Controlling Monoterpene Isomerization by Guiding Challenging Carbocation Rearrangement Reactions in Engineered Squalene‐Hopene Cyclases

• Published in: Angewandte Chemie 2024-03, Vol.136 (12), p.n/a
• Main Authors: Ludwig, Julian, Curado‐Carballada, Christian, Hammer, Stephan C., Schneider, Andreas, Diether, Svenja, Kress, Nico, Ruiz‐Barragán, Sergi, Osuna, Sílvia, Hauer, Bernhard
• Format: Article
• Language: English
• Subjects: Aromatic compounds; Biocatalysis; Borneol; Brønsted Acid Catalysis; Catalysts; Cations; Isomerization; Iterative methods; Lewis acid; Monoterpenes; Mutagenesis; Saturation; Saturation mutagenesis; Secondary Carbocations; Squalene; Steering; Substrates; Sustainable Chemistry; Terpenes; Terpenoids
• ISSN: 0044-8249; 1521-3757
• DOI: 10.1002/ange.202318913

The interconversion of monoterpenes is facilitated by a complex network of carbocation rearrangement pathways. Controlling these isomerization pathways is challenging when using common Brønsted and Lewis acid catalysts, which often produce product mixtures that are difficult to separate. In contrast, natural monoterpene cyclases exhibit high control over the carbocation rearrangement reactions but are reliant on phosphorylated substrates. In this study, we present engineered squalene‐hopene cyclases from Alicyclobacillus acidocaldarius (AacSHC) that catalyze the challenging isomerization of monoterpenes with unprecedented precision. Starting from a promiscuous isomerization of (+)‐β‐pinene, we first demonstrate noticeable shifts in the product distribution solely by introducing single point mutations. Furthermore, we showcase the tuneable cation steering by enhancing (+)‐borneol selectivity from 1 % to >90 % (>99 % de) aided by iterative saturation mutagenesis. Our combined experimental and computational data suggest that the reorganization of key aromatic residues leads to the restructuring of the water network that facilitates the selective termination of the secondary isobornyl cation. This work expands our mechanistic understanding of carbocation rearrangements and sets the stage for target‐oriented skeletal reorganization of broadly abundant terpenes. Carbocation‐based rearrangements are vital for terpene versatility. AacSHC triterpene cyclase from Alicyclobacillus acidocaldarius acted as a promiscuous catalyst for multiple monoterpene rearrangements. Tailored AacSHC variants directed the specific isomerization of pinene with exceptional selectivities. Success involved restructuring the aromatic active pocket and reorganizing water clusters to stabilize demanding carbocationic intermediates.