title: Structure-guided recombination creates an artificial family of cytochromes P450.
abstract: Creating artificial protein families affords new opportunities to explore the determinants of structure and biological function free from many of the constraints of natural selection. We have created an artificial family comprising 3,000 P450 heme proteins that correctly fold and incorporate a heme cofactor by recombining three cytochromes P450 at seven crossover locations chosen to minimize structural disruption. Members of this protein family differ from any known sequence at an average of 72 and by as many as 109 amino acids. Most (>73%) of the properly folded chimeric P450 heme proteins are catalytically active peroxygenases; some are more thermostable than the parent proteins. A multiple sequence alignment of 955 chimeras, including both folded and not, is a valuable resource for sequence-structure-function studies. Logistic regression analysis of the multiple sequence alignment identifies key structural contributions to cytochrome P450 heme incorporation and peroxygenase activity and suggests possible structural differences between parents CYP102A1 and CYP102A2.
authors.affiliation: Biochemistry and Molecular Biophysics, California Institute of Technology, Pasadena, California, USA.
authors.forename: Christopher R
authors.initials: CR
authors.lastname: Otey
authors.forename: Marco
authors.initials: M
authors.lastname: Landwehr
authors.forename: Jeffrey B
authors.initials: JB
authors.lastname: Endelman
authors.forename: Kaori
authors.initials: K
authors.lastname: Hiraga
authors.forename: Jesse D
authors.initials: JD
authors.lastname: Bloom
authors.forename: Frances H
authors.initials: FH
authors.lastname: Arnold
mesh_terms: D000595:Amino Acid Sequence; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability; D006358:Hot Temperature; D007848:Laurates / Q000737:chemistry / Q000378:metabolism; D016015:Logistic Models; D008958:Models, Molecular; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D017510:Protein Folding; D011994:Recombinant Proteins; D011995:Recombination, Genetic / Q000235:genetics*; D016415:Sequence Alignment
chemical_list: D007848:Laurates; D011994:Recombinant Proteins; C425970:p-nitrophenoxydodecanoic acid; D003577:Cytochrome P-450 Enzyme System