Abstract
Terpene synthases generate terpenes employing diversified carbocation chemistry, including highly specific ring formations, proton and hydride transfers, and 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 the 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.
Original language | English |
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Pages (from-to) | 21562-21574 |
Number of pages | 13 |
Journal | Journal of the American Chemical Society |
Volume | 142 |
Issue number | 51 |
DOIs | |
State | Published - 23 Dec 2020 |
Bibliographical note
Publisher Copyright:© 2020 American Chemical Society.
Funding
T.B. and M.R. gratefully acknowledge funding of the Werner Siemens Foundation for establishing the field of Synthetic Biotechnology at the Technical University of Munich. R.D. was supported by Elsa-Neumann and Nüsslein-Volhard stipends. We acknowledge support of A. de Amorim and B. V. Draegert in protein production and crystallization. We accessed beamlines of the BESSY II (Berliner Elektronenspeicherring-Gesellschaft für Synchrotronstrahlung II) storage ring (Berlin, Germany) via the Joint Berlin MX-Laboratory sponsored by the Helmholtz Zentrum Berlin für Materialien und Energie, the Freie Universität Berlin, the Humboldt-Universität zu Berlin, the Max-Delbrück-Centrum, the Leibniz-Institut für Molekulare Pharmakologie and Charité – Universitätsmedizin Berlin. Parts of this research were carried out at PETRA III at DESY, a member of the Helmholtz Association (HGF). We would like to thank A. Burkhardt for assistance in using beamline P11 and G. Bourenkov for assistance in using beamline P14. We are grateful to M. Wahl for continuous encouragement and support. This work was supported by a grant from the Israeli Science Foundation (Grant 1683/18) (D.T.M.) and by a grant from the German-Israeli Foundation for Scientific Research and Development (Grant I-85-302.14-2018) (D.T.M., T.B., B.L.).
Funders | Funder number |
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Israeli Science Foundation | 1683/18 |
Werner Siemens Foundation | |
German-Israeli Foundation for Scientific Research and Development | I-85-302.14-2018 |