Squalene-hopene cyclases (SHCs) are the biocatalytic pendant to asymmetric Brønsted-acid catalysis and thus comprise enormous synthetic potential. Nevertheless, their substrate scope is currently limited to terpenes. Herein, we present how to tailor the SHC's cation cage for an enantioselective semipinacol rearrangement of bicyclic allylic alcohols to produce valuable oxa-carbon spirocyclic compounds. Exploiting the subtle divergence of SHC active sites combined with structure-guided semirational engineering, we designed a biocatalyst with a high catalytic performance of ∼4500 TTN and excellent enantioselectivity of 99.5% enantiomeric excess (ee). In silico studies suggest that a broadened active site is pivotal for catalysis. This intricate cationic rearrangement is easily scalable, employing lyophilized cell powder in water. Furthermore, our substrate scope studies demonstrate the acceptance of diverse ring-sized substrates but also reveal that the naturally confined active site limits the function as a general "semipinacolase." This study showcases the ability to harness the SHC's cation cage to tap into the broader field of asymmetric Brønsted-acid catalysis.
Terpene 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 (À )-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 membranebound 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 10 5 total turnovers. We believe that our results may bridge the gap for broader application of SHCs in synthetic chemistry.
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.
Terpen-Zyklasen bergen enormes synthetisches Potenzial, da sie in der Lage sind, komplexe Kohlenwasserstoffgerüste aus achiralen Ausgangsstoffen in einer einzigen kationischen Umlagerungskaskade zu generieren. Dieses synthetische Potenzial nutzbar zu machen, erwies sich jedoch aufgrund ihrer allgemein geringen katalytischen Leistung als schwierig. In dieser Studie enthüllen wir das katalytische Potenzial der Squalen-Hopen-Zyklase (SHC), indem wir uns ihre Proteinstruktur und Dynamik zunutze machen. Zunächst haben wir das aktive Zentrum und den Eingangstunnel des Enzyms synergistisch evolviert, um damit eine 397-fach verbesserte (À )-Ambroxid-Synthase zu erzeugen. Unsere computergestützten Untersuchungen erklären, wie die eingeführten Mutationen zusammenarbeiten, um die Substrataufnahme, den Fluss und die Vorfaltung zu verbessern. Kinetische Untersuchungen zeigten jedoch eine Terpen-induzierte Inaktivierung der membrangebundenen SHC als Hauptlimitierung für die Biokatalyse in vivo. Indem diese Erkenntnisse mit der verbesserten und stereoselektiven Katalyse des Enzyms kombinierten wurden, konnte eine Fütterungsstrategie angewendet werden, um 10 5 katalytische Umsätze zu übertreffen. Diese Ergebnisse haben das Potenzial die Lücke für eine breitere Anwendung von SHCs in der synthetischen Chemie zu schließen.
Ambergris is a highly valued natural product in the fragrance industry and is also known as "floating gold" because of its biosynthesis in the intestines of sperm whales. Owing to this scarce natural resource, the stereoselective synthesis of its most prominent olfactory component, (−)‐ambroxide, via alternative routes is attractive. In their Research Article (e202301607), Sílvia Osuna, Bernhard Hauer et al. demonstrate the catalytic potential of an engineered squalene‐hopene cyclase in the intricate cyclization of the precursor homofarnesol. Cover art: Verena Resch, Graz.
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