Comparative analysis and validation of the malachite green assay for the high throughput biochemical characterization of terpene synthases.
Comparative analysis and validation of the malachite green assay for the high throughput biochemical characterization of terpene synthases.
复制标题
DOI：
10.1016/j.mex.2014.08.007
复制
发表时间：
2014
期刊：
影响因子：
1.9
通讯作者：
O'Maille, Paul E
中科院分区：
文献类型：
--
作者：
Vardakou, Maria;Salmon, Melissa;Faraldos, Juan A;O'Maille, Paul E
萜烯是最大的一类天然产物，具有重要而多样的生物学作用，同时作为香料、香精和药剂具有巨大的经济价值。一类萜烯合成酶（TPS）是TPS酶的主要类型，催化二磷酸戊烯基转化为结构多样的生物活性萜烯烃和无机焦磷酸（PPi）。为了测量它们的动力学性质，目前的生物分析方法通常依赖于通过放射性测量或气相色谱-质谱（GC-MS）直接检测碳氢化合物产物。我们描述了MG测定对tps的周转和催化效率测量的适应性。我们通过与已建立的测定法的直接比较来验证该方法。不同方法间kcat/KM的一致性使得这种适应方法最适合于tps的快速评价。我们展示了MG测定在TPS基因文库高通量筛选中的应用。在这项研究中，我们采用了一种已建立的快速比色法，焦磷酸盐/孔雀石绿法（MG），作为I类tps活性生化表征的替代方法。
Terpenes are the largest group of natural products with important and diverse biological roles, while of tremendous economic value as fragrances, flavours and pharmaceutical agents. Class-I terpene synthases (TPSs), the dominant type of TPS enzymes, catalyze the conversion of prenyl diphosphates to often structurally diverse bioactive terpene hydrocarbons, and inorganic pyrophosphate (PPi). To measure their kinetic properties, current bio-analytical methods typically rely on the direct detection of hydrocarbon products by radioactivity measurements or gas chromatography–mass spectrometry (GC–MS). We describe the adaptation of the MG assay for turnover and catalytic efficiency measurements of TPSs. We validate the method by direct comparison with established assays. The agreement of kcat/KM among methods makes this adaptation optimal for rapid evaluation of TPSs. We demonstrate the application of the MG assay for the high-throughput screening of TPS gene libraries. In this study we employed an established, rapid colorimetric assay, the pyrophosphate/malachite green assay (MG), as an alternative means for the biochemical characterization of class I TPSs activity.