72 research outputs found
Use of Radioactive Precursors for Biochemical Characterization the Biosynthesis of Isoprenoids in Intraerythrocytic Stages of Plasmodium falciparum
Effect of fosmidomycin on metabolic and transcript profiles of the methylerythritol phosphate pathway in Plasmodium falciparum
Non-phenolic method of dna purification from bacteria, blood samples and other biological sources for restriction enzyme assays and the polymerase chain reaction
Plasmodium falciparum uses vitamin E to avoid oxidative stress
Abstract Background Plasmodium falciparum is sensitive to oxidative stress in vitro and in vivo, and many drugs such as artemisinin, chloroquine and cercosporin interfere in the parasite’s redox system. To minimize the damage caused by reactive radicals, antioxidant enzymes and their substrates found in parasites and in erythrocytes must be functionally active. It was shown that P. falciparum synthesizes vitamin E and that usnic acid acts as an inhibitor of its biosynthesis. Vitamin E is a potent antioxidant that protects polyunsaturated fatty acids from lipid peroxidation, and this activity can be measured by detecting its oxidized product and by evaluating reactive oxygen species (ROS) levels. Results Here, we demonstrated that ROS levels increased in P. falciparum when vitamin E biosynthesis was inhibited by usnic acid treatment and decreased to basal levels if exogenous vitamin E was added. Furthermore, we used metabolic labelling to demonstrate that vitamin E biosynthesized by the parasite acts as an antioxidant since we could detect its radiolabeled oxidized product. The treatment with chloroquine or cercosporin of the parasites increased the ratio between α-tocopherolquinone and α-tocopherol. Conclusions Our findings demonstrate that vitamin E produced endogenously by P. falciparum is active as an antioxidant, probably protecting the parasite from the radicals generated by drugs
Effect of fosmidomycin on metabolic and transcript profiles of the methylerythritol phosphate pathway in Plasmodium falciparum
In Plasmodium falciparum , the formation of isopentenyl diphosphate
and dimethylallyl diphosphate, central intermediates in the
biosynthesis of isoprenoids, occurs via the methylerythritol phosphate
(MEP) pathway. Fosmidomycin is a specific inhibitor of the second
enzyme of the MEP pathway, 1-deoxy-D-xylulose-5-phosphate
reductoisomerase. We analyzed the effect of fosmidomycin on the levels
of each intermediate and its metabolic requirement for the isoprenoid
biosynthesis, such as dolichols and ubiquinones, throughout the
intraerythrocytic cycle of P. falciparum. The steady-state RNA levels
of the MEP pathway-associated genes were quantified by real-time
polymerase chain reaction and correlated with the related metabolite
levels. Our results indicate that MEP pathway metabolite peak precede
maximum transcript abundance during the intraerythrocytic cycle.
Fosmidomycin-treatment resulted in a decrease of the intermediate
levels in the MEP pathway as well as in ubiquinone and dolichol
biosynthesis. The MEP pathway associated transcripts were modestly
altered by the drug, indicating that the parasite is not strongly
responsive at the transcriptional level. This is the first study that
compares the effect of fosmidomycin on the metabolic and transcript
profiles in P. falciparum, which has only the MEP pathway for
isoprenoid biosynthesis
Active isoprenoid pathway in the intra-erythrocytic stages of Plasmodium falciparum: presence of dolichols of 11 and 12 isoprene units
Cloning and characterization of bifunctional enzyme farnesyl diphosphate/geranylgeranyl diphosphate synthase from Plasmodium falciparum
BACKGROUND: Isoprenoids are the most diverse and abundant group of natural products. In Plasmodium falciparum, isoprenoid synthesis proceeds through the methyl erythritol diphosphate pathway and the products are further metabolized by farnesyl diphosphate synthase (FPPS), turning this enzyme into a key branch point of the isoprenoid synthesis. Changes in FPPS activity could alter the flux of isoprenoid compounds downstream of FPPS and, hence, play a central role in the regulation of a number of essential functions in Plasmodium parasites. METHODS: The isolation and cloning of gene PF3D7_18400 was done by amplification from cDNA from mixed stage parasites of P. falciparum. After sequencing, the fragment was subcloned in pGEX2T for recombinant protein expression. To verify if the PF3D7_1128400 gene encodes a functional rPfFPPS protein, its catalytic activity was assessed using the substrate [4-(14)C] isopentenyl diphosphate and three different allylic substrates: dimethylallyl diphosphate, geranyl diphosphate or farnesyl diphosphate. The reaction products were identified by thin layer chromatography and reverse phase high-performance liquid chromatography. To confirm the product spectrum formed of rPfFPPS, isoprenic compounds were also identified by mass spectrometry. Apparent kinetic constants K(M) and V(max) for each substrate were determined by Michaelis–Menten; also, inhibition assays were performed using risedronate. RESULTS: The expressed protein of P. falciparum FPPS (rPfFPPS) catalyzes the synthesis of farnesyl diphosphate, as well as geranylgeranyl diphosphate, being therefore a bifunctional FPPS/geranylgeranyl diphosphate synthase (GGPPS) enzyme. The apparent K(M) values for the substrates dimethylallyl diphosphate, geranyl diphosphate and farnesyl diphosphate were, respectively, 68 ± 5 μM, 7.8 ± 1.3 μM and 2.06 ± 0.4 μM. The protein is expressed constitutively in all intra-erythrocytic stages of P. falciparum, demonstrated by using transgenic parasites with a haemagglutinin-tagged version of FPPS. Also, the present data demonstrate that the recombinant protein is inhibited by risedronate. CONCLUSIONS: The rPfFPPS is a bifunctional FPPS/GGPPS enzyme and the structure of products FOH and GGOH were confirmed mass spectrometry. Plasmodial FPPS represents a potential target for the rational design of chemotherapeutic agents to treat malaria
Cloning and characterization of bifunctional enzyme farnesyl diphosphate/geranylgeranyl diphosphate synthase from Plasmodium falciparum
Abstract
Background
Isoprenoids are the most diverse and abundant group of natural products. In Plasmodium falciparum, isoprenoid synthesis proceeds through the methyl erythritol diphosphate pathway and the products are further metabolized by farnesyl diphosphate synthase (FPPS), turning this enzyme into a key branch point of the isoprenoid synthesis. Changes in FPPS activity could alter the flux of isoprenoid compounds downstream of FPPS and, hence, play a central role in the regulation of a number of essential functions in Plasmodium parasites.
Methods
The isolation and cloning of gene PF3D7_18400 was done by amplification from cDNA from mixed stage parasites of P. falciparum. After sequencing, the fragment was subcloned in pGEX2T for recombinant protein expression. To verify if the PF3D7_1128400 gene encodes a functional rPfFPPS protein, its catalytic activity was assessed using the substrate [4-14C] isopentenyl diphosphate and three different allylic substrates: dimethylallyl diphosphate, geranyl diphosphate or farnesyl diphosphate. The reaction products were identified by thin layer chromatography and reverse phase high-performance liquid chromatography. To confirm the product spectrum formed of rPfFPPS, isoprenic compounds were also identified by mass spectrometry. Apparent kinetic constants K
M
and V
max
for each substrate were determined by Michaelis–Menten; also, inhibition assays were performed using risedronate.
Results
The expressed protein of P. falciparum FPPS (rPfFPPS) catalyzes the synthesis of farnesyl diphosphate, as well as geranylgeranyl diphosphate, being therefore a bifunctional FPPS/geranylgeranyl diphosphate synthase (GGPPS) enzyme. The apparent K
M
values for the substrates dimethylallyl diphosphate, geranyl diphosphate and farnesyl diphosphate were, respectively, 68 ± 5 μM, 7.8 ± 1.3 μM and 2.06 ± 0.4 μM. The protein is expressed constitutively in all intra-erythrocytic stages of P. falciparum, demonstrated by using transgenic parasites with a haemagglutinin-tagged version of FPPS. Also, the present data demonstrate that the recombinant protein is inhibited by risedronate.
Conclusions
The rPfFPPS is a bifunctional FPPS/GGPPS enzyme and the structure of products FOH and GGOH were confirmed mass spectrometry. Plasmodial FPPS represents a potential target for the rational design of chemotherapeutic agents to treat malaria
Intraerythrocytic stages of Plasmodium falciparum biosynthesize menaquinone
Herein, we show that intraerythrocytic stages of Plasmodium falciparum have an active pathway for biosynthesis of menaquinone. Kinetic assays confirmed that plasmodial menaquinone acts at least in the electron transport. Similarly to Escherichia coli, we observed increased levels of menaquinone in parasites kept under anaerobic conditions. Additionally, the mycobacterial inhibitor of menaquinone synthesis Ro 48-8071 also suppressed menaquinone biosynthesis and growth of parasites, although off-targets may play a role in this growth-inhibitory effect. Due to its absence in humans, the menaquinone biosynthesis can be considered an important drug target for malaria. (c) 2010 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)FAPESPConselho Nacional de Desenvolvimento CientÃfico e Tecnológico (CNPq)Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq)National Institutes of Health, NIAID/NIH, USA[AI049151]U.S. National Institutes of Health (NIH
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