Tropical malaria caused by the unicellular apicomplexan parasite Plasmodium is still a major threat to human health and welfare in tropical and subtropical regions of the world. In the past decades, both the emergence of antimalarial resistance of Plasmodium and insecticide-resistance in the mosquito vector made the situation more and more severe. The progress in developing a malaria vaccine, in identifying new drug targets, and in developing novel antimalarials is slow. Hexokinase represents a central enzyme of glucose metabolism in the malaria parasite P. falciparum. Due to the high glucose dependence of the parasite, studying hexokinase can enhance our knowledge of central metabolic processes in Plasmodium and contribute to the search for new antimalarial drug targets. In this thesis, I have therefore studied hexokinase (PfHK) from P. falciparum including its kinetic properties as well as its redox regulation.First, PfHK has been successfully cloned, heterologously overexpressed in Escherichia coli and purified to homogeneity. Gel filtration indicated that recombinant PfHK is present as a tetramer. Kinetic studies showed relatively low affinities for the substrates glucose and ATP when compared with the human homologues. In contrast to the common 50-kDa hexokinases in other species, PfHK can be inhibited by G6P. Two constructs of GFP fused PfHK (full-length and C-terminally truncated) were generated and revealed that the sub-cellular localization of PfHK is cytosolic in P. falciparum. The data furthermore showed that the C-terminal hydrophobic region in PfHK does not seem to lead to membrane association of the protein. In order to obtain crystals of PfHK and solve its three-dimensional structure, more than 500 crystallization conditions were tested. Although no high quality crystals have been obtained so far, the screening seems to be worth of further optimization. To gain first structural insights, a model of PfHK was generated based on the structure of human hexokinase I (PDB ID: 1DGK). Three insertions were found on the surface of PfHK when comparing the structure with its human counterpart, which might provide a basis for selective inhibitor development.In a second focus of my work, I studied the redox regulation of PfHK, which had been shown to be a target of members of the thioredoxin superfamily and of S-glutathionlylation. Thioredoxin-related proteins (thioredoxin 1, glutaredoxin and plasmoredoxin of P. falciparum) slightly enhanced the activity of PfHK. Similar results were obtained with DTT incubation which indicates an underlying reductive mechanism. Furthermore PfHK was inhibited by S-glutathionylation, an effect that could be partially reversed by DTT. Different incubation conditions with glutathione were tested and anti-GSH antibodies were used to probe S-glutathionylation. After trypsin digestion, a clear mass increase of ~305 Da was observed in several cysteine- containing peptides by MALDI-TOF. S-glutathionylation was reproducibly found on Cys21, Cys249, Cys236, and Cys346. Thirdly, to validate PfHK as a drug target in P. falciparum, I started to generate a PfHK knockout strain. For this purpose, first a merodiploid strain was constructed which can episomally express PfHK. The transgenic parasites were obtained and in a next step the knockout of endogenous PfHK will be performed.The experiments performed in this study represent important steps towards characterizing PfHK as a potential target of novel antimalarial compounds.
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