Characterization of Plasmodium falciparum ATP-dependent DNA helicase RuvB3.
BACKGROUND
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. 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.


METHODS
ATP-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.


RESULTS
Recombinant 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+2or 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.


CONCLUSIONS
Purified 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.
Department of Protozoology, Faculty of Tropical Medicine, Mahidol University, 420/6 Rajvithi Road, Bangkok, 10400, Thailand.
Paviga
P
Limudomporn
Department of Protozoology, Faculty of Tropical Medicine, Mahidol University, 420/6 Rajvithi Road, Bangkok, 10400, Thailand.
Saengduen
S
Moonsom
National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, 113 Thailand Science Park, Pahonyothin Rd, Pathumthani, 12120, Thailand.
Ubolsree
U
Leartsakulpanich
Department of Biomedical Sciences, Faculty of Science, Rangsit University, Lak Hok, Pathumthani, 12000, Thailand.
Pattra
P
Suntornthiticharoen
Department of Molecular Tropical Medicine and Genetics, Faculty of Tropical Medicine, Mahidol University, Bangkok, 10400, Thailand.
Songsak
S
Petmitr
Department of Oncology, Cross Cancer Institute, University of Alberta, Edmonton, AB, T6G 1Z2, Canada.
Michael
M
Weinfeld
Department of Protozoology, Faculty of Tropical Medicine, Mahidol University, 420/6 Rajvithi Road, Bangkok, 10400, Thailand. porntip.pet@mahidol.ac.th.
Porntip
P
Chavalitshewinkoon-Petmitr
D015153:Blotting, Western; D002413:Cations, Divalent / Q000378:metabolism; D003001:Cloning, Molecular; D003067:Coenzymes / Q000032:analysis; D004265:DNA Helicases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000032:analysis; D015870:Gene Expression; D008670:Metals / Q000378:metabolism; D008970:Molecular Weight; D010963:Plasmodium falciparum / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity; D053719:Tandem Mass Spectrometry
D002413:Cations, Divalent; D003067:Coenzymes; D004791:Enzyme Inhibitors; D008670:Metals; D011994:Recombinant Proteins; D004265:DNA Helicases
ATP-dependent DNA helicase; ATPase activity; Helicase activity; PfRuvB3; Plasmodium falciparum