title: A P450 fusion library of heme domains from Rhodococcus jostii RHA1 and its evaluation for the biotransformation of drug molecules.
abstract: The actinomycete Rhodococcus jostii RHA1 contains a multitude of oxygenase enzymes, consonant with its remarkable activities in the catabolism of hydrophobic xenobiotic compounds. In the interests of identifying activities for the transformation of drug molecules, we have cloned genes encoding 23 cytochrome P450 heme domains from R. jostii and expressed them as fusions with the P450 reductase domain (RhfRED) of cytochrome P450Rhf from Rhodococcus sp. NCIMB 9784. Fifteen of the fusions were expressed in the soluble fraction of Escherichia coli Rosetta (DE3) cells. Strains expressing the fusions of RhfRED with genes ro02604, ro04667, ro11069, ro11320, ro11277, ro08984 and ro04671 were challenged with 48 commercially available drugs revealing many different activities commensurate with P450-catalyzed hydroxylation and demethylation reactions. One recombinant strain, expressing the fusion of P450 gene ro11069 (CYP257A1) with RhfRED, and named Ro07-RhfRED, catalyzed the N-demethylation of diltiazem and imipramine. This observation was in accord with previous reports of this enzyme's activity as a demethylase of alkaloid substrates. Ro07-RhfRED was purified and characterised, and applied in cell-free biotransformations of imipramine (7 μM) giving a 63% conversion to the N-desmethyl product.
authors.affiliation: CVMD iMed DMPK, AstraZeneca R&D Mölndal, 431 83 Mölndal, Sweden.
authors.forename: Justyna K
authors.initials: JK
authors.lastname: Kulig
authors.affiliation: York Structural Biology Laboratory, Department of Chemistry, University of York, YO10 5DD York, United Kingdom.
authors.forename: Claudia
authors.initials: C
authors.lastname: Spandolf
authors.affiliation: York Structural Biology Laboratory, Department of Chemistry, University of York, YO10 5DD York, United Kingdom.
authors.forename: Ralph
authors.initials: R
authors.lastname: Hyde
authors.affiliation: Department of Microbiology and Immunology, 2350 Health Sciences Mall, Life Sciences Centre, University of British Columbia, V6T 1Z3 Vancouver, BC, Canada.
authors.forename: Antonio C
authors.initials: AC
authors.lastname: Ruzzini
authors.affiliation: Department of Microbiology and Immunology, 2350 Health Sciences Mall, Life Sciences Centre, University of British Columbia, V6T 1Z3 Vancouver, BC, Canada.
authors.forename: Lindsay D
authors.initials: LD
authors.lastname: Eltis
authors.affiliation: RIA iMed MedChem, AstraZeneca R&D Mölndal, 431 83 Mölndal, Sweden.
authors.forename: Gunnar
authors.initials: G
authors.lastname: Grönberg
authors.affiliation: CVMD iMed DMPK, AstraZeneca R&D Mölndal, 431 83 Mölndal, Sweden.
authors.forename: Martin A
authors.initials: MA
authors.lastname: Hayes
authors.affiliation: York Structural Biology Laboratory, Department of Chemistry, University of York, YO10 5DD York, United Kingdom. Electronic address: gideon.grogan@york.ac.uk.
authors.forename: Gideon
authors.initials: G
authors.lastname: Grogan
mesh_terms: D055162:Biocatalysis; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D015723:Gene Library; D006418:Heme / Q000378:metabolism*; D015394:Molecular Structure; D012240:Rhodococcus / Q000378:metabolism*
chemical_list: D006418:Heme; D003577:Cytochrome P-450 Enzyme System
keywords: Biotransformation; Diltiazem; Drug metabolism; Imipramine; N-demethylation; P450; P450Rhf