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 obtai... More
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 [2,2-2
H2]-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.
