Evaluation of coumarin-based fluorogenic P450 BM3 substrates and prospects for competitive inhibition screenings

Fluorescence-based assays for the cytochrome P450 BM3 monooxygenase from Bacillus megaterium address an attractive biotechnological challenge by facilitating enzyme engineering and the identification of potential substrates of this highly promising biocatalyst. In the current study, we used the scarcity of corresponding screening systems as an opportunity to evaluate a novel and continuous high-throughput assay for this unique enzyme. A set of nine catalytically diverse P450 BM3 variants was constructed and tested toward the native substrate-inspired fluorogenic substrate 12-(4-trifluoromethylcoumarin-7-yloxy)dodecanoic acid. Particularly high enzyme-mediated O-dealkylation yielding the fluorescent product 7-hydroxy-4-trifluoromethylcoumarin was observed with mutants containing the F87V substitution, with A74G/F87V showing the highest catalytic efficiency (0.458 min⁻¹ μM⁻¹). To simplify the assay procedure and show its versatility, different modes of application were successfully demonstrated, including (i) the direct use of NADPH or its oxidized form NADP⁺ along with diverse NADPH recycling systems for electron supply, (ii) the use of cell-free lysates and whole-cell preparations as the biocatalyst source, and (iii) its use for competitive inhibition screens to identify or characterize substrates and inhibitors. A detailed comparison with known, fluorescence-based P450 BM3 assays finally emphasizes the relevance of our contribution to the ongoing research.