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 from both experimental and computational perspectives. Firstly, experimental evolution was performed starting from a promiscuous isomerization of (+)-β-pinene, we 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. We combined the experimental data with Molecular Dynamics simulations and Quantum Mechanical calculations to elucidate  the role of the introduced mutations in the (+)-borneol selectivity. By means of MD simulations, we determined the rearrangement of the whole active site architecture in the most evolved variant compared to WT, revealing a different water arrangement in the active site. QM calculations determined that the presence of water molecules in the enzyme variant redistribute the populations of the different carbocations present in the isomerization process, thus, being determinant for (+)-borneol production.

This work has been led by Dr. Christian Curado-Carballada, Dr. Sergi Ruiz-Barragán and Prof. Sílvia Osuna in collaboration with Prof. Bernhard Hauer (University of Stuttgart).

It has been recently published open access in Angewandte Chemie International Edition:
J. Ludwig, C. Curado-Carballada, S. C. Hammer, A. Schneider, S. Diether, N. Kress, S. Ruiz-Barragán, S. Osuna*, B. Hauer*
“Controlling Monoterpene Isomerization by Guiding Challenging Carbocation Rearrangement Reactions in Engineered Squalene-Hopene Cyclases“
Angew. Chem. Int. Ed.., 2024, e202318913
DOI: 10.1002/anie.202318913

Girona, February 19, 2024
For more info: ges.iqcc@udg.edu