Cloning and characterization of bifunctional enzyme farnesyl diphosphate/geranylgeranyl diphosphate synthase from Plasmodium falciparum

Abstract

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 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.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.

Isoprenoid Biosynthesis in Plasmodium falciparum

“…In P. falciparum, the condensation of GPP and IPP (to produce FPP) and of FPP and IPP (to produce geranylgeranyl diphosphate [GGPP]) appears to be catalyzed by a bifunctional FPP-GGPP synthase (PlasmoDB ID PF3D7_1128400) (49). The crystal structure of the P. vivax enzyme has been solved (50,51).…”

Andrographolide and Its Derivative - A Story of Antimalarial Drug Design and Synthesis

“…Geranylgeranyl pyrophosphate synthase (GGPPS) is a key enzyme in PF isoprenoid biosynthesis [3]. It catalyzes the synthesis of GGPP, 3 as well as farnesyl pyrophosphate [3]. In the presence of Mg 2+ cofactors [4].…”

“…This report presents the basis for a new antimalarial drug development in Indonesia. Geranylgeranyl pyrophosphate synthase (GGPPS) is a key enzyme in PF isoprenoid biosynthesis [3]. It catalyzes the synthesis of GGPP, 3 as well as farnesyl pyrophosphate [3].…”

Predicted Binding Mode of Andrographolide and Its Derivatives Bound to Plasmodium Falciparum Geranylgeranyl Pyrophosphate Synthase

“…The present report, therefore, offers the basis for the development of a new antimalarial drug. Geranylgeranyl pyrophosphate synthase (GGPPS) is a key enzyme in P. falciparum isoprenoid biosynthesis [2]. It catalyzes the synthesis of geranylgeranyl pyrophosphate (GGPP) [2], as well as farnesyl pyrophosphate [2], in the presence of Mg 2+ cofactors [3].…”

“…Geranylgeranyl pyrophosphate synthase (GGPPS) is a key enzyme in P. falciparum isoprenoid biosynthesis [2]. It catalyzes the synthesis of geranylgeranyl pyrophosphate (GGPP) [2], as well as farnesyl pyrophosphate [2], in the presence of Mg 2+ cofactors [3]. This enzyme is recommended as an antimalarial drug target due to its essentiality, druggability, and amenability to high-throughput screening [4].…”