Expanding the Biocatalytic Toolbox: A Singular P450 Enzyme Efficiently Produces Diverse Pharmaceutical Metabolites

Abstract

P450 BM3 is a natural fusion protein with a P450 domain fused to its necessary redox partner and capable of binding a range of fatty acids at high catalytic rates. The flexibility of the active site and catalytic efficiency have led to extensive research aimed at modifying substrate and product profiles for its utilization as a biocatalyst. The introduction of a double mutation (DM) at the base of the active site (A82F/F87V) allows the binding of a broad range of structurally diverse small molecules, including pharmaceutical drugs. Herein, we describe screening with an FDA-approved drug compound library, and the results exhibit the promiscuous nature of the DM BM3 variant. Of the 978 compounds screened; 59% of the library elicited UV–vis spectral shifts indicative of binding with a range of structurally diverse drugs. The interaction of the DM BM3 variant with a range of structurally diverse drugs, with a particular focus on the antidiabetic glitazone (thiazolidinedione) class, was investigated to observe ligand binding modes and metabolite production. BM3-derived metabolites were successfully produced and structurally elucidated for several structurally diverse drugs in the steroid, glitazone, meglitinide, terpene lactone, retinoid, and fibrate classes. The range of modifications performed includes some that are human P450-derived metabolites. Structural elucidation of the troglitazone-bound DM variant P450 domain shows an unusually close crystallographic interaction between the substrate and heme cofactor. We report here the use of the DM BM3 variant as a model for the main human metabolizing P450 enzymes CYP1A1, CYP2B6, CYP2C8, CYP2C9, and CYP3A4, as well as a biotechnological tool for the production of metabolites with diverse functionality.