The diterpene cyclase CotB2 catalyzes the cyclization of geranylgeranyl diphosphate (GGPP) to the tricyclic cyclooctat-9-en-7-ol, which is characterized by a 5-8-5-fused ring skeleton. We have previously proposed a cyclization cascade involving a unique carbon-carbon bond rearrangement combined with multiple hydride shifts, all occurring at a single active site. Here, we report the first high-resolution X-ray crystal structure of CotB2 with bound substrate analog geranylgeranyl thiodiphosphate (GGSPP). In the GGSPP-bound form, GGSPP folds into a unique S-shaped conformation that probably reflects the substrate-bound state prior to ionization of the substrate GGPP. The folded framework of GGSPP is surrounded by hydrophobic residues and several aromatic and asparagine residues that are well-positioned to stabilize a series of reactive carbocation intermediates through a combination of cation-π and dipole charge interactions. The combined crystal structures and mutagenesis-based biochemical assays provide a structural basis for exquisite control of ring formation and stereochemistry during CotB2 catalysis.

Terpene cyclization reactions are fascinating owing to the precise control of connectivity and stereochemistry during the catalytic process. Cyclooctat-9-en-7-ol synthase (CotB2) synthesizes an unusual 5-8-5 fused-ring structure with six chiral centers from the universal diterpene precursor, the achiral C20 geranylgeranyl diphosphate substrate. An unusual new mechanism for the exquisite CotB2-catalyzed cyclization that involves a carbon-carbon backbone rearrangement and three long-range hydride shifts is proposed, based on a powerful combination of in vivo studies using uniformly (13)C-labeled glucose and in vitro reactions of regiospecifically deuterium-substituted geranylgeranyl diphosphate substrates. This study shows that CotB2 elegantly demonstrates the synthetic virtuosity and stereochemical control that evolution has conferred on terpene synthases.

Terpene cyclization reactions are fascinating owing to the precise control of connectivity and stereochemistry during the catalytic process. Cyclooctat-9-en-7-ol synthase (CotB2) synthesizes an unusual 5-8-5 fused-ring structure with six chiral centers from the universal diterpene precursor, the achiral C 20 geranylgeranyl diphosphate substrate. An unusual new mechanism for the exquisite CotB2-catalyzed cyclization that involves a carbon-carbon backbone rearrangement and three long-range hydride shifts is proposed, based on a powerful combination of in vivo studies using uniformly 13 C-labeled glucose and in vitro reactions of regiospecifically deuteriumsubstituted geranylgeranyl diphosphate substrates. This study shows that CotB2 elegantly demonstrates the synthetic virtuosity and stereochemical control that evolution has conferred on terpene synthases.