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

Abstract

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.

Alternative Active Site Confinement in Squalene–Hopene Cyclase Enforces Substrate Preorganization for Cyclization

“…Therefore, the generated variants are discriminating between isomers and demonstrate a highly selective active site for the E -isomer. The recently discovered convergent cyclization of linear terpenes was not observed …”

“…Known SHC homologs also convert substrates predominantly following the usual and native isoprene rule, with variations mostly observed in the form of chain length, epoxide groups at the terminal isoprene unit, or nucleophilic groups at the final cyclization unit. ,,, In contrast, class I terpene cyclases demonstrated greater tolerance to substrates deviating from the common isoprene unit, e.g., in the methylation pattern, but these enzymes are dependent on activated substrates and are less suitable for cost-effective broad applications. − …”

A Career in Catalysis: Bernhard Hauer

“…By reaching the milestone of synthetic application, Bernhard′s group was able to demonstrate how to transition an enzyme from paper to industry by discovering its promiscuity, rationally evolving its active site (Figure 3D), and paying attention to its topology. However, the journey is not over yet for the topic, as open questions remain about enzyme-controlled stereoconvergent cyclizations, 79 substrate-induced enzyme conformations, and their interconnectivity.…”

“…This protocol further enabled the establishment of the desired highly atom-economical hybrid synthetic routes for various terpenes, e.g., ambrinol, 67 pyrone meroterpenes, or biopolymer precursors, within two to four steps. 82 Notably, AacSHC activity toward its natural substrate squalene has also been demonstrated in bicontinuous microemulsions. 83 In collaboration with the groups of Engelskirchen and Stubenrauch from the University of Stuttgart, with whom studies on lipase B from Candida antarctica in bicontinuous microemulsions 84−87 were previously performed, AacSHC was tested in a microemulsion consisting of buffer−n-octane− nonionic surfactant.…”