Journal
BIOORGANIC & MEDICINAL CHEMISTRY
Volume 23, Issue 17, Pages 5603-5609Publisher
PERGAMON-ELSEVIER SCIENCE LTD
DOI: 10.1016/j.bmc.2015.07.025
Keywords
P450; Biotransformation; P450Rhf; Imipramine; N-demethylation; Drug metabolism; Diltiazem
Funding
- European Union FP7 People Inititiative as the Marie-Curie International Training Network [P4FIFTY 289217]
- Industrial Affiliates of the Centre of Excellence for Biocatalysis, Biotransformation and Biomanufacture (CoEBio3)
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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 P450(Rhf) 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 mu M) giving a 63% conversion to the N-desmethyl product. (C) 2015 Elsevier Ltd. All rights reserved.
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