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The biosynthesis of pyrimidines in Pseudomonas putida was investigated. In this study, pyrimidine requiring mutants were isolated by conventional mutagenesis and enrichment. The strains required exogenously supplied pyrimidines for growth and were found by enzyme assays to be deficient for the product of the pyrB gene encoding the enzyme aspartate transcarbamoylase. None of the intermediates of the pathway could supply the auxotrophic requirement of the strain; only preformed pyrimidines, metabolized via salvage pathways could suffice. Pyrimidine limitation in the mutant caused a slight but significant fifty per cent increase in expression of all the de novo biosynthetic enzymes. Pyrimidine starvation's effect on nucleotide pool levels was examined in the mutant and caused a marked swelling of the purine nucleotide pools.

Plasmodium falciparum causes the most severe form of human malaria, and the continual development of resistance of this parasite to current anti-malarial drugs underpins a pressing need for the discovery of novel chemotherapeutic approaches. Polyamines and their biosynthetic enzymes are present at high levels in rapidly proliferating cells, including cancer cells and protozoan parasites. Inhibition of the malaria parasite’s polyamine biosynthesis pathway causes cytostatic arrest in the trophozoite stage, but does not cure infections in vivo. This may be due to the salvage of exogenous polyamines from the host, replenishing the intracellular polyamine pool; however the mechanism(s) of polyamine uptake by the intraerythrocytic parasite are not well understood. In this study the uptake of the polyamines putrescine and spermidine into P. falciparum-infected erythrocytes (iRBC) well as into P. falciparum parasites functionally isolated from their host cell by saponin-permeabilisation of the erythrocyte membrane was investigated using radioisotope flux techniques. While the characteristics of transport of putrescine into infected erythrocytes were similar to those of transport into uninfected erythrocytes, spermidine entered iRBC in part via the ‘new permeation pathways’ induced by the parasite in the erythrocyte membrane. Both putrescine and spermidine were taken up across the plasma membrane of isolated parasites via a saturable, temperature-dependent process that showed competition between different polyamines as well as the polyamine precursor ornithine and basic amino acids. Inhibition of polyamine biosynthesis led to increased total uptake of both putrescine and spermidine. The influx of putrescine and spermidine into isolated parasites was independent of Na+ but increased with increasing pH and showed a marked dependence on the membrane potential, decreasing with membrane depolarisation and increasing with membrane hyperpolarisation. Both anthracene and polyamine derivatives have been shown to have anti-malarial activity. Anthracene-polyamine conjugates have been developed with the aim of utilising the polyamine uptake mechanisms of cancer cells to deliver the cytotoxic anthracene moieties to these cells. Here, several anthracene-polyamine conjugates showed promising anti-malarial activity. These compounds inhibited parasite proliferation with IC50 values in the nM range, and caused an arrest in the cell cycle, as well as a decrease in the mitochondrial membrane potential. Cytotoxicity could not be reversed by the addition of exogenous polyamines, nor did the conjugates have an effect on intracellular polyamine levels. This doctoral study showed that P. falciparum parasites not only synthesise polyamines, but can also acquire putrescine and spermidine from the extracellular environment and paves the way for interfering with polyamine metabolism as an anti-parasitic strategy. / Thesis (PhD)--University of Pretoria, 2012. / Biochemistry / unrestricted

Malaria is a life-threatening vector borne infectious disease caused by protozoan parasites of the genus Plasmodium. More than 100 species of Plasmodium can infect numerous animal species such as reptiles, birds and various mammals. However, human malaria is caused by four Plasmodium species -Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale and Plasmodium malariae, and occasionally by the simian malaria parasite, Plasmodium knowlesi. Of these, P. falciparum and P. vivax are the major causative agents and P. falciparum is the most virulent. About 300-500 million malaria infections occur every year leading to over 1-2 million deaths, of which 75% occur in African children of less than 5 years infected with P. falciparum. In spite of major global efforts to eliminate this disease over the past few decades, it continues to persist as a major affliction of human-kind imposing serious health and economic burden, especially to the poor countries. In India, the present scenario is about 2 million malaria positive cases every year, with almost 50% being caused by P. falciparum. Although remarkable attempts have been made over the years to develop vaccines against sexual and asexual stages of malaria parasite, an effective vaccine is still not in sight and remains as a distant goal. Hence, highly potent, less toxic and affordable antimalarial drugs remain as a first line therapy for malaria. Unfortunately, these parasites have been evolving against every known antimalarial drug and many of these drugs have lost their potency due to rapid emergence and spread of drug resistant strains. With development of resistance against frontline antimalarials such as chloroquine and antifolates, artemisinin and its derivatives seem to be the only effective antimalarials. However, recent reports on the possible emergence of artemisinin resistant strains, have led to the implementation of artemisinin-based combination therapies as a strategy to prevent drug resistance. Also, this continuous emergence of drug resistance has necessitated the development of new antimalarial drugs to combat this disease. While, Anopheles mosquitoes transmit parasites that infect humans, monkeys and rodents, Culex and Aedes mosquitoes predominate in the natural transmission to birds, and vectors of reptilian parasites are largely unknown. Of the approximately 400 species of Anopheles throughout the world, about 60 are malaria vectors under natural conditions, and 30 of which are of major importance. Ironically, the strategies implemented for controlling Anopheles, have also been hampered by insecticide resistance and other practical difficulties that exist in the scope of their applicability. In the past few years several milestones have been achieved in parasite genome, transcriptome and proteome studies, which could be exploited for the development of new drugs and drug targets. One such promising target includes the metabolic pathways of the malaria parasite which differ significantly from its human host. This thesis entitled “Unique Features of the Heme-Biosynthetic Pathway in Human Malaria Parasite, Plasmodium falciparum” unravels the unique biochemical features of heme-biosynthetic enzymes of P. falciparum, which have the potential for being drug targets. This pathway was first identified in this laboratory over 15 years ago. In the present study, five of the 7 enzymes of this pathway have been cloned, expressed, properties studied and sites of localization identified. With the knowledge on the first two enzymes coming from earlier studies, it is now possible to depict the unique hybrid pathway for heme biosynthesis in P. falciparum with full experimental validation.

3-Deoxy-D-arabino-heptulosonate 7-phosphate (DAH7P) synthase and 3-deoxy-Dmanno- octulosonate 8-phosphate synthase (KDO8P) synthase are two enzymes that catalyse very similar reactions. DAH7P synthase is the first enzyme of the shikimate pathway and catalyses the condensation reaction between the four-carbon sugar erythrose 4-phosphate (E4P) 1 and the three-carbon sugar phosphoenolpyruvate (PEP) 2 to give the seven-carbon sugar DAH7P 3. KDO8P synthase catalyses a similar condensation reaction between the five-carbon sugar arabinose 5-phosphate (A5P) 8 and PEP 2 to give the eight-carbon sugar KDO8P 9. Early mechanistic studies have shown the reaction mechanisms of these two enzymes to be very similar and structural and phylogenic analysis has suggested that the two enzymes share a common ancestor. However, there are differences between the two enzymes that have not been explained by the current literature. Whereas all DAH7P synthases require a divalent metal ion for activity, there exists both metallo and non-metallo KDO8P synthases. As well as this, there is the difference in substrate specificity. The natural substrate of KDO8P synthase, A5P, is one carbon longer and has the opposite C2 stereochemistry to E4P, the natural substrate of DAH7P synthase. This study investigates the role of the C2 and C3 hydroxyl groups of E4P and A5P in the enzyme catalysed reactions. The E4P analogues 2-deoxyE4P 38 and 3-deoxyE4P 39 have been synthesised from [beta]-hydroxy-[gamma]-butyrolactone and malic acid respectively. The two analogues were tested as substrates for DAH7P synthase from a variety of organisms, including N. meningitidis, the purification and characterisation of which was carried out during the course of these studies. It was found that both analogues were substrates for DAH7P synthase. 2-DeoxyE4P was found to be the best alternative substrate for DAH7P synthase to date. The analogous study was carried out on KDO8P synthase from N. meningitidis with 2- deoxyR5P 34 and 3-deoxyA5P 40. It was found that removal of the C2 and C3 hydroxyl groups of A5P was much more catastrophic for the KDO8P synthase catalysed reaction. Commercially available 2-deoxyR5P was found to be a very poor substrate, whereas 3-deoxyA5P, which was prepared according to a literature procedure was not a substrate. The difference in substrate specificities of DAH7P synthase and KDO8P synthase is consistent with the hypothesis that despite their similarities, these two related enzymes have different mechanisms. The key step for DAH7P synthase appears to be coordination of the E4P carbonyl to the divalent metal. The metal appears to play a less important role in the KDO8P synthase reaction and the key step is the correct orientation of A5P in the active site.