Harnessing the Structure and Dynamics of the Squalene‐Hopene Cyclase for (−)‐Ambroxide Production**

Author:

Schneider Andreas1,Curado Christian2ORCID,Lystbaek Thomas B.1,Osuna Sílvia23ORCID,Hauer Bernhard1ORCID

Affiliation:

1. Department of Technical Biochemistry Institute of Biochemistry and Technical Biochemistry University of Stuttgart Stuttgart-Vaihingen Germany

2. CompBioLab Group Institut de Química Computacional i Catàlisi and Departament de Química Universitat de Girona Girona Spain

3. ICREA Barcelona Spain

Abstract

AbstractTerpene cyclases offer enormous synthetic potential, given their unique ability to forge complex hydrocarbon scaffolds from achiral precursors within a single cationic rearrangement cascade. Harnessing their synthetic power, however, has proved to be challenging owing to their generally low catalytic performance. In this study, we unveiled the catalytic potential of the squalene‐hopene cyclase (SHC) by harnessing its structure and dynamics. First, we synergistically tailored the active site and entrance tunnel of the enzyme to generate a 397‐fold improved (−)‐ambroxide synthase. Our computational investigations explain how the introduced mutations work in concert to improve substrate acquisition, flow, and chaperoning. Kinetics, however, showed terpene‐induced inactivation of the membrane‐bound SHC to be the major turnover limitation in vivo. Merging this insight with the improved and stereoselective catalysis of the enzyme, we applied a feeding strategy to exceed 105 total turnovers. We believe that our results may bridge the gap for broader application of SHCs in synthetic chemistry.

Funder

Deutsche Forschungsgemeinschaft

Generalitat de Catalunya

H2020 European Research Council

Publisher

Wiley

Subject

General Chemistry,Catalysis

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