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

Malaria Journal

Research

Claudia B Angeli¹  Emilia A Kimura¹  Thaís D Bifano¹  Fabiana M Jordão¹  Heloisa B Gabriel¹  Gerhard Wunderlich¹  Alejandro M Katzin¹  João MP Alves¹  Luiz A Basso²  Ardala Breda²  Mauro F de Azevedo³ 

[1] Department of Parasitology, Institute of Biomedical Sciences, University of São Paulo, Av. Lineu Prestes 1374, CEP 05508-000, São Paulo, SP, Brazil;Research Center for Molecular Biology and Functional, National Institute of Science and Technology on Tuberculosis, Pontifical Catholic University of Rio Grande do Sul, Rio Grande do Sul, Brazil;The Macfarlane Burnet Institute for Medical Research and Public Health, Melbourne, Victoria, Australia;

关键词: Plasmodium falciparum;    Malaria;    Isoprenoids;    Farnesyl diphosphate;    Farnesyl diphosphate synthase;    Geranylgeranyl diphosphate;    Geranylgeranyl diphosphate synthase;   

DOI  :  10.1186/1475-2875-12-184

 received in 2013-03-22, accepted in 2013-05-29,  发布年份 2013

来源: Springer

【 摘 要 】

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.

【 授权许可】

Unknown   
© Jordão et al.; licensee BioMed Central Ltd. 2013. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.