Molecular Characterization of Plasmodium Falciparum DNA-3-Methyladenine Glycosylase

Abstract

Background The emergence of artemisinin-resistant malaria parasite highlights the need for novel drugs and their targets. Alkylation in purine bases can hinder DNA replication if remained unsolved would eventually resulting in cell death. DNA-3-methyladenine glycosylase is an enzyme responsible for the repair of those alkylated lesions. Based on the AT-rich genome of Plasmodium falciparum and unexplored Plasmodium falciparum DNA-3-methyladenine glycosylase (PfMAG), therefore PfMAG should be characterized for its potential candidate for antimalarial drug development.Methods Bioinformatics analysis of PfMAG was performed. The native PfMAG from crude extract of chloroquine and pyrimethamine resistant Plasmodium falciparum strain K1 was partially purified using three columns. The existence of PfMAG activity leads to the cloning and expression of PfMAG for further characterization. The PfMAG was amplified, cloned to expression vector (pBAD202/D-TOPO), and expressed in Escherichia coli. The molecular weight of recombinant PfMAG was analyzed by SDS-PAGE and Western blot. Both functional and biochemical properties of the recombinant enzyme were characterized.Results PfMAG activity was most prominent in schizont. Native PfMAG was partially purified with a specific activity of 147.36 units/mg protein. The DNA sequence of amplified PfMAG showed an insertion of three nucleotides coding for asparagine compared to strain 3D7 and only 16% similarity compared to the human enzyme was observed. After cloning, the 74-kDa recombinant PfMAG was expressed and identified. PfMAG showed a larger size than its human counterpart twice. PfMAG preferred a double-stranded DNA substrate. Glycosylase activity was detected in a broad range of pH 5–8. The quite narrow optimal salt concentration was observed between 100–200 mM NaCl, whereas 250 mM NaCl reduces its activity. Divalent cations are not required for its glycosylase activity; on the contrary, they inhibited glycosylase activity in a concentration-dependent manner.Conclusion PfMAG activity increases according to parasite development. Native PfMAG was partially purified, the recombinant PfMAG was successfully expressed and characterized. An insertion of AAT coding for asparagine was found compared with that of strain 3D7. PfMAG differs from the human counterpart in a twice larger size and a wide range of optimal pH. Results obtained from this study will be a benefit for exploring and investigating of candidate targets toward antimalarial drug design in the future.