title: Evaluation of alkoxyresorufins as fluorescent substrates for cytochrome P450 BM3 and site-directed mutants.
abstract: In this study, the first fluorescent assay for bacterial cytochrome P450 BM3 (BM3) and mutants is described. BM3 mutants are potentially very versatile biocatalysts for the production of fine chemicals. A fluorescent assay would be very useful for the identification of nonnatural ligands in high-throughput inhibition assays. Because of the ease and sensitivity of alkoxyresorufin O-dealkylation assays, four different alkoxyresorufins were evaluated as substrates. Wild-type BM3 showed extremely low activity toward all four alkoxyresorufins tested. Five different BM3 mutants were constructed, carrying different combinations of mutations R47L, F87V, and L188Q, which were previously shown to increase activity toward nonnatural substrates. For all mutants, a high benzyloxyresorufin O-dealkylation (BROD) activity was found. The triple mutant of BM3, R47L/F87V/L188Q, showed the highest activity, increasing 900-fold compared to wild-type BM3. The BROD assay could also be applied in whole Escherichia coli cells; permeabilization by lipopolysaccharide deficiency strongly increased activity. To demonstrate the applicability of the BROD assay to screening for novel ligands of BM3 R47L/F87V/L188Q, a library of 45 drug-like compounds was tested for inhibition. Of these compounds, 8 showed strong inhibition of the BROD activity, demonstrating for the first time that drug-like molecules also can bind with high affinity to BM3 mutants.
authors.affiliation: LACDR/Division of Molecular Toxicology, Department of Pharmacochemistry, Vrije Universiteit, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands.
authors.forename: Barbara M A
authors.initials: BM
authors.lastname: Lussenburg
authors.forename: Lloyd C
authors.initials: LC
authors.lastname: Babel
authors.forename: Nico P E
authors.initials: NP
authors.lastname: Vermeulen
authors.forename: Jan N M
authors.initials: JN
authors.lastname: Commandeur
mesh_terms: D000478:Alkylation; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics*; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics*; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism*; D006899:Mixed Function Oxygenases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009251:NADPH-Ferrihemoprotein Reductase; D010078:Oxazines / Q000737:chemistry / Q000378:metabolism*; D013050:Spectrometry, Fluorescence / Q000379:methods; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D010078:Oxazines; C014180:resorufin; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases