An isopentenol utilization pathway-based method for the investigation of the cyclization mechanism of terpene cyclases (TCs) is developed. By feeding deuterium-labeled prenols/isoprenols in combination with unlabeled ones to engineered E. coli hosts, terpene products with certain deuterium labeling patterns at hydrogen-bearing positions were obtained that can be used for deducing the cyclization processes, especially for those steps involving stereoselective hydride/proton shifts. Different types of TCs of varied origins for the biosynthesis of six known terpenes were used to test the scope and limitations of this method. Reliable results without significant deuterium dilution and scrambling are obtained by using this “deuterium-scanning” method and are consistent with those obtained previously. Limitations exist in the deuterium transfer process between those positions that are derived from the same labeled position of isoprenol, as exemplified by the failure of precisely tracking the origin of each deuterium in the labeled fusicocca-2,10(14)-diene obtained by feeding [2,2-²H₂]-isoprenol. Nonetheless, the newly developed method could be used as an alternate to those using custom-labeled oligoprenyl diphosphates for probing the cyclization mechanism of TCs.

中文翻译：

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一种基于异戊烯醇利用途径的“氘扫描”方法，用于萜烯环化酶的机理研究


开发了一种基于异戊烯醇利用途径的方法，用于研究萜烯环化酶 （TCs） 的环化机制。通过将氘标记的异戊二醇/异戊二醇与未标记的异戊二醇/异戊二醇联合喂食给工程化的大肠杆菌宿主，获得了在含氢位置具有某些氘标记模式的萜烯产物，可用于推断环化过程，特别是对于那些涉及立体选择性氢化物/质子位移的步骤。使用不同来源的不同类型的 TCs 来测试该方法的范围和局限性，用于 6 种已知萜烯的生物合成。使用这种“氘扫描”方法获得了可靠的结果，没有明显的氘稀释和加扰，并且与以前获得的结果一致。从异戊二醇的相同标记位置得出的那些位置之间的氘转移过程存在局限性，例如未能精确跟踪通过喂食 [2,2-2 H₂]-异戊二烯获得的标记梭球菌-2,10（14）-二烯中每个氘的来源。尽管如此，新开发的方法可以用作使用定制标记的寡戊二烯基二磷酸的替代方法，以探测 TC 的环化机制。