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Dissertation zugänglich unter
URN: urn:nbn:de:gbv:18-27909
URL: http://ediss.sub.uni-hamburg.de/volltexte/2006/2790/


The characterization of the spermidine synthase from Plasmodium falciparum (Welch 1897) and Caenorhabditis elegans (Maupas, 1900)

Haider, Nashya

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Freie Schlagwörter (Englisch): spermidine synthase , polyamine , plasmodium
Basisklassifikation: 42.36 , 35.74
Institut: Biologie
DDC-Sachgruppe: Biowissenschaften, Biologie
Dokumentart: Dissertation
Hauptberichter: Walter, Rolf D. (Prof. Dr.)
Sprache: Englisch
Tag der mündlichen Prüfung: 16.12.2005
Erstellungsjahr: 2005
Publikationsdatum: 13.02.2006
Kurzfassung auf Englisch: The naturally occurring polyamines spermidine and spermine are involved in numerous cellular processes and are found to be essential for cell proliferation and differentiation of prokaryotes and eukaryotes. In the polyamine synthesis pathway, spermidine is formed by the transfer of an aminopropyl group from decarboxylated S-adenosylmethionine (dcAdoMet) to a terminal amino group of putrescine. This reaction is catalyzed by spermidine synthase, where by the precursors putrescine and dcAdoMet are provided by the enzymes ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC), respectively. In contrast to spermidine synthases, the two key enzymes ODC and AdoMetDC have been extensively investigated in many organisms. In the current study, the spermidine synthase from the human malaria parasite Plasmodium falciparum and the model organism Caenorhabditis elegans was cloned, recombinantly expressed and characterized. Malaria remains one of the most devastating tropical diseases with 300-500 million infections annually, resulting in 1-3 million deaths. There are only a few chemotherapeutics available that serve as treatment against malaria. This is compounded by the increasing prevalence of multi-resistant P. falciparum worldwide that has become a serious public health threat to the global control of the disease. Thus there is an urgent and pressing need for new drugs attacking novel targets in the metabolism of the malaria parasite. Inhibition of polyamine synthesis correlates with blockage of cell growth. Hence, the enzymes of the polyamine synthesis pathway represent attractive targets for the therapeutic intervention of rapidly proliferating cells such as tumour cells and parasites like P. falciparum. It is therefore conceivable that exploitation of this pathway could lead to the detection of inhibitors that will have a more adverse effect on the parasites than on the human host. The biochemical characterization of the polyamine metabolic pathway of P. falciparum is essential to illustrate differences between the parasite and its human host. Accordingly, previous studies have demonstrated that P. falciparum possesses a unique bifunctional ODC/AdoMetDC. However, the third enzyme of the polyamine biosynthetic pathway, spermidine synthase, has not been investigated. P. falciparum contains one copy of the spermidine synthase gene. Northern and Western blot analyses revealed a stage specific expression during the erythrocytic schizogony with the maximal amount of transcript and protein in mature trophozoites. The spermidine synthase polypeptide of 321 amino acids has a molecular mass of 36.6 kDa and contains an N-terminal extension that, similarly, is also found in certain plant but not in animal or bacterial orthologues. Immunofluorescence assays indicate a cytoplasmic localization of the spermidine synthase. Omitting the first 29 amino acids, a truncated form of P. falciparum spermidine synthase has been recombinantly expressed in Escherichia coli. The enzyme catalyses the transfer of an aminopropyl group from dcAdoMet onto putrescine with Km values of 35 and 52 µM, respectively. It is possible that the dcAdoMet and putrescine formed by the P. falciparum ODC/AdoMetDC-catalyzed reactions may be channelled effectively to spermidine synthase via protein-protein interaction, modulating the formation of the product spermidine. By applying site-directed mutagenesis it was shown that the highly conserved amino acid residues Y102 and D196 of P. falciparum spermidine synthase are possibly important for substrate binding. Among the spermidine synthase inhibitors tested against P. falciparum spermidine synthase, trans-4-methylcyclohexylamine (4MCHA) was found to be most potent with a Ki value of 0.18 µM. In contrast to the situation in mammals, where inhibition of spermidine synthase has no or only little effect on cell proliferation, 4MCHA was an efficient inhibitor of P. falciparum cell growth in vitro with an IC50 of 35 µM, indicating that P. falciparum spermidine synthase represents a putative drug target. Consistent with reports on other organisms, the free-living nematode C. elegans depends on polyamines, since ODC null mutants that do not have access to exogenous polyamines exhibit a stage-specific block of embryogenesis. The C. elegans ODC and AdoMetDC have been previously investigated at the protein level. Here, the spermidine synthase of C. elegans has been recombinantly expressed and biochemically characterised. The protein revealed a high degree of similarity to other eukaryotic spermidine synthases with the exception of a low affinity towards the substrate dcAdoMet (Km = 110 µM) and a less pronounced feedback inhibition by the second reaction product 5’-methylthioadenosine (IC50 = 430 µM).



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