An Isopentenol\nUtilization Pathway-Based “Deuterium-Scanning”\nMethod for Mechanistic Investigations of Terpene Cyclases

An isopentenol utilization pathway-based method for the\ninvestigation\nof the cyclization mechanism of terpene cyclases (TCs) is developed.\nBy feeding deuterium-labeled prenols/isoprenols in combination with\nunlabeled ones to engineered E. coli hosts, terpene products with certain deuterium labeling patterns\nat hydrogen-bearing positions were obtained that can be used for deducing\nthe cyclization processes, especially for those steps involving stereoselective\nhydride/proton shifts. Different types of TCs of varied origins for\nthe biosynthesis of six known terpenes were used to test the scope\nand limitations of this method. Reliable results without significant\ndeuterium dilution and scrambling are obtained by using this “deuterium-scanning”\nmethod and are consistent with those obtained previously. Limitations\nexist in the deuterium transfer process between those positions that\nare derived from the same labeled position of isoprenol, as exemplified\nby the failure of precisely tracking the origin of each deuterium\nin the labeled fusicocca-2,10(14)-diene obtained by feeding [2,2-<sup>2</sup>H<sub>2</sub>]-isoprenol. Nonetheless, the newly developed\nmethod could be used as an alternate to those using custom-labeled\noligoprenyl diphosphates for probing the cyclization mechanism of\nTCs.