Short Communication Generation of the Human Metabolite Piceatannol from the Anticancer-Preventive Agent Resveratrol by Bacterial Cytochrome P450 BM3

In recent studies, the wild-type and mutant forms of cytochrome P450 (P450) BM3 (CYP102A1) from Bacillus megaterium were found to metabolize various drugs through reactions similar to those catalyzed by human P450 enzymes. Therefore, it was suggested that CYP102A1 can be used to produce large quantities of the metabolites of human P450-catalyzed reactions. trans-Resveratrol (3,4 ,5-trihydroxystilbene), an anticancer-preventive agent, is oxidized by human P450 1A2 to produce two major metabolites, piceatannol (3,5,3 ,4 -tetrahydroxystilbene) and another hydroxylated product. In this report, we show that the oxidation of transresveratrol, a human P450 1A2 substrate, is catalyzed by wild-type and a set of CYP102A1 mutants. One major hydroxylated product, piceatannol, was produced as a result of the hydroxylation reaction. Other hydroxylated products were not produced. Piceatannol formation was confirmed by high-performance liquid chromatography and gas chromatograph-mass spectrometry by comparing the metabolite with the authentic piceatannol compound. These results demonstrate that CYP102A1 mutants can be used to produce piceatannol, a human metabolite of resveratrol. Resveratrol (3,4 ,5-trihydroxystilbene) (Fig. 1) is a phytoalexin found in a wide variety of dietary sources including grapes, plums, and peanuts. It exhibits pleiotropic health-beneficial effects including antioxidant, anti-inflammatory, cardioprotective, and antitumor activities (Athar et al., 2007; Kundu and Surh, 2008; Pirola and Fröjdö, 2008). Currently, numerous preclinical findings suggest resveratrol as a promising part of nature’s arsenal for cancer prevention and treatment. As a potential anticancer agent, resveratrol has been shown to inhibit or retard the growth of various cancer cells in culture and implanted tumors in vivo. The compound significantly inhibits experimental tumorigenesis in a wide range of animal models. The biological activities of resveratrol are found to relate to its ability to modulate various targets and signaling pathways. Resveratrol exists as cisand trans-isomers. trans-Resveratrol is the preferred steric form and is relatively stable. Piceatannol (3,5,3 ,4 -tetrahydroxystilbene) (Fig. 1) is a polyphenol found in grapes and other plants. It is known as a protein kinase inhibitor that modifies multiple cellular targets, exerting immunosuppressive and antitumorigenic activities in several cell lines. Piceatannol has been shown to exert various pharmacological effects on immune and cancer cells (Kim et al., 2008b and references therein). In humans, piceatannol is produced as a major metabolite of resveratrol by CYP1B1 and CYP1A2 (Potter et al., 2002; Piver et al., 2004). In addition, the metabolism of trans-resveratrol into two major metabolites, piceatannol and another tetrahydroxystilbene, is catalyzed by recombinant human CYP1A1, CYP1A2, and CYP1B1 (Piver et al., 2004). It is also known that trans-resveratrol can inhibit reactions catalyzed by human CYP1A1 and CYP1A2 (Chun et al., 1999). If prodrugs are converted to biologically “active metabolites” by human liver cytochromes P450 (P450s) during the drug development process (Johnson et al., 2004), large quantities of the pure metabolites are required to understand the drug’s efficacy, toxic effect, and pharmacokinetics. Because the pure metabolites may be difficult to synthesize, an alternative is to use P450s to generate the metabolites of drugs or drug candidates. Although hepatic microsomes can be a source of human P450s, their limited availability makes their use in preparative-scale metabolite synthesis impractical. Some human enzymes can also be obtained by expression in recombinant hosts (Yun et al., 2006). Metabolite preparation has been demonstrated using human P450s expressed in Escherichia coli and in insect cells (Parikh et al., 1997; Rushmore et al., 2000; Vail et al., 2005), but these systems are costly and have low productivities due to limited stabilities and slow reaction rates (Guengerich et al., 1996). An alternative approach to preparing the human metabolites is to use an engineered bacterial P450 that has the desired catalytic activities. Several mutants of Bacillus megaterium P450 BM3 (CYP102A1) generated through rational design or directed evolution could oxidize several human P450 substrates to produce authentic metabolites with higher activities (Otey et al., 2005; Yun et al., 2007 and references therein; Kim et al., 2008a; Stjernschantz et al., 2008). These recent This work was supported in part by the 21C Frontier Microbial Genomics and the Application Center Program of the Ministry of Education, Science and Technology of the Republic of Korea; the Korea Science and Engineering Foundation [Grant R01-2008-000-21072-02008]; and the Second Stage BK21 Project from the Ministry of Education, Science and Technology of the Republic of Korea. Article, publication date, and citation information can be found at http://dmd.aspetjournals.org. doi:10.1124/dmd.108.026484. □S The online version of this article (available at http://dmd.aspetjournals.org) contains supplemental material. ABBREVIATIONS: P450, cytochrome P450; HPLC, high-performance liquid chromatography; TTNs, total turnover numbers; GC-MS, gas chromatograph-mass spectrometry. 0090-9556/09/3705-932–936$20.00 DRUG METABOLISM AND DISPOSITION Vol. 37, No. 5 Copyright © 2009 by The American Society for Pharmacology and Experimental Therapeutics 26484/3464889 DMD 37:932–936, 2009 Printed in U.S.A. 932 http://dmd.aspetjournals.org/content/suppl/2009/02/23/dmd.108.026484.DC1 Supplemental material to this article can be found at: at A PE T Jornals on Jauary 0, 2018 dm d.aspurnals.org D ow nladed from advances suggest that CYP102A1 can be developed as biocatalysts for drug discovery and synthesis (Bernhardt, 2006; Di Nardo et al., 2007). Very recently, it was reported that some selected mutations enabled the CYP102A1 enzyme to catalyze O-deethylation and 3-hydroxylation of 7-ethoxycoumarin, which are the same reactions catalyzed by human P450s (Kim et al., 2008a). In this study, we tested whether CYP102A1 mutants could be used to produce piceatannol, the major human metabolite of trans-resveratrol. Piceatannol is more expensive than trans-resveratrol, its substrate. Some of the tested mutations enabled the CYP102A1 enzyme to catalyze the hydroxylation of trans-resveratrol to generate its human metabolite, piceatannol. Materials and Methods Chemicals. trans-Resveratrol, piceatannol, and NADPH were purchased from Sigma-Aldrich (St. Louis, MO). Other chemicals were of the highest grade commercially available. Construction of BM3 Mutants by Site-Directed Mutagenesis. Seventeen different site-directed mutants of CYP102A1 were prepared as described previously (Kim et al., 2008a and references therein). The CYP102A1 mutants used in this study were selected based on earlier work showing their increased catalytic activity toward several human substrates. Each mutant bears the amino acid substitution(s) relative to wild-type CYP102A1, as summarized at Table 1 (Kim et al., 2008a). Expression and Purification of CYP102A1 Mutants. Wild-type and mutants of CYP102A1 were expressed in E. coli and purified as described previously (Kim et al., 2008a). The CYP102A1 concentrations were determined from CO-difference spectra as described by Omura and Sato (1964) using 91 mM/cm. For all of the wild-type and mutated enzymes, a typical culture yielded 300 to 700 nM P450. The expression level of CYP102A1 wild-type and mutants was typically in the range of 1.0 to 2.0 nmol P450/mg cytosolic protein. Hydroxylation of trans-Resveratrol. Typical steady-state reactions for the trans-resveratrol hydroxylation included 50 pmol of CYP102A1 in 0.25 ml of 100 mM potassium phosphate buffer (pH 7.4) containing a final concentration of 100 M trans-resveratrol. To determine the kinetic parameters of several CYP102A1 mutants, we used 2 to 500 M trans-resveratrol. An aliquot of a NADPH-generating system was used to initiate reactions (final concentrations: 10 mM glucose 6-phosphate, 0.5 mM NADP , and 1 IU yeast glucose 6-phosphate per ml). A stock solution of trans-resveratrol (20 mM) was prepared in dimethyl sulfoxide and diluted into the enzyme reactions with a final organic solvent concentration of 1% (v/v). The reaction mixtures (final volume of 0.25 ml) were incubated for 10 min at 37°C and terminated with 0.50 ml of ice-cold ethyl acetate. After centrifugation of the reaction mixture, organic phases were evaporated under a nitrogen gas. The product formation was analyzed by high-performance liquid chromatography (HPLC) as described previously (Piver et al., 2004). Samples (30 l) were injected onto a Gemini C18 column (4.6 mm 150 mm, 5 m; Phenomenex, Torrance, CA). The mobile phase A was water containing 0.5% acetic acid/acetonitrile (95:5, v/v), and the mobile phase B was acetonitrile/ 0.5% acetic acid (95:5, v/v); the mobile phase A/B (75:25, v/v) was delivered at a flow rate of 1 ml/min by a gradient pump (LC-20AD; Shimadzu, Kyoto, Japan). Eluates were detected by UV at 320 nm. To determine the total turnover numbers (TTNs) of several CYP102A1 mutants, 100 M trans-resveratrol was used. The reaction was initiated by the addition of the NADPH-generating system, incubated for 1 and 2 h at 30°C. The formation rate of piceatannol was determined by HPLC as described above. The kinetic parameters (Km and kcat) were determined using nonlinear regression analysis with GraphPad Prism software (GraphPad, Software Inc., San Diego, CA). The data were fit to the standard Michaelis-Menten equation: v kcat[E][S]/([S] Km), where the velocity of the reaction is a function of the turnover (kcat), which is the rate-limiting step, the enzyme concentration ([E]), substrate concentration ([S]), and the Michaelis constant (Km). Gas Chromatograph-Mass Spectrometry Analysis. For the identification of the trans-resveratrol metabolite produced by the CYP102A1 mutants, the gas chromatograph-mass spectrometry (GC-MS) analysis compared the GCprofile and fragmentation patterns of the authentic compounds, piceatannol and trans-resveratrol. The oxidation reaction of trans-resveratrol by CYP102A1 mutants was done as described above. The aqueous samples were extracted with ethyl acetate. After centrifugation, the organic phase, standard transresveratrol, and authentic piceatannol solution (10 mM in ethanol) were each dried under nitrogen. Then trimethylsilyl derivatives were prepared as follows: 100 l of a solution of N,O-bis-(trimethylsilyl)trifluoroacetamide/trimethylchlorosilane (99:1, v/v) (Supelco, Bellefonte, PA) was added to the dry residue, and the mixture was left for 60 min at 60°C. GC-MS analysis was performed on a GC-2010 gas chromatograph (Shimadzu) with an Rtx-5 column (5% diphenyl/95% dimethyl polysiloxane capillary column) (30 m 0.32 mm i.d. 0.25 m film thickness). The injector temperature was 250°C. The derivatized samples of resveratrol and piceatannol were separated by GC under GC oven conditions of 60°C for 5 min, followed by an increase of 50°C for min 1 up to 200°C, and then 2°C for min 1 to 300°C. The gas chromatography was combined with a GCMSQP2010 Shimazu mass spectrometer operating in electron ionization mode (70 eV) (Piver et al., 2004). Results and Discussion Oxidation of trans-Resveratrol by P450 BM3 Wild-Type and Its Mutants. We examined whether CYP102A1 can oxidize trans-resveratrol. First, the ability of wild-type and a set of P450 BM3 mutants to oxidize trans-resveratrol was measured at a fixed substrate concentration (100 M). The metabolites were analyzed by HPLC and compared with those of human CYP1A2 (Table 1; Fig. 2). Whereas human CYP1A2 oxidized resveratrol to produce two major metabolites, CYP102A1 mutants produced only one major metabolite. In the case of the human metabolites, one is piceatannol and the other is also hydroxylated product, as reported previously (Piver et al., 2004). Wild-type and catalytically active mutants of CYP102A1 produced only one major product, which had a retention time that exactly matched the piceatannol standard, but not the other hydroxylated product. The turnover numbers for the entire set of the 17 mutants for the trans-resveratrol oxidation (piceatannol formation) varied over a FIG. 1. Molecular structures of trans-resveratrol and piceatannol. The conversion of trans-resveratrol to piceatannol is catalyzed by P450 enzyme in the presence of NADPH. 933 OXIDATION OF RESVERATROL CATALYZED BY BACTERIAL P450 BM3 at A PE T Jornals on Jauary 0, 2018 dm d.aspurnals.org D ow nladed from