title: Mutating a Highly Conserved Residue in Diverse Cytochrome P450s Facilitates Diastereoselective Olefin Cyclopropanation.
abstract: Cytochrome P450s and other heme-containing proteins have recently been shown to have promiscuous activity for the cyclopropanation of olefins using diazoacetate reagents. Despite the progress made thus far, engineering selective catalysts for all possible stereoisomers for the cyclopropanation reaction remains a considerable challenge. Previous investigations of a model P450 (P450BM3 ) revealed that mutation of a conserved active site threonine (Thr268) to alanine transformed the enzyme into a highly active and selective cyclopropanation catalyst. By incorporating this mutation into a diverse panel of P450 scaffolds, we were able to quickly identify enantioselective catalysts for all possible diastereomers in the model reaction of styrene with ethyl diazoacetate. Some alanine variants exhibited selectivities that were markedly different from the wild-type enzyme, with a few possessing moderate to high diastereoselectivity and enantioselectivities up to 97 % for synthetically challenging cis-cyclopropane diastereomers.
authors.affiliation: Department of Chemistry, University of North Carolina at Chapel Hill, 125 South Rd CB 3290, Chapel Hill, NC, 27599-3290, USA.
authors.forename: Joshua G
authors.initials: JG
authors.lastname: Gober
authors.affiliation: Department of Chemistry, University of North Carolina at Chapel Hill, 125 South Rd CB 3290, Chapel Hill, NC, 27599-3290, USA.
authors.forename: Amy E
authors.initials: AE
authors.lastname: Rydeen
authors.affiliation: Department of Chemistry, University of North Carolina at Chapel Hill, 125 South Rd CB 3290, Chapel Hill, NC, 27599-3290, USA.
authors.forename: Evan J
authors.initials: EJ
authors.lastname: Gibson-O'Grady
authors.affiliation: Department of Molecular Genetics and Genomics, Wake Forest School of Medicine, Medical Center Boulevard, Winston-Salem, NC, 27157, USA.
authors.forename: Janelle B
authors.initials: JB
authors.lastname: Leuthaeuser
authors.affiliation: Department of Chemistry, Gottwald Center for the Sciences, University of Richmond, 28 Westhampton Way, Richmond, VA, 23173, USA.
authors.forename: Jacquelyn S
authors.initials: JS
authors.lastname: Fetrow
authors.affiliation: Department of Chemistry, University of North Carolina at Chapel Hill, 125 South Rd CB 3290, Chapel Hill, NC, 27599-3290, USA. brustad@email.unc.edu.|Carolina Center for Genome Sciences, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599, USA. brustad@email.unc.edu.
authors.forename: Eric M
authors.initials: EM
authors.lastname: Brustad
mesh_terms: D000475:Alkenes / Q000737:chemistry*; D020134:Catalytic Domain; D017124:Conserved Sequence; D003521:Cyclopropanes / Q000737:chemistry*; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics*; D009154:Mutation; D013237:Stereoisomerism
chemical_list: D000475:Alkenes; D003521:Cyclopropanes; D003577:Cytochrome P-450 Enzyme System; C030797:cyclopropane
keywords: biocatalysis; cyclopropanation; cytochromes; protein engineering