Mechanistic Characterization of the Fusicoccane-type Diterpene Synthase for Myrothec-15(17)-en-7-ol
Fu-Long Lin, Lukas Lauterbach, Jian Zou, Yongheng Wang, Jian‐Ming Lv, Guo‐Dong Chen, Dan Hu, Hao Gao, Xin‐Sheng Yao, Jeroen S. Dickschat
Abstract
Fusicoccane (FC)-type diterpenes, featuring a common 5–8–5 tricyclic skeleton, possess diverse biological functions. Currently, only FC-type diterpene synthases (DTSs) for 3 of the 16 possible stereochemically distinct subtypes of the FC skeleton have been discovered. Herein, a (2si,6si,10re,14si)-FC-type DTS from Myrothecium gramineum is reported, which produces the diterpene alcohol myrothec-15(17)-en-7-ol. On the basis of homology modelings, the I189F variant was obtained by site-directed mutagenesis that prevents the penultimate carbocation from water attack, resulting in the deprotonation product myrotheca-7,15(17)-diene. Vibrational circular dichroism calculations in combination with chemical correlations through an enantioselective labeling strategy established the absolute configurations of these diterpenes. Density functional theory calculations revealed a water-mediated proton transfer mechanism, which is supported by isotope labeling experiments, in contrast to the deprotonation/reprotonation mechanism reported in previous studies.