title: Functional evolution and structural conservation in chimeric cytochromes p450: calibrating a structure-guided approach.
abstract: Recombination generates chimeric proteins whose ability to fold depends on minimizing structural perturbations that result when portions of the sequence are inherited from different parents. These chimeric sequences can display functional properties characteristic of the parents or acquire entirely new functions. Seventeen chimeras were generated from two CYP102 members of the functionally diverse cytochrome p450 family. Chimeras predicted to have limited structural disruption, as defined by the SCHEMA algorithm, displayed CO binding spectra characteristic of folded p450s. Even this small population exhibited significant functional diversity: chimeras displayed altered substrate specificities, a wide range in thermostabilities, up to a 40-fold increase in peroxidase activity, and ability to hydroxylate a substrate toward which neither parent heme domain shows detectable activity. These results suggest that SCHEMA-guided recombination can be used to generate diverse p450s for exploring function evolution within the p450 structural framework.
authors.affiliation: Biochemistry and Molecular Biophysics Option, Mail Code 210-41, Pasadena, CA 91125, USA.
authors.forename: Christopher R
authors.initials: CR
authors.lastname: Otey
authors.forename: Jonathan J
authors.initials: JJ
authors.lastname: Silberg
authors.forename: Christopher A
authors.initials: CA
authors.lastname: Voigt
authors.forename: Jeffrey B
authors.initials: JB
authors.lastname: Endelman
authors.forename: Geethani
authors.initials: G
authors.lastname: Bandara
authors.forename: Frances H
authors.initials: FH
authors.lastname: Arnold
mesh_terms: D001407:Bacillus / Q000737:chemistry / Q000201:enzymology / Q000235:genetics; D002138:Calibration; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability; D019143:Evolution, Molecular; D015723:Gene Library; D006418:Heme / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009195:Peroxidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D040681:Structural Homology, Protein; D013696:Temperature
chemical_list: D011993:Recombinant Fusion Proteins; D006418:Heme; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D010088:Oxidoreductases; D009195:Peroxidase; C022333:peroxygenase