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Dissertation
Plasmodium falciparum enzymes involved in redox balancing of nicotinamide nucleotides
Doctor of Philosophy (Ph.D.), Drexel University
Sep 2003
DOI:
https://doi.org/10.17918/etd-477
Abstract
Because nicotinamide nucleotides play a central role in numerous metabolic steps, the ratio of their reduced and oxidized states needs to be tightly regulated. We are particularly interested in the regulation of NAD/NADP levels in malaria mitochondria. Biochemical and bioinformatics data suggest an absence of the standard complex I NADH dehydrogenase in malaria parasites. Instead, we have identified an NADH dehydrogenase gene in Plasmodium falciparum that greatly differs from its mammalian counterpart. Unlike Complex I of mammalian mitochondria with ca. 43 subunits, P. falciparum NADH dehydrogenase (PfND) is a single-subunit enzyme with no obvious proton translocation capability. Sequence analysis indicated that this 533 amino acid protein has an N-terminal mitochondrial targeting signal sequence. This is analogous to Saccharomyces cerevisiae , which has been shown to be deficient of complex I but possesses three single subunit NADH dehydrogenases (NDI-1, NDE-1 and NDE-2). We are attempting to complement yeast mutants deficient in these enzymes with P. falciparum gene. In order to optimize expression in yeast, we synthesized the entire PfND gene with yeast codons, using 40 nucleotide long oligomers. In addition, we have also identified an energy-transducing enzyme, nicotinamide nucleotide transhydrogenase (PfTH), in P. falciparum . Transhydrogenases transfer hydride ions between NAD(H) and NADP(H) with concomitant proton translocation across a membrane. The protonmotive force alters the affinity of the transhydrogenase for substrates and shifts the equilibrium of the reaction towards NADPH production. This reaction is also reversible, the enzyme can produce a proton gradient by consuming NADPH. Our evidence indicates that PfTH mRNA is not expressed during the asexual erythrocytic stages of malaria parasites, but is present in gametocytes. Proteomics data also indicate that the protein is expressed in sporozoite stage. However, the antibodies we raised against PfTH-domain III were unable to detect TH in sporozoites by immunofluorescence. We have expressed the soluble NAD(H) and NADP(H) binding domains as a single polypeptide from both Plasmodium falciparum and Entamoeba histolytica . Cyclic reaction catalyzed by the soluble domain III+I fragment of P. falciparum enzyme appeared very similar to that of E. histolytica enzyme. We also constructed knock out clones for P. falciparum and P. berghei transhydrogenases. However, our initial transfections have revealed no indication of gene disruption. At this point, whether the TH function is essential to parasitic survival remains elusive.
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Details
- Title
- Plasmodium falciparum enzymes involved in redox balancing of nicotinamide nucleotides
- Creators
- Devrim Eren - DU
- Contributors
- Akhil B. Vaidya (Advisor) - Drexel University (1970-)
- Awarding Institution
- Drexel University
- Degree Awarded
- Doctor of Philosophy (Ph.D.)
- Publisher
- Drexel University; Philadelphia, Pennsylvania
- Resource Type
- Dissertation
- Language
- English
- Academic Unit
- Microbiology and Immunology; College of Medicine; Drexel University
- Other Identifier
- 477; 991014632050604721