title: High throughput assay for cytochrome P450 BM3 for screening libraries of substrates and combinatorial mutants.
abstract: A rapid method for identifying compounds that are potential substrates for the drug metabolising enzyme cytochrome P450 is described. The strategy is based on the detection of a degradation product of NAD(P)H oxidation during substrate turnover by the enzyme expressed in Escherichia coli cells spontaneously lysed under the experimental conditions. The performance of the method has been tested on two known substrates of the wild-type cytochrome P450 BM3, arachidonic (AA) and lauric (LA) acids, and two substrates with environmental significance, the anionic surfactant sodium dodecyl sulfate (SDS), and the solvent 1,1,2,2-tetrachloroethane (TCE). The minimal background signal given from cells expressing cytochrome P450 BM3 in the absence of added substrate is only 3% of the signal in the presence of saturating substrate. Control experiments have proven that this method is specifically detecting NADPH oxidation by catalytic turnover of P450 BM3. The assay has been adapted to a microtitre plate format and used to screen a series of furazan derivatives as potential substrates. Three derivatives were identified as substrates. The method gave a significant different signal for two isomeric furazan derivatives. All results found on the cell lysate were verified and confirmed with the purified enzyme. This strategy opens the way to automated high throughput screening of NAD(P)H-linked enzymatic activity of molecules of pharmacological and biotechnological interest and libraries of random mutants of NAD(P)H-dependent biocatalysts.
authors.affiliation: Department of Biological Sciences, Biochemistry building, Imperial College of Science, Technology and Medicine, London SW7 2AY, UK.
authors.forename: Georgia Eleni
authors.initials: GE
authors.lastname: Tsotsou
authors.forename: Anthony Edward George
authors.initials: AE
authors.lastname: Cass
authors.forename: Gianfranco
authors.initials: G
authors.lastname: Gilardi
mesh_terms: D001426:Bacterial Proteins; D020650:Combinatorial Chemistry Techniques; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D009154:Mutation; D009249:NADP / Q000378:metabolism*; D009251:NADPH-Ferrihemoprotein Reductase; D010084:Oxidation-Reduction; D019151:Peptide Library
chemical_list: D001426:Bacterial Proteins; D019151:Peptide Library; D009249:NADP; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases