Abstract
BackgroundMalaria is one of the most serious and widespread parasitic diseases affecting humans. Because of the spread of resistance in both parasites and the mosquito vectors to anti-malarial drugs and insecticides, controlling the spread of malaria is becoming difficult. Thus, identifying new drug targets is urgently needed. Helicases play key roles in a wide range of cellular activities involving DNA and RNA transactions, making them attractive anti-malarial drug targets.MethodsATP-dependent DNA helicase gene (PfRuvB3) of Plasmodium falciparum strain K1, a chloroquine and pyrimethamine-resistant strain, was inserted into pQE-TriSystem His-Strep 2 vector, heterologously expressed and affinity purified. Identity of recombinant PfRuvB3 was confirmed by western blotting coupled with tandem mass spectrometry. Helicase and ATPase activities were characterized as well as co-factors required for optimal function.ResultsRecombinant PfRuvB3 has molecular size of 59 kDa, showing both DNA helicase and ATPase activities. Its helicase activity is dependent on divalent cations (Cu2+, Mg2+, Ni+2 or Zn+2) and ATP or dATP but is inhibited by high NaCl concentration (>100 mM). PfPuvB3 is unable to act on blunt-ended duplex DNA, but manifests ATPase activity in the presence of either single- or double-stranded DNA. PfRuvB3.is inhibited by doxorubicin, daunorubicin and netropsin, known DNA helicase inhibitors.ConclusionsPurified recombinant PfRuvB3 contains both DNA helicase and ATPase activities. Differences in properties of RuvB between the malaria parasite obtained from the study and human host provide an avenue leading to the development of novel drugs targeting specifically the malaria form of RuvB family of DNA helicases.
Highlights
Malaria is one of the most serious and widespread parasitic diseases affecting humans
In 2015 an estimated 3.2 billion people were at risk of being infected with malaria parasites and contracting the disease, with approximately 214 million people becoming infected resulting in a mortality of 438,000 [1]
Based on amino acid sequence analysis, all 13 peptides obtained from mass spectrometry were matched with the sequence of P. falciparum 3D7 PfRuvB3 with ion scores of 4512, indicating an extensive homology (p value
Summary
Malaria is one of the most serious and widespread parasitic diseases affecting humans. Because of the spread of resistance in both parasites and the mosquito vectors to anti-malarial drugs and insecticides, controlling the spread of malaria is becoming difficult. With the emergence of drug-resistant parasites to all available antimalarials, control of malaria is becoming difficult [1, 2]. This has led to efforts in developing novel strategies and. The availability of the complete genome sequence of Plasmodium falciparum, the causative agent of fatal malaria, has opened new avenues to identify genes important for parasite survival. Many potential chemotherapeutic targets involved in various metabolic pathways at different malaria parasite life stages have been identified recently [3]. Helicases constitute a highly conserved group of enzymes important in all aspects of nucleic acid metabolism, such as replication, recombination, repair, transcription and (RNA) stability [3,4,5,6]
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