Max Planck Institute for Chemical Ecology, Beutenberg Campus, Hans-Knöll-Str. 8, 07745, Jena, Germany.
Plant Cell Environ. 2018 Jan;41(1):39-49. doi: 10.1111/pce.12901. Epub 2017 Mar 9.
During the process of terpene biosynthesis, C-C bond breaking and forming steps are subjected to kinetic carbon isotope effects, leading to distinct carbon isotopic signatures of the products. Accordingly, carbon isotopic signatures could be used to reveal the 'biosynthetic history' of the produced terpenoids. Five known sesquiterpene cyclases, regulating three different pathways, representing simple to complex biosynthetic sequences, were heterologously expressed and used for in vitro assays with farnesyl diphosphate as substrate. Compound specific isotope ratio mass spectrometry measurements of the enzyme substrate farnesyl diphosphate (FDP) and the products of all the five cyclases were performed. The calculated δ C value for FDP, based on δ C values and relative amounts of the products, was identical with its measured δ C value, confirming the reliability of the approach and the precision of measurements. The different carbon isotope ratios of the products reflect the complexity of their structure and are correlated with the frequency of carbon-carbon bond forming and breaking steps on their individual biosynthetic pathways. Thus, the analysis of carbon isotopic signatures of terpenes at natural abundance can be used as a powerful tool in elucidation of associated biosynthetic mechanisms of terpene synthases and in future in vivo studies even without 'touching' the plant.
在萜类生物合成过程中，C-C 键的断裂和形成步骤受到动力学碳同位素效应的影响，导致产物具有明显的碳同位素特征。因此，碳同位素特征可用于揭示所产生的萜类化合物的“生物合成历史”。五种已知的倍半萜环化酶，调节三种不同的途径，代表简单到复杂的生物合成序列，通过异源表达并使用法呢基二磷酸作为底物进行体外测定。对酶底物法呢基二磷酸（FDP）和所有五种环化酶产物的化合物特异性同位素比质谱测量进行了分析。基于δ C 值和产物的相对量计算出的 FDP 的δ C 值与实测的δ C 值相同，证实了该方法的可靠性和测量的精度。产物的不同碳同位素比值反映了它们结构的复杂性，并与它们各自生物合成途径中碳-碳键形成和断裂步骤的频率相关。因此，萜类化合物天然丰度下的碳同位素特征分析可作为阐明萜烯合酶相关生物合成机制的有力工具，并且在未来的体内研究中甚至无需“接触”植物即可进行。
