title: LICRED: a versatile drop-in vector for rapid generation of redox-self-sufficient cytochrome P450s.
abstract: Cytochromes P450 (P450s) are a family of haem-containing oxidases with considerable potential as tools for industrial biocatalysis. Organismal genomes are revealing thousands of gene sequences that encode P450s of as yet unknown function, the exploitation of which will require high-throughput tools for their isolation and characterisation. In this report, a ligationindependent cloning vector "LICRED" is described that enables the high-throughput generation of libraries of redox-self-sufficient P450s by fusing a range of P450 haem domains to the reductase of P450RhF (RhF-Red) in a robust and generically applicable way. Cloning and expression of fusions of RhF-Red with the haem domains of P450cam and P450-XplA resulted in soluble, active, redox-self-sufficient, chimeric enzymes. In vitro studies also revealed that electron transfer from NADPH to haem was primarily intramolecular. The general applicability of the LICRED platform was then demonstrated through the creation of a library of RhF-Red fusion constructs by using the diverse complement of P450 haem domains identified in the genome of Nocardia farcinica. The resultant fusion-protein library was then screened against a panel of substrates; this revealed chimeric enzymes competent for the hydroxylation of testosterone and methyltestosterone, and the dealkylation of 7-ethoxycoumarin.
authors.affiliation: Centre for Novel Agricultural Products, Department of Biology, University of York, YO10 5YW York, UK.
authors.forename: Federico
authors.initials: F
authors.lastname: Sabbadin
authors.forename: Ralph
authors.initials: R
authors.lastname: Hyde
authors.forename: Aelig
authors.initials: A
authors.lastname: Robin
authors.forename: Eva-Maria
authors.initials: EM
authors.lastname: Hilgarth
authors.forename: Marie
authors.initials: M
authors.lastname: Delenne
authors.forename: Sabine
authors.initials: S
authors.lastname: Flitsch
authors.forename: Nicholas
authors.initials: N
authors.lastname: Turner
authors.forename: Gideon
authors.initials: G
authors.lastname: Grogan
authors.forename: Neil C
authors.initials: NC
authors.lastname: Bruce
mesh_terms: D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D004579:Electron Transport; D004798:Enzymes; D005822:Genetic Vectors; D006418:Heme / Q000737:chemistry; D007700:Kinetics; D009249:NADP / Q000737:chemistry; D010084:Oxidation-Reduction; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D004798:Enzymes; D011993:Recombinant Fusion Proteins; D006418:Heme; D009249:NADP; D003577:Cytochrome P-450 Enzyme System