title: Improved cyclopropanation activity of histidine-ligated cytochrome P450 enables the enantioselective formal synthesis of levomilnacipran.
abstract: 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.
authors.affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology, 1200 E. California Blvd. MC 210-41, Pasadena, CA 91125 (USA).
authors.forename: Z Jane
authors.initials: ZJ
authors.lastname: Wang
authors.forename: Hans
authors.initials: H
authors.lastname: Renata
authors.forename: Nicole E
authors.initials: NE
authors.lastname: Peck
authors.forename: Christopher C
authors.initials: CC
authors.lastname: Farwell
authors.forename: Pedro S
authors.initials: PS
authors.lastname: Coelho
authors.forename: Frances H
authors.initials: FH
authors.lastname: Arnold
mesh_terms: D001410:Bacillus megaterium / Q000201:enzymology; D020134:Catalytic Domain; D003521:Cyclopropanes / Q000138:chemical synthesis* / Q000737:chemistry; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D004798:Enzymes; D006639:Histidine / Q000737:chemistry / Q000378:metabolism*; D000078764:Milnacipran; D016297:Mutagenesis, Site-Directed; D015202:Protein Engineering; D013237:Stereoisomerism
chemical_list: D003521:Cyclopropanes; D004798:Enzymes; D006639:Histidine; D003577:Cytochrome P-450 Enzyme System; C030797:cyclopropane; D000078764:Milnacipran
keywords: biocatalysis; cyclopropanation; cytochrome P450; enzymes; protein engineering