Jordão Fabiana M, Gabriel Heloisa B, Alves João M P, Angeli Claudia B, Bifano Thaís D, Breda Ardala, de Azevedo Mauro F, Basso Luiz A, Wunderlich Gerhard, Kimura Emilia A, Katzin Alejandro M
Malar J. 2013 Jun 4;12:184. doi: 10.1186/1475-2875-12-184.
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.
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 KM and Vmax for each substrate were determined by Michaelis-Menten; also, inhibition assays were performed using risedronate.
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 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.
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.
类异戊二烯是种类最多且含量最丰富的天然产物类别。在恶性疟原虫中，类异戊二烯的合成通过甲基赤藓糖醇二磷酸途径进行，其产物由法尼基二磷酸合酶（FPPS）进一步代谢，这使得该酶成为类异戊二烯合成的关键分支点。FPPS活性的变化可能会改变FPPS下游类异戊二烯化合物的通量，从而在疟原虫多种基本功能的调节中发挥核心作用。
通过从恶性疟原虫混合阶段寄生虫的cDNA中扩增来完成基因PF3D7_18400的分离和克隆。测序后，将该片段亚克隆到pGEX2T中用于重组蛋白表达。为验证PF3D7_1128400基因是否编码功能性rPfFPPS蛋白，使用底物[4-14C]异戊烯基二磷酸和三种不同的烯丙基底物：二甲基烯丙基二磷酸、香叶基二磷酸或法尼基二磷酸来评估其催化活性。通过薄层色谱和反相高效液相色谱鉴定反应产物。为确认rPfFPPS形成的产物谱，还通过质谱法鉴定类异戊二烯化合物。通过米氏方程确定每种底物的表观动力学常数KM和Vmax；此外，使用利塞膦酸盐进行抑制试验。
恶性疟原虫FPPS（rPfFPPS）的表达蛋白催化法尼基二磷酸以及香叶基香叶基二磷酸的合成，因此是一种双功能的FPPS/香叶基香叶基二磷酸合酶（GGPPS）酶。二甲基烯丙基二磷酸、香叶基二磷酸和法尼基二磷酸底物的表观KM值分别为68±5μM、7.8±1.3μM和2.06±0.4μM。使用带有血凝素标签的FPPS转基因寄生虫证明，该蛋白在恶性疟原虫的所有红细胞内阶段均组成性表达。此外，目前的数据表明重组蛋白被利塞膦酸盐抑制。
rPfFPPS是一种双功能的FPPS/GGPPS酶，通过质谱法确认了产物FOH和GGOH的结构。疟原虫FPPS是合理设计抗疟化疗药物的潜在靶点。
