Supplemental material to this article can be found at: http://dmd.aspetjournals.org/content/suppl/2016/03/16/dmd.115.069104.DC1 1521-009X/44/8/1270–1276$25.00 http://dx.doi.org/10.1124/dmd.115.069104 DRUG METABOLISM AND DISPOSITION Drug Metab Dispos 44:1270–1276, August 2016 Copyright ª 2016 by The American Society for Pharmacology and Experimental Therapeutics Special Section on Emerging Novel Enzyme Pathways in Drug Metabolism Microbial Flavoprotein Monooxygenases as Mimics of Mammalian Flavin-Containing Monooxygenases for the Enantioselective Preparation of Drug Metabolites s Turan Gul, Marzena Krzek, Hjalmar P. Permentier, Marco W. Fraaije, and Rainer Bischoff Analytical Biochemistry, Groningen Research Institute of Pharmacy (T.G., H.P.P., R.B.), Molecular Enzymology Group, Groningen Biomolecular Sciences and Biotechnology Institute (M.K., M.W.F.), and Interfaculty Mass Spectrometry Center (H.P.P.), University Downloaded from of Groningen, Groningen, the Netherlands Received December 21, 2015; accepted March 15, 2016 ABSTRACT Mammalian flavin-containing monooxygenases, which are difficult to sulfoxides are formed in good to excellent enantiomeric excess (e.e.)by dmd.aspetjournals.org obtain and study, play a major role in detoxifying various xenobiotics. To several of the tested monooxygenases. Intriguingly, depending on the provide alternative biocatalytic tools to generate flavin-containing mono- chosen microbial monooxygenase, either the (R)- or (S)-sulfoxide was oxygenases (FMO)–derived drug metabolites, a collection of microbial formed. For example, when using a monooxygenase from Rhodococcus flavoprotein monooxygenases, sequence-related to human FMOs, was jostii the (S)-sulfoxide of albendazole (ricobendazole) was obtained with a tested for their ability to oxidize a set of xenobiotic compounds. For all 95% e.e. whereas a fungal monooxygenase yielded the respective tested xenobiotics [nicotine, lidocaine, 3-(methylthio)aniline, albendazole, (R)-sulfoxide in 57% e.e. For nicotine and lidocaine, monooxygenases and fenbendazole], one or more monooxygenases were identified capable could be identified that convert the amines into their respective N-oxides. at ASPET Journals on September 30, 2021 of converting the target compound. Chiral liquid chromatography with This study shows that recombinantly expressed microbial monooxyge- tandem mass spectrometry analyses of the conversions of 3-(methylthio) nases represent a valuable toolbox of mammalian FMO mimics that can be aniline, albendazole, and fenbendazole revealed that the respective exploited for the production of FMO-associated xenobiotic metabolites. Introduction (Cashman and Zhang, 2006). FMOs have been shown to be involved in Metabolism of xenobiotics in humans and other mammals often starts the oxygenation of heteroatom-containing compounds, such as amines and with oxidation of the target molecule. Most of the phase I metabolism sulfides (Cashman, 2004; Krueger and Williams, 2005; Cashman and reactions are catalyzed by cytochrome P450 monooxygenases (P450) Zhang, 2006). Different from P450s, which contain a heme cofactor, FMOs (Cashman, 2005). However, in addition to P450s, recent studies have use a flavin cofactor for oxidations which also translates into a different shown that the so-called flavin-containing monooxygenases (FMOs) oxidative mechanism. Furthermore, to discriminate between metabolism by play a crucial role in the biotransformation of a large variety of xenobi- human FMOs or P450s, often differences in stability and specific inhibitors otics, including pharmaceuticals and natural products. can be used (Taniguchi-Takizawa et al., 2015). FMO enzymes require Mammals typically employ several FMO isoforms. The human pro- nicotinamide adenine dinucleotide phosphate (NADPH) for reducing the teome contains five isoforms, FMO1–FMO5, all of which have their flavin adenine dinucleotide (FAD) flavin cofactor. The reduced flavin typical tissue-dependent expression patterns and roles in metabolism subsequently reacts with molecular oxygen, resulting in the formation of a reactive 4a-hydroperoxyflavin. This reactive flavin intermediate is able to perform a variety of oxygenation reactions, for example, sulfoxidations and This work was supported by the Dutch Technology Foundation (STW) [Grant N-hydroxylations (see Ziegler 1990, 1993; Malito et al., 2004; Cashman, 11957], and the Dutch Foundation for Scientific Research (NWO, ECHO) [Grant 711.012.006]. 2005; Krueger and Williams, 2005; Cashman and Zhang, 2006; van Berkel T.G. and M.K. contributed equally to this work. et al., 2006 for mechanistic details). dx.doi.org/10.1124/dmd.115.069104. It has been established that human FMOs are essential in oxidizing a s This article has supplemental material available at dmd.aspetjournals.org. variety of xenobiotics, but biochemical and metabolic studies on these ABBREVIATIONS: BVMO, Baeyer-Villiger monooxygenase; BVMOMt1, Baeyer-Villiger monooxygenase from Myceliophthora thermophila; BVMORj, Baeyer-Villiger monooxygenase from Rhodoccocus jostii; CD, circular dichroism; CHMOAc, cyclohexanone monooxygenase from Acinetobacter calcoaceticus; e.e., enantiomeric excess; FAD, flavin adenine dinucleotide; FMO, flavin-containing monooxygenase; HPLC, high-pressure liquid chromatography; LC, liquid chromatography; LC-MS/MS, liquid chromatography with tandem mass spectrometry; NADPH, nicotinamide adenine dinucleotide phosphate; OD, optical density; P450, cytochrome P450; PAMO, phenylacetone monooxygenase; PAMOM446G¸ phenylacetone monooxygenase Met446Gly mutant; SPE, solid phase extraction; SRM, selected reaction monitoring. 1270 Microbial Monooxygenases for Drug Metabolite Synthesis 1271 enzymes are hampered by their poor availability. Human FMOs and their Recombinant Expression of BVMOs and Preparation of Cell Extracts. mammalian orthologs are typically membrane associated and often ther- The enzymes were overexpressed in Escherichia coli using previously molabile, which appear to be the major reasons for their problematic established conditions and protocols. Cyclohexanone monooxygenase from Acinetobacter calcoaceticus isolation from tissue (Cashman et al., 1995; Wu et al., 2004) and inefficient (CHMOAc), the phenylacetone monooxygenase Met446Gly mutant (PAMO ), and BVMO were expressed using the recombinant production. Although human FMOs can be studied using M446G Rj24 pCRE2 expression vector (Torres Pazmiño et al., 2009), yielding the enzyme microsomal preparations and some human FMOs were expressed as func- fused to His-tagged phosphite dehydrogenase, which facilitates cofactor tional enzymes in heterologous hosts (Motika et al., 2009; Balke et al., regeneration. His-tagged PAMO and Strep-tagged FMORjE were expressed 2012; Geier et al., 2015; Shimizu et al., 2015), these enzyme preparations as described previously elsewhere (Fraaije et al., 2005). For expressing involve costly and cumbersome isolation procedures, and often suffer BVMOMt1, a pET_SUMO vector was used. Precultures were grown overnight from low activity and stability (Cashman et al., 1992; Cashman and at 37°C with shaking (180 rpm) in lysogeny broth medium containing m Zhang, 2006). ampicillin (50 g/ml). The exception was BVMOMt1 for which cells were Sequence comparison studies have revealed that FMOs are part of a grown in the presence of kanamycin (100 mg/ml). large family of monooxygenases, the so-called class B flavoprotein Flasks containing 200 ml of terrific broth medium with the respective antibiotic monooxygenases (Reddy et al., 2010). Intriguingly, many bacteria and were inoculated 1:100 (v/v) using the preculture and grown for another 4 hours at 30°C. After that, each flask was supplemented with inducer: 1.0 mM isopropyl fungi contain sequence-related class B flavin-containing monooxygenases b-D-thiogalactopyranoside for BVMO , and 0.02% arabinose for PAMO, (Mascotti et al., 2015), which are typically involved in catalyzing Baeyer- Mt1 PAMO , and CHMO , and 0.002% arabinose for the remaining enzymes. Villiger oxidations to form a subfamily of Baeyer-Villiger monooxy- M446G Ac After 48 hours of growth at 24°C with shaking (130 rpm), the cells were harvested Downloaded from genases (BVMOs). Biocatalytic studies on these microbial monooxygenases by centrifugation at 4°C, 17,000g and resuspended in 50 mM Tris buffer, pH 8.0. confirmed that they employ the same catalytic mechanism as FMOs The cells were diluted to an optical density of 600 nm (OD600) of 212 for (Torres Pazmiño et al., 2008); interestingly, they also can catalyze ox- terrific broth samples and an OD600 of 98 for lysogeny broth samples. Subse- ygenations of heteroatom-containing compounds. In contrast to human quently, the cells were disrupted by sonication for 90 seconds using 2-second FMOs, many microbial BVMOs are soluble enzymes and can be easily sonication pulses and 2-second breaks, while on ice. produced in recombinant form (de Gonzalo et al., 2010). The prepared cell extracts were supplemented with glycerol (15%), aliquoted Inspired by the observation that microbial BVMOs are sequence-related (100 ml in Eppendorf tubes), frozen in liquid nitrogen, and stored at 280°C. As dmd.aspetjournals.org to human FMOs and exhibit similar activities, we set out to explore the negative control, E. coli cells were grown without expression plasmid and their use as mammalian FMO mimics. By testing a panel of xenobi- used for the preparation of cell extract as described earlier. Overexpression of the otic compounds, including drug molecules, with a collection