Drug Oxidation by Cytochrome P450_BM3: Metabolite Synthesis and Discovering New P450 Reaction Types

Abstract

There is intense interest in late-stage catalytic C-H bond functionalization as an integral part of synthesis. Effective catalysts must have a broad substrate range and tolerate diverse functional groups. Drug molecules provide a good test of these attributes of a catalyst. A library of P450BM3 mutants developed from four base mutants with high activity for hydrocarbon oxidation produced human metabolites of a panel of drugs that included neutral (chlorzoxazone, testosterone), cationic (amitriptyline, lidocaine) and anionic (diclofenac, naproxen) compounds. No single mutant was active for all the tested drugs but multiple variants in the library showed high activity with each compound. The high conversions enabled full product characterization that led to the discovery of the new P450 reaction type of oxidative decarboxylation of an α-hydroxy carboxylic acid and the formation a protected imine from an amine, offering a novel route to α-functionalization of amines. The substrate range and varied product profiles suggest that this library of enzymes is a good basis for developing late-stage C-H activation catalysts.

CYP267A1 and CYP267B1 from Sorangium cellulosum So ce56 are Highly Versatile Drug Metabolizers

“…This progress can directly be of use for efficient and timesaving production of human drug metabolites. High yields and conversion rates can already be achieved by using corresponding human (Rushmore et al, 2000;Vail et al, 2005;Schroer et al, 2010;Geier et al, 2012;Schiffer et al, 2015) or suitable nonhuman (Taylor et al, 1999;Otey et al, 2006;Sawayama et al, 2009;Reinen et al, 2011;Di Nardo and Gilardi, 2012;Kiss et al, 2015;Ren et al, 2015) P450 enzymes in a whole-cell system to produce respective metabolites. The majority of published bacterial P450 enzymes used for the conversion of drugs are mutants of CYP102A1 (BM3) from Bacillus megaterium.…”

“…Since it is not mandatory to employ associated human P450 enzymes to synthesize human drug metabolites (Schroer et al, 2010;Geier et al, 2015), microbial, especially bacterial, P450 enzymes serve as a good alternative because they are convenient to handle and usually hold higher expression levels and activities, recommending the possibility to employ them as useful biocatalysts (Bernhardt, 2006). The genetic manipulation of bacterial P450 enzymes toward a drug metabolizing activity has been successfully demonstrated for several P450 enzymes including the most studied P450 102A1 (BM3), CYP102A1 (Whitehouse et al, 2012;Ren et al, 2015). However, the engineering of enzymes against their native, narrow substrate range, or in general the screening for enzymes to produce certain drug metabolites, is time consuming and complex, which can be overcome by employing versatile wild-type P450 enzymes showing an untypically broad substrate range (Yin et al, 2014).…”

Human Cytochrome P450 3A4 as a Biocatalyst: Effects of the Engineered Linker in Modulation of Coupling Efficiency in 3A4-BMR Chimeras

“…This has led to a wide range of protein engineering studies on this enzyme to widen its catalytic abilities (Tsotsou et al, 2012, 2013; Ryu et al, 2014; Di Nardo et al, 2015; Ren et al, 2015; Capoferri et al, 2016). …”

Steroids hydroxylation catalyzed by the monooxygenase mutant 139-3 from Bacillus megaterium BM3

“…BM3 is a soluble and stable fusion between a catalytic domain involved in substrate oxidation and a diflavin reductase domain responsible for electron transport, thereby making the electron transfer very efficient. These fine properties, together with the ease of over-expression in Escherichia coli ( E. coli ) made BM3 a promising candidate for the biocatalysis3, 4, 5. Therefore, BM3 was studied extensively and many BM3 mutants with broader substrate range and altered region- and stereo-selectivities were generated by laboratory evolution6, 7, 8.…”