Terpene synthases generate terpenes employing functional plasticity due to diversified carbocation chemistry, including highly specific ring formations, proton and hydride transfers, methyl as well as methylene migrations, followed by reaction quenching. In this enzyme family, the main catalytic challenge is not rate enhancement, but rather structural and reactive control of intrinsically unstable carbocations in order to guide the resulting product distribution. Here we employ multiscale modeling within classical and quantum dynamics frameworks to investigate the reaction mechanism in the diterpene synthase CotB2, commencing with the substrate geranyl geranyl diphosphate and terminating with the carbocation precursor to the final product cyclooctat-9-en-7-ol. The 11 step in-enzyme carbocation cascade is compared with the same reaction in absence of the enzyme. Remarkably, the free energy profiles in gas-phase and in CotB2 are surprisingly similar. This similarity contrasts the multitude of strong π-cation, dipole-cation and ion-pair interactions between all intermediates in the reaction cascade and the enzyme, suggesting a remarkable balance of interactions in CotB2. We ascribe this balance to the similar magnitude of the interactions between the carbocations along the reaction coordinate and the enzyme environment. The effect of CotB2 mutations is studied using multiscale mechanistic docking, machine learning, and X-ray crystallography, pointing the way for future terpene synthase design.

The impression of a non-existing catalytic effect: The role of CotB2 in guiding the complex biosynthesis of cyclooctat-9-en-7-ol

ACS Fall 2021

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