title: The self-sufficient P450 RhF expressed in a whole cell system selectively catalyses the 5-hydroxylation of diclofenac.
abstract: P450 monooxygenases are able to catalyze the highly regio- and stereoselective oxidations of many organic molecules. However, the scale-up of such bio-oxidations remains challenging due to the often-low activity, level of expression and stability of P450 biocatalysts. Despite these challenges they are increasingly desirable as recombinant biocatalysts, particularly for the production of drug metabolites. Diclofenac is a widely used anti-inflammatory drug that is persistent in the environment along with the 4'- and 5-hydroxy metabolites. Here we have used the self-sufficient P450 RhF (CYP116B2) from Rhodococcus sp. in a whole cell system to reproducibly catalyze the highly regioselective oxidation of diclofenac to 5-hydroxydiclofenac. The product is a human metabolite and as such is an important standard for environmental and toxicological analysis. Furthermore, access to significant quantities of 5-hydroxydiclofenac has allowed us to demonstrate further oxidative degradation to the toxic quinoneimine product. Our studies demonstrate the potential for gram-scale production of human drug metabolites through recombinant whole cell biocatalysis.
authors.affiliation: Institute of Technical Biochemistry, University of Stuttgart, Stuttgart, Germany.
authors.forename: Jan M
authors.initials: JM
authors.lastname: Klenk
authors.affiliation: Institute of Technical Biochemistry, University of Stuttgart, Stuttgart, Germany.
authors.forename: Bernd A
authors.initials: BA
authors.lastname: Nebel
authors.affiliation: School of Chemistry, Manchester Institute of Biotechnology, The University of Manchester, Manchester, UK.
authors.forename: Joanne L
authors.initials: JL
authors.lastname: Porter
authors.affiliation: Cardiovascular and Metabolic Diseases DMPK, Innovative Medicines and Early Development Biotech Unit, AstraZeneca, Mölndal, Sweden.|Present address: Crop Science Division, Bayer AG, Monheim am Rhein, Germany.
authors.forename: Justyna K
authors.initials: JK
authors.lastname: Kulig
authors.affiliation: School of Chemistry, Manchester Institute of Biotechnology, The University of Manchester, Manchester, UK.
authors.forename: Shaneela A
authors.initials: SA
authors.lastname: Hussain
authors.affiliation: Institute of Technical Biochemistry, University of Stuttgart, Stuttgart, Germany.
authors.forename: Sven M
authors.initials: SM
authors.lastname: Richter
authors.affiliation: School of Chemistry, Manchester Institute of Biotechnology, The University of Manchester, Manchester, UK.
authors.forename: Michele
authors.initials: M
authors.lastname: Tavanti
authors.affiliation: School of Chemistry, Manchester Institute of Biotechnology, The University of Manchester, Manchester, UK.
authors.forename: Nicholas J
authors.initials: NJ
authors.lastname: Turner
authors.affiliation: Cardiovascular and Metabolic Diseases DMPK, Innovative Medicines and Early Development Biotech Unit, AstraZeneca, Mölndal, Sweden.
authors.forename: Martin A
authors.initials: MA
authors.lastname: Hayes
authors.affiliation: Institute of Technical Biochemistry, University of Stuttgart, Stuttgart, Germany.
authors.forename: Bernhard
authors.initials: B
authors.lastname: Hauer
authors.affiliation: School of Chemistry, Manchester Institute of Biotechnology, The University of Manchester, Manchester, UK.
authors.forename: Sabine L
authors.initials: SL
authors.lastname: Flitsch
mesh_terms: D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000737:chemistry*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001673:Biodegradation, Environmental; D001711:Biotransformation; D002384:Catalysis; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D004008:Diclofenac / Q000031:analogs & derivatives* / Q000737:chemistry; D005285:Fermentation; D006900:Hydroxylation; D010084:Oxidation-Reduction; D011809:Quinones / Q000737:chemistry; D012240:Rhodococcus / Q000201:enzymology*
chemical_list: D000894:Anti-Inflammatory Agents, Non-Steroidal; D001426:Bacterial Proteins; D011809:Quinones; D004008:Diclofenac; D003577:Cytochrome P-450 Enzyme System; C117715:5-hydroxydiclofenac
keywords: Biocatalysis; Cytochrome P450; Diclofenac; Drug metabolism; Fermentation; Regioselectivity