Cloning and characterization of bifunctional enzyme farnesyl diphosphate/geranylgeranyl diphosphate synthase from Plasmodium falciparum.
Cloning and characterization of bifunctional enzyme farnesyl diphosphate/geranylgeranyl diphosphate synthase from Plasmodium falciparum.
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DOI：
10.1186/1475-2875-12-184
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发表时间：
2013-06-04
期刊：
影响因子：
3
通讯作者：
Katzin AM
中科院分区：
文献类型：
--
作者：
Jordão FM;Gabriel HB;Alves JM;Angeli CB;Bifano TD;Breda A;de Azevedo MF;Basso LA;Wunderlich G;Kimura EA;Katzin AM
类异戊二烯是最多样化和丰富的天然产物。在恶性疟原虫中，类异戊二烯的合成通过甲基异戊二磷酸途径进行，并且产物进一步被法呢基二磷酸合酶（FPPS）代谢，使该酶成为类异戊二烯合成的关键分支点。FPPS活性的变化可以改变FPPS下游类异戊二烯化合物的通量，因此，在疟原虫寄生虫的一些基本功能的调节中发挥核心作用。通过从恶性疟原虫混合期寄生虫的cDNA扩增完成基因PF3D7_18400的分离和克隆。测序后，将该片段亚克隆到pGEX 2 T中用于重组蛋白表达。为了验证PF3D7_1128400基因是否编码功能性rPfFPPS蛋白，使用底物[4- 14 C]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、香叶基二磷酸或法呢基二磷酸）评估其催化活性。反应产物经薄层色谱和反相高效液相色谱鉴定。为了确认由rPfFPPS形成的产物谱，还通过质谱法鉴定了异戊二烯化合物。表观动力学常数KM和Vmax为每种底物测定Michaelis-Menten，也进行了抑制试验，使用利塞膦酸盐。恶性疟原虫FPPS（rPfFPPS）的表达蛋白催化法呢基二磷酸以及香叶基香叶基二磷酸的合成，因此是双功能FPPS/香叶基香叶基二磷酸合酶（GGPPS）酶。底物二甲基烯丙基二磷酸、香叶基二磷酸和法呢基二磷酸的表观KM值分别为68 ± 5 μM、7.8 ± 1.3 μM和2.06 ± 0.4 μM。该蛋白质在恶性疟原虫的所有红细胞内阶段组成型表达，通过使用具有血凝素标记的FPPS版本的转基因寄生虫来证明。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FPPS/GGPPS酶，产物FOH和GGOH的结构经质谱证实。疟原虫FPPS是合理设计治疗疟疾的化疗药物的潜在靶点。
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.
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影响因子：
4.8
作者：
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