Rai Avanish, Nagegowda Dinesh A
Molecular Plant Biology and Biotechnology Lab, CSIR-Central Institute of Medicinal and Aromatic Plants, Research Centre, Bengaluru - 560065, India.
Bio Protoc. 2021 Jan 5;11(1):e3874. doi: 10.21769/BioProtoc.3874.
Isoprenoids represent the largest class of metabolites with amazing diversities in structure and function. They are involved in protecting plants against pathogens or herbivores or involved in attracting pollinators. Isoprenoids are derived from geranyl diphosphate (GPP; C), farnesyl diphosphate (FPP; C), geranylgeranyl diphosphate (GGPP; C), and geranylfarnesyl diphosphate (GFPP; C) that are in turn formed by sequential condensations of isopentenyl diphosphate (IPP; C) with an allylic acceptor such as dimethylallyl diphosphate (DMAPP; C), GPP, FPP, or GGPP in a reaction catalyzed by isoprenyl diphosphate synthases (IDSs). IDS enzyme assay for determination of prenyl diphosphate products is generally performed using radiolabelled substrates, and the products formed are identified by employing expensive instruments such as phosphor imager, radio-GC, or radio-HPLC. Though a non-radioactive assay for measuring IDS activity in crude plant extract has been reported, it requires a complex methodology utilizing chromatography coupled with tandem mass spectrometry (LC/MS-MS). Here, we describe a non-radioactive and simple inexpensive assay for determining the IDS assay products using non-radiolabeled IPP and its co-allylic substrates DMAPP, GPP, and FPP. The detection of prenyl diphosphate products generated in the assay was highly efficient and spots corresponding to prenyl alcohols were visible at >40 µM concentrations of IPP and DMAPP/GPP/FPP substrates. The protocol described here is sensitive, reliable, and technically simple, which could be used for functional characterization of IDS candidates.
类异戊二烯是最大的一类代谢产物，其结构和功能具有惊人的多样性。它们参与保护植物免受病原体或食草动物侵害，或参与吸引传粉者。类异戊二烯由香叶基二磷酸（GPP；C）、法尼基二磷酸（FPP；C）、香叶基香叶基二磷酸（GGPP；C）和香叶基法尼基二磷酸（GFPP；C）衍生而来，而这些又依次由异戊烯基二磷酸（IPP；C）与烯丙基受体如二甲基烯丙基二磷酸（DMAPP；C）、GPP、FPP或GGPP在异戊烯基二磷酸合酶（IDSs）催化的反应中连续缩合形成。用于测定异戊二烯基二磷酸产物的IDS酶分析通常使用放射性标记的底物进行，所形成的产物通过使用诸如磷成像仪、放射性气相色谱或放射性高效液相色谱等昂贵仪器来鉴定。尽管已经报道了一种用于测量粗植物提取物中IDS活性的非放射性分析方法，但它需要一种利用色谱与串联质谱联用（LC/MS-MS）的复杂方法。在这里，我们描述了一种使用非放射性标记的IPP及其共烯丙基底物DMAPP、GPP和FPP来测定IDS分析产物的非放射性且简单廉价的分析方法。该分析中产生的异戊二烯基二磷酸产物的检测效率很高，在IPP和DMAPP/GPP/FPP底物浓度>40µM时，对应于异戊烯醇的斑点清晰可见。这里描述的方案灵敏、可靠且技术简单，可用于IDS候选物的功能表征。
