challenge for cellular death research remains the identification of the molecular cell death machinery of this protist, such as caspases pathway, nuclear abnormalities, morphology cell changes, cellular death in this parasite and the prospects in the future research. Although, the possibility of the existence of different pathways to cell death in trichomonads is discussed and a model for possible executioners pathways during T. foetus cell death is proposed

Citric acid cycle enzymes of Methylophilus methylotrophus

SIGLEAvailable from British Library Document Supply Centre- DSC:DX79219 / BLDSC - British Library Document Supply CentreGBUnited Kingdo

Metabolism of methane and propane and the role of the glyoxylate bypass enzymes in Methylocella silvestris BL2

Methylocella silvestris BL2 is a moderately acidophilic facultative methanotroph isolated from forest soil in 2003. Uniquely, it has the ability to grow on a wide range of multi-carbon compounds in addition to methane. An analysis of growth conditions identified the requirements for robust and predictable growth on a wide range of substrates. A simple and effective method of targeted mutagenesis was developed, which relies on electroporation with a linear DNA fragment, and several strains with deletions of key enzymes were constructed using this method. Deletion of isocitrate lyase demonstrated that this enzyme is required for growth on both one-carbon and two-carbon compounds. The second enzyme of the glyoxylate cycle, malate synthase, was shown to be essential for growth on two-carbon compounds. However, surprisingly, deletion of glyoxylate cycle enzymes had a dramatic effect on expression of methanol dehydrogenase. Possible causes of this effect are discussed. Surprisingly, M. silvestris was able to grow on propane and the presence and expression of a gene cluster encoding a putative propane monooxygenase was confirmed. This enzyme was found to be a second soluble diiron monooxygenase (SDIMO) with homology to the propane monooxygenase from Gordonia TY5, identifying M. silvestris as the first known methanotroph to contain SDIMOs from more than one group. Deletion of these enzymes in turn was used to determine the requirement for each during growth on methane or propane. The soluble methane monooxygenase (sMMO) was found to be capable of oxidising propane, whereas the propane monooxygenase (PrMO) was unable to oxidise methane. However, although a strain lacking the PrMO was capable of growth on 2.5% (v/v) propane, it was unable to grow on this gas at 20% (v/v), and at 2.5%, assimilation into biomass was less efficient in comparison to the wild-type. Evidence is presented that products of oxidation of propane by the sMMO may be toxic to the cell or inhibitory to growth in the absence of the PrMO. Both the sMMO and the PrMO were found to be capable of oxidation of a wide range of aliphatic and aromatic compounds, including xenobiotics, suggesting a possible role in bioremediation. M. silvestris BL2 was found to oxidise propane at both terminal and sub-terminal positions, resulting in 1- propanol and 2-propanol respectively, and biochemical methods were used to assay the enzymes of terminal and sub-terminal pathways. Assimilation of 1-propanol was found to be by the methylmalonyl-CoA pathway, and the data suggested that 2- propanol was oxidised to acetone and acetol. The final gene of the PrMO genecluster, predicted to encode a flavin adenine dinucleotide (FAD)-containing enzyme with homology to characterised membrane-bound D-gluconate dehydrogenase from Gluconobacter spp., was found to be essential for growth on 2-propanol and acetone and may be involved in the oxidation of acetol during propane metabolism by the sub-terminal pathway

Haem Biosynthesis in Isolated Human Erythroblasts

Hepatic haem biosynthesis has been well characterised. The first enzyme of the haem biosynthetic pathway, 5-aminolaevulinic acid (ALA) synthase, is rate-limiting and under negative feedback control by haem. Liver is, however, a relatively minor site of haem synthesis as 80% of haem is formed in the erythroid cells of the bone marrow. Within developing erythroid cells haem is required both for its specific complexing with globin and for the coordinate regulation of erythroblast metabolism and differentiation. The study of erythroid haem synthesis has been hampered by the heterogeneous nature of the marrow cell population, the various differentiation stages of the erythroblast population, the small sample sizes available and the lack of sensitive enzyme assays. A variety of models and techniques have been used to study erythroid haem synthesis. Accordingly, the results have been inconclusive. This work describes human bone marrow fractionation to provide four purified age-matched erythroblast cell populations. Myeloid (white) cells were removed from the sample by monoclonal antibody mediated cell lysis. The remaining cells were then fractionated by equilibrium density gradient centrifugation. These 'in vivo' human erythroblasts were used to examine the effects of normal and abnormal erythroid differentiation on the first and last enzymes of the haem pathway, ALA synthase and ferrochelatase. A sensitive radiochemical assay for ALA synthase was adopted and improved. This was then used to examine the temperature-dependent inactivation of erythroid ALA synthase. A novel radiochemical assay for ferrochelatase was developed. Both assays utilise HPLC for isolation of the radioactive product and are capable of detecting picomoles of activity. The pattern of ALA synthase and ferrochelatase activities during normoblastic erythropoiesis was established. ALA synthase activity was maximal in the most immature erythroid cells and diminished as the cells matured. Ferrochelatase activity was maximal in the intermediate erythroid cells. Hence, the development of enzyme activity appears to be sequential rather than simultaneous. On a quantitative basis, however, it is uncertain whether the development of ferrochelatase activity is limiting for haem formation. The effects of iron and haem deficiency on haem enzyme activity were studied. In iron deficient erythroblasts ALA synthase activity was significantly reduced, particularly in the most immature erythroid cells. This enzyme reduction is likely to result from consequent haem deficiency rather than iron deficiency per se as an iron replete patient with haem deficiency associated with hepatoerythropoietic porphyria also showed reduced enzyme activity. This suggests that erythroid haem synthesis is controlled in a different manner to hepatic haem synthesis - a view compatible with the recent identification of specific genes and tissue-specific isoenzymes for ALA synthase. ALA synthase and ferrochelatase activities were measured in patients with sideroblastic anaemia (SA). Haem synthesis is impaired in SA and ALA synthase is generally believed to be the site of the primary defect. ALA synthase activity was markedly impaired in 3 patients with congenital SA (CSA) and 7 patients with primary acquired SA (PASA). One patient with secondary SA (SSA) demonstrated normal activity. Ferrochelatase activity was measured in six patients (1 CSA, 5 PASA) and was found to be significantly reduced. The latter 6 patients (1 CSA, 5 PASA) were treated with haem arginate in an attempt to correct the haem deficiency and to stimulate haem enzyme activity. Haem arginate was clinically ineffective (Hb levels did not change in any of the patients). However, the study demonstrated that exogenous haem is able to enter erythroblasts and influence haem enzyme activity. In two patients (1 CSA, 1 PASA), both ALA synthase and ferrochelatase activities returned to normal. This indicates a potential role for haem arginate in the treatment of haematological disorders other than SA which may respond to stimulated haem biosynthesis. The results also suggest that the primary abnormality in these cases of CSA and PASA is not ALA synthase (or ferrochelatase) deficiency. Furthermore, the stimulatory effect of exogenous haem on enzyme activity and the reduced ALA synthase activity in haem deficiency provide strong evidence of independent regulatory mechanisms for haem biosynthesis in erythroid and hepatic tissue

Morphological and physiological adaptations of prosthecate bacteria to growth in low nutrient environments

Prosthecate bacteria are usually observed in oligotrophic (low nutrient) environments and have been proposed as "model" oligotrophic bacteria since they possess a number of adaptations for growth under very low nutrient conditions. This project was designed to study selected prosthecate bacteria and attempt to relate their ecology to physiological and morphological adaptations, in particular the production of a motile swarmer cell stage. Ecological investigations showed that the prosthecate bacteria were ubiquitous in the oligotrophic fresh water environments examined. A variety of morphological types were observed in populations which also included large numbers of morphologically "typical" cells, demonstrating that there exists a large variety of oligotrophic bacteria. The responses of these bacteria to increasing nutrient concentrations suggested that currently accepted definitions of oligotrophy are in need of reconsideration. Caulobacter crescentus CB1S, Hyphomicrobium X and Rhodomicrobium vanniel1i Rm5 were studied in detail in both batch and continuous culture with respect to nutrient effects on cell morphology and cell type expression. All three organisms produced elongated prosthecae with increasing nutrient stress under both carbon and phosphate—1imi ted conditions, an observation somewhat in contradiction with reports suggesting that prosthecae function as specialised phosphate uptake sites. The production of «warmer cells was enhanced under conditions of nutrient stress, supporting the proposition that swarmer cells function as specialised survival and dispersal cells. R, vannlelli was chosen for investigations into the intermediary metabolism of prosthecate bacteria with respect to adaptions to oligotrophy and differential cell type expression. Unlike other Rhodospirillaceae this organism was shown to possess an incomplete tricarboxyllc acid (TCA) cycle under anaerobic conditions broken at 2-oxoglutarate dehydrogenase and lacked NADH oxidase activity but these enzymes were present under aerobic conditions. Of the glyoxylate shunt enzymes, malate synthase activity was detected but isocltrate lyase was absent. The TCA cycle enzymes, Ribulose—1,5-bl«phosphate carboxylase/oxygenase (RUBISCO) and phosphoenolpyruvate carboxylase possessed similar activities and Inhibitor patterns in both swarmer and prosthecate cells and therefore the physiological adaptations of the swarmer cells remain largely unknown. This work has demonstrated the success and ubiquity of prosthecate bacteria in the oligotrophic fresh water ecoystem although it must be emphasised that there exist a large number of non-prosthecate bacteria in these environments. The roles of the prosthecae and swarmer cells in this competitiveness are as yet not fully clear but appear to be of importance in view of observed responses to nutrient limitation

Incomplete tricarboxylic acid cycle and proton gradient in Pandoravirus massiliensis: is it still a virus?

The discovery of Acanthamoeba polyphaga Mimivirus, the first isolated giant virus of amoeba, challenged the historical hallmarks defining a virus. Giant virion sizes are known to reach up to 2.3µm, making them visible by optical microscopy. Their large genome sizes of up to 2.5Mb can encode proteins involved in the translation apparatus. We have investigated possible energy production in Pandoravirus massiliensis. Mitochondrial membrane markers allowed for the detection of a membrane potential in purified virions and this was enhanced by a regulator of the tricarboxylic acid cycle but abolished by the use of a depolarizing agent. Bioinformatics was employed to identify enzymes involved in virion proton gradient generation and this approach revealed that 8 putative P. massiliensis proteins exhibited low sequence identities with known cellular enzymes involved in the universal tricarboxylic acid cycle. Further, all 8 viral genes were transcribed during replication. The product of one of these genes, ORF132, was cloned and expressed in Escherichia coli, and shown to function as an isocitrate dehydrogenase, a key enzyme of the tricarboxylic acid cycle. Our findings show for the first time that a membrane potential can exist in Pandoraviruses, and this may be related to tricarboxylic acid cycle. The presence of a proton gradient in P. massiliensis makes this virus a form of life for which it is legitimate to ask the question ‘what is a virus?’

Metabolism of methane and propane and the role of the glyoxylate bypass enzymes in Methylocella silvestris BL2

Methylocella silvestris BL2 is a moderately acidophilic facultative methanotroph isolated from forest soil in 2003. Uniquely, it has the ability to grow on a wide range of multi-carbon compounds in addition to methane. An analysis of growth conditions identified the requirements for robust and predictable growth on a wide range of substrates. A simple and effective method of targeted mutagenesis was developed, which relies on electroporation with a linear DNA fragment, and several strains with deletions of key enzymes were constructed using this method. Deletion of isocitrate lyase demonstrated that this enzyme is required for growth on both one-carbon and two-carbon compounds. The second enzyme of the glyoxylate cycle, malate synthase, was shown to be essential for growth on two-carbon compounds. However, surprisingly, deletion of glyoxylate cycle enzymes had a dramatic effect on expression of methanol dehydrogenase. Possible causes of this effect are discussed. Surprisingly, M. silvestris was able to grow on propane and the presence and expression of a gene cluster encoding a putative propane monooxygenase was confirmed. This enzyme was found to be a second soluble diiron monooxygenase (SDIMO) with homology to the propane monooxygenase from Gordonia TY5, identifying M. silvestris as the first known methanotroph to contain SDIMOs from more than one group. Deletion of these enzymes in turn was used to determine the requirement for each during growth on methane or propane. The soluble methane monooxygenase (sMMO) was found to be capable of oxidising propane, whereas the propane monooxygenase (PrMO) was unable to oxidise methane. However, although a strain lacking the PrMO was capable of growth on 2.5% (v/v) propane, it was unable to grow on this gas at 20% (v/v), and at 2.5%, assimilation into biomass was less efficient in comparison to the wild-type. Evidence is presented that products of oxidation of propane by the sMMO may be toxic to the cell or inhibitory to growth in the absence of the PrMO. Both the sMMO and the PrMO were found to be capable of oxidation of a wide range of aliphatic and aromatic compounds, including xenobiotics, suggesting a possible role in bioremediation. M. silvestris BL2 was found to oxidise propane at both terminal and sub-terminal positions, resulting in 1- propanol and 2-propanol respectively, and biochemical methods were used to assay the enzymes of terminal and sub-terminal pathways. Assimilation of 1-propanol was found to be by the methylmalonyl-CoA pathway, and the data suggested that 2- propanol was oxidised to acetone and acetol. The final gene of the PrMO genecluster, predicted to encode a flavin adenine dinucleotide (FAD)-containing enzyme with homology to characterised membrane-bound D-gluconate dehydrogenase from Gluconobacter spp., was found to be essential for growth on 2-propanol and acetone and may be involved in the oxidation of acetol during propane metabolism by the sub-terminal pathway.EThOS - Electronic Theses Online ServiceGBUnited Kingdo