Tailoring the squalene-hopene cyclase for stereoconvergent and efficient cationic cyclization cascades

Asymmetric catalysis has witnessed paramount lessons from terpene cyclase enzymology such as the ability to control dynamic carbocations or cationic cyclization cascades. In general, these cascades are stereodivergent and thus rely on the terpene’s double-bond geometry. In this work, we illuminate how the dynamic supramolecular framework of squalene-hopene cyclases (SHCs) can be tailored to break with this paradigm. Creating a locally electron-enriched confined active site, we enabled the stereoconvergent cationic cyclization of a cis/trans terpene mixture into one isomer. Our results suggest that a priorly unknown active site “memory” effect of the SHC aids this intricate transformation. Based on these findings, we employed synergistic active site and tunnel engineering to generate a most efficient (–)-ambroxide cyclase. Broad computational investigations evidently explain how the introduced mutations work in concert to improve substrate acquisition, flow and chaperoning. Nonetheless, kinetics disclosed a substrate-induced downregulation of the membrane-bound SHC as the major turnover limitation in vivo. Merging these new insights with the improved and stereoconvergent catalysis of the enzyme, we applied a feeding strategy to exceed 106 TTN with the SHC.