Sen Stephanie E, Cusson Michel, Trobaugh Corey, Béliveau Catherine, Richard Thenesha, Graham Wendy, Mimms Adrian, Roberts Gregory
Department of Chemistry, Indiana U.-Purdue U. Indianapolis (IUPUI), 402 North Blackford Street, Indianapolis, IN 46202, USA.
Insect Biochem Mol Biol. 2007 Aug;37(8):819-28. doi: 10.1016/j.ibmb.2007.05.012. Epub 2007 Jun 3.
Two forms of farnesyl diphosphate synthase (FPPS) from the spruce budworm, Choristoneura fumiferana, and one from the armyworm Pseudaletia unipuncta, have been cloned and their catalytic properties assessed. The type-2 FPPS of C. fumiferana (CfFPPS2) was efficient in the prenyl coupling of DMAPP or GPP with [(14)C]IPP, producing FPP as its final product; however, type-1 FPPSs (CfFPPS1, PuFPPS1, as well as Agrotis ipsilon FPPS1) were essentially inactive. A variety of purification methods was employed to purify the type-1 enzymes. Under mild chromatographic conditions, the isolated type-1 enzyme showed modest activity, but was apparently contaminated with endogenous prenyltransferase derived from the Escherichia coli host cells. Similarly, unpurified extracts of PuFPPS1 expressed in an E. coli FPPS-null mutant, had low FPPS activity. When equimolar amounts of homogenous CfFPPS1 and CfFPP2 were combined, a sharp synergistic enhancement of activity was observed, and the coupling of several homologous substrates, which are precursors to ethyl-branched JHs, was enhanced. Association between CfFPPS1 and CfFPPS2 was confirmed by both protein interaction chromatography and competitive ELISA. These data suggest that type-1 and type-2 FPPSs can form a heteromer, which may play a role in sesquiterpene biosynthesis, such as JH homologue formation, in moths.
已克隆了云杉芽卷叶蛾（Choristoneura fumiferana）的两种法尼基二磷酸合酶（FPPS）形式以及草地贪夜蛾（Pseudaletia unipuncta）的一种法尼基二磷酸合酶形式，并评估了它们的催化特性。云杉芽卷叶蛾的2型FPPS（CfFPPS2）能有效地将二甲基烯丙基二磷酸（DMAPP）或牻牛儿基二磷酸（GPP）与[¹⁴C]异戊烯基二磷酸（IPP）进行异戊烯基偶联，最终生成法尼基二磷酸（FPP）；然而，1型FPPS（CfFPPS1、PuFPPS1以及小地老虎（Agrotis ipsilon）FPPS1）基本无活性。采用了多种纯化方法来纯化1型酶。在温和的色谱条件下，分离出的1型酶表现出适度的活性，但显然被源自大肠杆菌宿主细胞的内源性异戊烯基转移酶污染。同样，在大肠杆菌FPPS基因缺失突变体中表达的未纯化的PuFPPS1提取物，其FPPS活性较低。当将等摩尔量的纯CfFPPS1和CfFPP2混合时，观察到活性急剧协同增强，并且几种作为乙基支链保幼激素前体的同源底物的偶联增强。通过蛋白质相互作用色谱法和竞争性酶联免疫吸附测定法（ELISA）证实了CfFPPS1和CfFPPS2之间的关联。这些数据表明，1型和2型FPPS可以形成异源二聚体，这可能在蛾类的倍半萜生物合成中发挥作用，例如保幼激素同系物的形成。
