title: Identification of Mechanism-Based Inactivation in P450-Catalyzed Cyclopropanation Facilitates Engineering of Improved Enzymes.
abstract: Following the recent discovery that heme proteins can catalyze the cyclopropanation of styrenyl olefins with high efficiency and selectivity, interest in developing new enzymes for a variety of non-natural carbene transfer reactions has burgeoned. The fact that diazo compounds and other carbene precursors are known mechanism-based inhibitors of P450s, however, led us to investigate if they also interfere with this new enzyme function. We present evidence for two inactivation pathways that are operative during cytochrome P450-catalyzed cyclopropanation. Using a combination of UV-vis, mass spectrometry, and proteomic analyses, we show that the heme cofactor and several nucleophilic side chains undergo covalent modification by ethyl diazoacetate (EDA). Substitution of two of the affected residues with less-nucleophilic amino acids led to a more than twofold improvement in cyclopropanation performance (total TTN). Elucidating the inactivation pathways of heme protein-based carbene transfer catalysts should aid in the optimization of this new biocatalytic function.
authors: Division of Chemistry and Chemical Engineering, MC 210-41, and ‡Proteome Exploration Laboratory, Division of Biology and Biological Engineering, Beckman Institute, MC 139-74, California Institute of Technology , 1200 E California Blvd, Pasadena, California 91125, United States.
authors: Hans
authors: H
authors: Renata
authors: Division of Chemistry and Chemical Engineering, MC 210-41, and ‡Proteome Exploration Laboratory, Division of Biology and Biological Engineering, Beckman Institute, MC 139-74, California Institute of Technology , 1200 E California Blvd, Pasadena, California 91125, United States.
authors: Russell D
authors: RD
authors: Lewis
authors: Division of Chemistry and Chemical Engineering, MC 210-41, and ‡Proteome Exploration Laboratory, Division of Biology and Biological Engineering, Beckman Institute, MC 139-74, California Institute of Technology , 1200 E California Blvd, Pasadena, California 91125, United States.
authors: Michael J
authors: MJ
authors: Sweredoski
authors: Division of Chemistry and Chemical Engineering, MC 210-41, and ‡Proteome Exploration Laboratory, Division of Biology and Biological Engineering, Beckman Institute, MC 139-74, California Institute of Technology , 1200 E California Blvd, Pasadena, California 91125, United States.
authors: Annie
authors: A
authors: Moradian
authors: Division of Chemistry and Chemical Engineering, MC 210-41, and ‡Proteome Exploration Laboratory, Division of Biology and Biological Engineering, Beckman Institute, MC 139-74, California Institute of Technology , 1200 E California Blvd, Pasadena, California 91125, United States.
authors: Sonja
authors: S
authors: Hess
authors: Division of Chemistry and Chemical Engineering, MC 210-41, and ‡Proteome Exploration Laboratory, Division of Biology and Biological Engineering, Beckman Institute, MC 139-74, California Institute of Technology , 1200 E California Blvd, Pasadena, California 91125, United States.
authors: Z Jane
authors: ZJ
authors: Wang
authors: Division of Chemistry and Chemical Engineering, MC 210-41, and ‡Proteome Exploration Laboratory, Division of Biology and Biological Engineering, Beckman Institute, MC 139-74, California Institute of Technology , 1200 E California Blvd, Pasadena, California 91125, United States.
authors: Frances H
authors: FH
authors: Arnold
mesh_terms: D000595:Amino Acid Sequence; D002384:Catalysis; D003067:Coenzymes; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000145:classification / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D006418:Heme; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*
chemical_list: D003067:Coenzymes; D065607:Cytochrome P-450 Enzyme Inhibitors; D006418:Heme; D003577:Cytochrome P-450 Enzyme System