title: An Evolved Orthogonal Enzyme/Cofactor Pair.
abstract: We introduce a strategy that expands the functionality of hemoproteins through orthogonal enzyme/heme pairs. By exploiting the ability of a natural heme transport protein, ChuA, to promiscuously import heme derivatives, we have evolved a cytochrome P450 (P450BM3) that selectively incorporates a nonproteinogenic cofactor, iron deuteroporphyrin IX (Fe-DPIX), even in the presence of endogenous heme. Crystal structures show that selectivity gains are due to mutations that introduce steric clash with the heme vinyl groups while providing a complementary binding surface for the smaller Fe-DPIX cofactor. Furthermore, the evolved orthogonal enzyme/cofactor pair is active in non-natural carbenoid-mediated olefin cyclopropanation. This methodology for the generation of orthogonal enzyme/cofactor pairs promises to expand cofactor diversity in artificial metalloenzymes.
authors: Department of Chemistry, University of North Carolina-Chapel Hill , 125 South Road, CB 3290, Chapel Hill, North Carolina 27599, United States.
authors: Evan W
authors: EW
authors: Reynolds
authors: Department of Chemistry, University of North Carolina-Chapel Hill , 125 South Road, CB 3290, Chapel Hill, North Carolina 27599, United States.
authors: Matthew W
authors: MW
authors: McHenry
authors: Department of Chemistry, University of North Carolina-Chapel Hill , 125 South Road, CB 3290, Chapel Hill, North Carolina 27599, United States.
authors: Fabien
authors: F
authors: Cannac
authors: Department of Chemistry, University of North Carolina-Chapel Hill , 125 South Road, CB 3290, Chapel Hill, North Carolina 27599, United States.
authors: Joshua G
authors: JG
authors: Gober
authors: Department of Chemical and Biological Engineering, Colorado State University , Fort Collins, Colorado 80523, United States.
authors: Christopher D
authors: CD
authors: Snow
authors: Department of Chemistry, University of North Carolina-Chapel Hill , 125 South Road, CB 3290, Chapel Hill, North Carolina 27599, United States.
authors: Eric M
authors: EM
authors: Brustad
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D020134:Catalytic Domain; D003067:Coenzymes; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000378:metabolism*; D019020:Directed Molecular Evolution; D007501:Iron / Q000737:chemistry*; D008665:Metalloporphyrins / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D015394:Molecular Structure; D009154:Mutation; D009251:NADPH-Ferrihemoprotein Reductase / Q000737:chemistry* / Q000378:metabolism*; D010084:Oxidation-Reduction
chemical_list: D001426:Bacterial Proteins; D003067:Coenzymes; D008665:Metalloporphyrins; D003577:Cytochrome P-450 Enzyme System; D007501:Iron; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases