Abstract
The cyclization mechanisms involved in the biosynthesis of sesterterpenes are not fully understood. For example, there are two plausible reaction pathways for sesterfisherol biosynthesis, which differ in the order of ring cyclization: A-D-B/C (Path a) and A-B-C/D (Path b). It is difficult to capture intermediates of terpene cyclization, which is a complex, domino-type reaction, and so here we employed a combination of experimental and computational methods. Density functional theory calculations revealed unexpected intermediates and transition states, and implied that C-H···π interaction between a carbocation intermediate and an aromatic residue of sesterfisherol synthase (NfSS) plays a critical role, serving to accelerate the 1,2-H shift (thereby preventing triquinane carbocation formation) and to protect reactive carbocation intermediates from bases such as pyrophosphate or water in the active site. Site-directed mutagenesis of NfSS guided by docking simulations confirmed that phenylalanine F191 is a critical amino acid residue for sesterfisherol synthase, as the F191A mutant of NfSS produces novel sesterterpenes, but not sesterfisherol. Although both pathways are energetically viable, on the basis of our computational and experimental results, NfSS-mediated sesterfisherol biosynthesis appears to proceed via Path a. These findings may also provide new insight into the cyclization mechanisms in related sesterterpene synthases.