title: Engineering substrate recognition in catalysis by cytochrome P450cam.
abstract: We have a continuing interest in applying the current knowledge of cytochrome P450cam substrate recognition to engineer the enzyme for the biotransformation of unnatural substrates with the long-term aim of applications in the synthesis of fine chemicals and bioremediation of environmental contaminants. Comparisons of the structure of target substrates with that of camphor, the natural substrate, led to the design of active-site mutants with greatly enhanced activity for the oxidation of chlorinated benzenes and selectivity of (+)-alpha-pinene oxidation. The crystal structures of the F87W/Y96F/V247L mutant with 1,3,5-trichlorobenzene or (+)-alpha-pinene bound have revealed the enzyme-substrate contacts and provided insights into the activity and selectivity patterns. The structures have also provided a novel basis for further engineering of P450cam for increased activity in the oxidation of the highly inert pentachlorobenzene and hexachlorobenzene, and increased selectivity of (+)-alpha-pinene oxidation.
authors.affiliation: Department of Chemistry, Inorganic Chemistry Laboratory, University of Oxford, South Parks Road, Oxford OX1 3QR, UK.
authors.forename: S G
authors.initials: SG
authors.lastname: Bell
authors.forename: X
authors.initials: X
authors.lastname: Chen
authors.forename: F
authors.initials: F
authors.lastname: Xu
authors.forename: Z
authors.initials: Z
authors.lastname: Rao
authors.forename: L-L
authors.initials: LL
authors.lastname: Wong
mesh_terms: D019943:Amino Acid Substitution; D019475:Camphor 5-Monooxygenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D018360:Crystallography, X-Ray; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D011994:Recombinant Proteins; D019475:Camphor 5-Monooxygenase