Improved Cyclopropanation Activity of Histidine-Ligated Cytochrome P450 Enables the Enantioselective Formal Synthesis of Levomilnacipran

• Published in: Angewandte Chemie International Edition Vol. 53; no. 26; pp. 6810 - 6813
• Main Authors: Wang, Z. Jane, Renata, Hans, Peck, Nicole E., Farwell, Christopher C., Coelho, Pedro S., Arnold, Frances H.
• Format: Journal Article
• Language: English
• Published: Weinheim WILEY-VCH Verlag 23.06.2014; WILEY‐VCH Verlag; Wiley
• Subjects: Bacillus megaterium - enzymology; biocatalysis; Catalytic Domain; Chemistry; Chemistry, Multidisciplinary; cyclopropanation; Cyclopropanes - chemical synthesis; Cyclopropanes - chemistry; Cytochrome P-450 Enzyme System - genetics; Cytochrome P-450 Enzyme System - metabolism; cytochrome P450; Enzymes; Histidine - chemistry; Histidine - metabolism; Milnacipran; Mutagenesis, Site-Directed; Physical Sciences; Protein Engineering; Science & Technology; Stereoisomerism; CARBENE TRANSFER; enzymes; ISO-1-CYTOCHROME-C; cytochrome P450; AXIAL-LIGAND; SACCHAROMYCES-CEREVISIAE; biocatalysis; protein engineering; cyclopropanation; IN-VIVO; P450BM3; SELECTIVITY; C-H AMINATION; TRANSFORMATIONS; cytochrome P450
• ISSN: 1433-7851; 1521-3773
• DOI: 10.1002/anie.201402809

Engineering enzymes capable of modes of activation unprecedented in nature will increase the range of industrially important molecules that can be synthesized through biocatalysis. However, low activity for a new function is often a limitation in adopting enzymes for preparative‐scale synthesis, reaction with demanding substrates, or when a natural substrate is also present. By mutating the proximal ligand and other key active‐site residues of the cytochrome P450 enzyme from Bacillus megaterium (P450‐BM3), a highly active His‐ligated variant of P450‐BM3 that can be employed for the enantioselective synthesis of the levomilnacipran core was engineered. This enzyme, BM3‐Hstar, catalyzes the cyclopropanation of N,N‐diethyl‐2‐phenylacrylamide with an estimated initial rate of over 1000 turnovers per minute and can be used under aerobic conditions. Cyclopropanation activity is highly dependent on the electronic properties of the P450 proximal ligand, which can be used to tune this non‐natural enzyme activity. Old cytochrome, new tricks: Mutation of the proximal Cys residue in the cytochrome P450 enzyme from Bacillus megaterium (P450‐BM3) leads to highly active, oxygen tolerant, and enantioselective catalysts for the cyclopropanation of N,N‐diethyl‐2‐phenylacrylamide. Directed evolution of a histidine‐ligated P450‐BM3 enabled the enantioselective formal synthesis of levomilnacipran.