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Remarkable similarity in Plasmodium falciparum and Plasmodium vivax geranylgeranyl diphosphate synthase dynamics and its implication for antimalarial drug design.

ABSTRACT: Malaria, mainly caused by Plasmodium falciparum and Plasmodium vivax, has been a growing cause of morbidity and mortality. P. falciparum is more lethal than is P. vivax, but there is a vital need for effective drugs against both species. Geranylgeranyl diphosphate synthase (GGPPS) is an enzyme involved in the biosynthesis of quinones and in protein prenylation and has been proposed to be a malaria drug target. However, the structure of P. falciparumGGPPS (PfGGPPS) has not been determined, due to difficulties in crystallization. Here, we created a PfGGPPS model using the homologous P.vivaxGGPPS X-ray structure as a template. We simulated the modeled PfGGPPS as well as PvGGPPS using conventional and Gaussian accelerated molecular dynamics in both apo- and GGPP-bound states. The MD simulations revealed a striking similarity in the dynamics of both enzymes with loop 9-10 controlling access to the active site. We also found that GGPP stabilizes PfGGPPS and PvGGPPS into closed conformations and via similar mechanisms. Shape-based analysis of the binding sites throughout the simulations suggests that the two enzymes will be readily targeted by the same inhibitors. Finally, we produced three MD-validated conformations of PfGGPPS to be used in future virtual screenings for potential new antimalarial drugs acting on both PvGGPPS and PfGGPPS.

SUBMITTER: Venkatramani A

PROVIDER: S-EPMC6707526 | biostudies-literature | 2018 Jun

REPOSITORIES: biostudies-literature

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Remarkable similarity in Plasmodium falciparum and Plasmodium vivax geranylgeranyl diphosphate synthase dynamics and its implication for antimalarial drug design.

Venkatramani Aishwarya A Gravina Ricci Clarisse C Oldfield Eric E McCammon J Andrew JA

Chemical biology & drug design 20180204 6

Malaria, mainly caused by Plasmodium falciparum and Plasmodium vivax, has been a growing cause of morbidity and mortality. P. falciparum is more lethal than is P. vivax, but there is a vital need for effective drugs against both species. Geranylgeranyl diphosphate synthase (GGPPS) is an enzyme involved in the biosynthesis of quinones and in protein prenylation and has been proposed to be a malaria drug target. However, the structure of P. falciparumGGPPS (PfGGPPS) has not been determined, due to ...[more]

PMID: 29345110

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Project description:BackgroundIsoprenoids 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.MethodsThe 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 KM and Vmax for each substrate were determined by Michaelis-Menten; also, inhibition assays were performed using risedronate.ResultsThe 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 KM values for the substrates dimethylallyl diphosphate, geranyl diphosphate and farnesyl diphosphate were, respectively, 68?±?5 ?M, 7.8?±?1.3 ?M and 2.06?±?0.4 ?M. 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.ConclusionsThe 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.

Project description:Background:Hypusination is an essential post-translational modification in eukaryotes. The two enzymes required for this modification, namely deoxyhypusine synthase (DHS) and deoxyhypusine hydrolase are also conserved. Plasmodium falciparum human malaria parasites possess genes for both hypusination enzymes, which are hypothesized to be targets of antimalarial drugs. Methods:Transgenic P. falciparum parasites with modification of the PF3D7_1412600 gene encoding PfDHS enzyme were created by insertion of the glmS riboswitch or the M9 inactive variant. The PfDHS protein was studied in transgenic parasites by confocal microscopy and Western immunoblotting. The biochemical function of PfDHS enzyme in parasites was assessed by hypusination and nascent protein synthesis assays. Gene essentiality was assessed by competitive growth assays and chemogenomic profiling. Results:Clonal transgenic parasites with integration of glmS riboswitch downstream of the PfDHS gene were established. PfDHS protein was present in the cytoplasm of transgenic parasites in asexual stages. The PfDHS protein could be attenuated fivefold in transgenic parasites with an active riboswitch, whereas PfDHS protein expression was unaffected in control transgenic parasites with insertion of the riboswitch-inactive sequence. Attenuation of PfDHS expression for 72 h led to a significant reduction of hypusinated protein; however, global protein synthesis was unaffected. Parasites with attenuated PfDHS expression showed a significant growth defect, although their decline was not as rapid as parasites with attenuated dihydrofolate reductase-thymidylate synthase (PfDHFR-TS) expression. PfDHS-attenuated parasites showed increased sensitivity to N 1-guanyl-1,7-diaminoheptane, a structural analog of spermidine, and a known inhibitor of DHS enzymes. Discussion:Loss of PfDHS function leads to reduced hypusination, which may be important for synthesis of some essential proteins. The growth defect in parasites with attenuated Pf DHS expression suggests that this gene is essential. However, the slower decline of PfDHS mutants compared with PfDHFR-TS mutants in competitive growth assays suggests that PfDHS is less vulnerable as an antimalarial target. Nevertheless, the data validate PfDHS as an antimalarial target which can be inhibited by spermidine-like compounds.

Dataset Information

Remarkable similarity in Plasmodium falciparum and Plasmodium vivax geranylgeranyl diphosphate synthase dynamics and its implication for antimalarial drug design.

Publications

Remarkable similarity in Plasmodium falciparum and Plasmodium vivax geranylgeranyl diphosphate synthase dynamics and its implication for antimalarial drug design.

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