Reprogramming the Cyclization of the Sesquiterpene Synthase BcBOT2 Using 2,3-<i>Z</i>-Configured FPP Derivatives and by Means of “Methyl Mapping”

The fungal sesquiterpene synthase BcBOT2 shows unique substrate promiscuity. It transforms farnesyl pyrophosphate (FPP) into presilphiperfolan-8β-ol via a cationic cascade that is initiated by a (1 → 11) cyclization. Here, it is shown that BcBOT2 also accepts (2,3-Z)-configured FPP derivatives, which provide terpenoids that result from an initial (1 → 6) cyclization. “Methyl mapping” was conducted by shifting the position of one or more methyl groups, and it was found that the location of methyl groups has a profound effect on the efficacy of cyclizations. In particular, the shift of the methyl group at C3 to the C2 position has the most profound effect on cyclohexane formation. Molecular modeling studies show that (1 → 6) cyclization took place due to adopting a different catalytically competent docking pose of FPP derivatives compared to natural FPP within the active site of the BcBOT2, which is mainly due to the (2,3-Z)-configuration. This docking pose leads to a C1–C6 distance shorter (3.3–3.5 Å) than the typical association with a near-attack conformation required for cyclization. Finally, these biotransformation results were benchmarked by a comprehensive study of the “hydrolysis” of eight different FPP derivatives. Under enzyme-free conditions, cyclohexene and cycloheptene terpenoids are formed. The ring size is mainly determined by the position of the methyl group at C6 or C7. The study reveals that in addition to protein engineering, unnatural substrates can also be used to specifically manipulate the mode of cyclization of terpene synthases.