Biosynthetic formation of the 6/7/5-odyverdiene diterpenes via a 6/10-trans-eunicellanyl intermediate.

Odyverdienes are a rare class of bacterial diterpenes possessing 6/8/4- or 6/7/5-tricyclic skeletons and the cyclization mechanism underlying their formation remains unknown. Based on their skeletons, we hypothesized that their tricyclic skeletons pass through a neutral or cationic 6/10-eunicellane intermediate. Here, we investigated the mechanism of odyverdiene B2 synthase, alkS from , using site-directed mutagenesis, isotopic labeling experiments, and quantum chemical calculations. Two key shunt products, the 10-membered monocyclic 13-deoxolobophytumin A and a -eunicellane named alkacellene, isolated from alkS variants and quantum chemical calculations supported the proposed mechanism to odyverdiene B2 via initial 1,10-cyclization and a cationic eunicellanyl intermediate. Deuterium labeling studies also supported the occurrence of two sequential 1,2-hydride shifts that set distinctive stereocenters during eunicellanyl formation. Alkacellene was also shown to undergo acid-catalyzed cyclization to the 6/6/6/6-tetracyclic isohydropyrene, an isomer of the bacterial diterpene synthase product hydropyrene. This mechanistic study of alkS highlights the frequent formation and use of eunicellanyl intermediates in diversifying bacterial diterpene biosynthesis and provides a model system to address fundamental questions involving 6/10-bicyclic ring systems.