Characterization of Plasmodium falciparum ATP-dependent DNA helicase RuvB3

Limudomporn, Paviga; Moonsom, Saengduen; Leartsakulpanich, Ubolsree; Suntornthiticharoen, Pattra; Petmitr, Songsak; Weinfeld, Michael; Chavalitshewinkoon-Petmitr, Porntip

doi:10.1186/s12936-016-1573-2

Cited by 8 publications

(11 citation statements)

References 39 publications

(53 reference statements)

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“…RuvB homologs are conserved in all the Plasmodium species (http://www.PlasmoDB.org) . In the chloroquine and pyrimethamine‐resistant P. falciparum K1 strain, PfRuvB3 K1 has been shown to contain ATPase and DNA unwinding activities and its ATPase activity is reduced by netropsin, daunorubicin, and doxorubicin .…”

Section: Ruvb Family Of Plasmodium Falciparummentioning

confidence: 99%

Unraveling the importance of the malaria parasite helicases

Tuteja

2017

The FEBS Journal

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Malaria is a human parasitic disease caused by infection from Plasmodium species, particularly Plasmodium falciparum. Each year millions of people are infected with malaria and large numbers of deaths result due to this deadly infection. P. falciparum contains 14 chromosomes, nearly 5400 genes and a multistage life cycle in humans and mosquitoes. The control of malaria is still a challenge as the parasite is continuously developing resistance to available antimalarial drugs and the mosquito vector is developing resistance to insecticides. The availability of P. falciparum genome has resulted in the identification of parasite‐specific proteins that can be targeted without harmful effects to the human host. Toward this goal, we have been working on the identification and characterization of helicases in order to find parasite‐specific helicases, which can be used as novel drug targets to tackle the rising problem of drug resistance. Helicases are ATP‐dependent nucleic acid unwinding enzymes. The P. falciparum genome analysis depicts that it contains some parasite‐specific helicases and homologs to most of the human helicases. Here, we present an overview of P. falciparum helicases and their importance in parasite growth and survival.

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Section: Ruvb Family Of Plasmodium Falciparummentioning

confidence: 99%

Unraveling the importance of the malaria parasite helicases

Tuteja

2017

The FEBS Journal

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“…Among the various parasite targets being studied for drug development, enzymes in P. falciparum DNA repair pathway present potential drugable targets, including P. falciparum uracil DNA glycosylase (PfUGD) [6], P. falciparum DNA polymerase delta (PfPold) [7] and P. falciparum ATP-dependent DNA helicase RuvB3 (PfRuvB3) [8]. The parasite genome lacks genes encoding DNA repair enzymes in the non-homologous end joining pathway, but previous identi cation of PfPold [7] suggests parasite base excision repair mechanism might rely mainly on a long patch repair pathway [9].…”

Section: Introductionmentioning

confidence: 99%

Molecular characterization of Plasmodium falciparum DNA-3-methyladenine glycosylase

Pinthong

Limudomporn

Vasuvat

et al. 2020

Preprint

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Background The emergence of artemisinin-resistant malaria parasites highlights the need for novel drugs and their targets. Alkylation of purine bases can hinder DNA replication and if unresolved would eventually result in cell death. DNA-3-methyladenine glycosylase (MAG) is responsible for the repair of those alkylated bases. Plasmodium falciparum (Pf)MAG was characterized for its potential for development as an anti-malarial candidate.Methods Native PfMAG from crude extract of chloroquine- and pyrimethamine-resistant P. falciparum K1 strain was partially purified using three chromatographic procedures. From bio-informatics analysis, primers were designed for amplification, insertion into pBAD202/D-TOPO and heterologous expression in Escherichia coli of recombinant PfMAG. Functional and biochemical properties of the recombinant enzyme were characterized. Results PfMAG activity was most prominent in parasite schizont stages, with a specific activity of 147 U/mg (partially purified) protein. K1 PfMAG contained an insertion of AAT (coding for asparagine) compared to 3D7 strain and 16% similarity to the human enzyme. Recombinant PfMAG (74 kDa) was twice as large as the human enzyme, preferred double-stranded DNA substrate, and demonstrated glycosylase activity over a pH range of 4-9, optimal salt concentration of 100-200 mM NaCl but reduced activity at 250 mM NaCl, no requirement for divalent cations, which were inhibitory in a dose-dependent manner.Conclusio PfMAG activity increased with parasite development being highest in the schizont stages. K1 PfMAG contained an indel AAT (asparagine) not present in 3D7 strain and the native enzyme was twice as large as the human enzyme. Recombinant PfMAG had a wide range of optimal pH activity, and was inhibited at high (250 mM) NaCl concentration as well as by divalent cations. The properties of PfMAG provide basic data that should be of assistance in developing anti-malarials against this potential parasite target.

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“…Among several targets being studied for antimalarial development, enzymes in DNA repair pathway of P. falciparum were investigated as potentials targets for drug development including P. falciparum uracil DNA glycosylase (PfUGD) [6], P. falciparum DNA polymerase delta (PfPolδ) [7], and P. falciparum ATPdependent DNA helicase RuvB3 (PfRuvB3) [8]. The parasite complete genome showed the absence of DNA repair enzymes in the non-homologous end joining pathway.…”

Section: Introductionmentioning

confidence: 99%

Molecular Characterization of Plasmodium Falciparum DNA-3-Methyladenine Glycosylase

Pinthong

Limudomporn

Vasuvat

et al. 2020

Preprint

Self Cite

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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.

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Characterization of Plasmodium falciparum ATP-dependent DNA helicase RuvB3

Cited by 8 publications

References 39 publications

Unraveling the importance of the malaria parasite helicases

Unraveling the importance of the malaria parasite helicases

Molecular characterization of Plasmodium falciparum DNA-3-methyladenine glycosylase

Molecular Characterization of Plasmodium Falciparum DNA-3-Methyladenine Glycosylase

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