Introduction

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Flavin-containing monooxygenase (FMO,³ EC 1.14.13.8) is a family of xenobiotic-metabolizing enzymes that catalyze the oxygenation of a wide variety of xenobiotics that contain a soft nucleophile, most commonly nitrogen and sulfur (Ziegler, 1993; Cashman, 1995).

Mammals express five FMOs that are developmentally regulated in a sex-, tissue-, and species-specific manner (Hines et al., 1994; Lawton et al., 1994; Falls et al., 1995, 1997; Lee et al., 1995; Shehin-Johnson et al., 1995; Dolphin et al., 1996; Larsen-Su et al., 1999). A sixth isoform (FMO6) has been putatively identified between FMO2 and FMO3 on human chromosome 1, from genomic DNA (ENTREZ accession AL021026, submitted by the Sanger Center); the gene is constitutively expressed but it is not known if functional protein is produced (Shephard et al., 1999).

From its initial discovery as a unique form of FMO in rabbit lung (Williams et al., 1984; Tynes et al., 1985), FMO2 has been identified as a major pulmonary isoform in a number of species including guinea pig, mouse, and monkey (Lawton et al., 1990; Nikbakht et al., 1992; Yueh et al., 1997). Although the FMO2 isoform can metabolize many general substrates of FMO, it is inactive toward certain tertiary amines such as imipramine and chlorpromazine that are metabolized by FMO1 (Williams et al., 1984). In addition, FMO2 is capable of N-oxygenation of some primary alkylamines (Poulsen et al., 1986; Tynes et al., 1986) and exhibits isoform-specific stereoselectivity in S-oxygenation of alkyl-substituted p-tolyl sulfides (Rettie et al., 1995). We previously reported that rabbit FMO2 is developmentally regulated by age and pregnancy and that FMO2 levels were correlated with plasma concentration of progesterone and corticosterone at late gestation and early postpartum, respectively (Lee et al., 1995; Larsen-Su et al., 1999), indicating possible hormonal regulation. Elucidation of the 5' regulatory region of rabbit FMO2 suggests that tissue-specific transcription factors may regulate lung-specific expression of FMO2 (Shehin-Johnson et al., 1996).

Characterization of pulmonary FMO is of interest because of its potential role in the metabolism of environmental chemicals for which lung is a target organ or portal of entry. Early attempts by our laboratory to detect FMO2 protein (by Western analysis and enzyme assay) from human lung microsomes were not successful (unpublished). Therefore, we focused instead on a close human relative, the rhesus monkey lung FMO2 (mFMO2), for the study of pulmonary FMO2. We previously cloned mFMO2 from a rhesus monkey lung cDNA library. Positive identification as an ortholog of rabbit FMO2 (rFMO2) was made by cDNA comparison, immunochemical analysis of protein, and FMO substrate specificity (Yueh et al., 1997). Recently, human FMO2 (hFMO2) cDNA was isolated and characterized (Dolphin et al., 1998). Their sequence analysis indicated that the major allele encodes a polypeptide that lacks 64 amino acid residues, resulting from a C to T amber mutation at codon 472 (FMO2*2A), compared with the FMO2 ortholog in other mammals. The C allele (FMO2*1), which encodes full-length protein (FMO2.1), occurs in 26% of African-Americans, but it is absent in Caucasians and Asian-Americans (Whetstine et al., 2000).

In this study, we established a heterologous expression system for production of functional mFMO2-535 protein, and then went on to create a truncated mFMO2 (mFMO2-471) analogous to FMO2*2A, by site-directed mutagenesis. Expressed mFMO2-535 resembled the rabbit ortholog immunochemically; however, in-depth characterization of mFMO2-535 using an assay for methimazole-dependent nitro-5-thiobenzoate (TNB) oxidation identified a number of differences compared with rFMO2-535. While mFMO2-471 was immunochemically detectable, our results confirm that the C terminus is essential for enzyme stability and detectable catalytic activity.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Triton N101, tergitol NP9, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) (catalog no. C3023, C5849), blue dextran, hydroxyapatite, flavin adenine dinucleotide (FAD), methimazole, NADPH, isocitrate, isocitrate dehydrogenase, EDTA, [UL-¹⁴C]N,N'-dimethylaniline (specific activity, 6.2 µCi/µmol) (DMA), 5,5-dithiobis-(2-nitrobenzoate) (DTNB), dithiothreitol, and phenylmethylsulfonyl fluoride (PMSF) were purchased from Sigma (St. Louis, MO). (S)-5[³H]Nicotine (32 Ci/mmol) was a gift from Dr. Mark Shigenaga (University of California, San Francisco, CA). Restriction endonucleases and T4 DNA ligase were from New England Biolabs (Beverly, MA). Taq polymerase, Expand High Fidelity enzyme, buffers, and 2'-deoxynucleoside-5'-triphosphate (dNTP) mixture for PCR were from Roche Molecular Biochemicals (Indianapolis, IN). DH5, and the components of the Bac-to-Bac baculovirus expression system, including Max Efficiency DH10bac competent cells, Spodoptera frugiperda (Sf9) insect cells, Sf-900 II serum-free medium, cellfectin reagent, bluo-gal, gentamicin, and Puc/M13 primers, were obtained from Life Technologies (Gaithersburg, MD). Acrylamide, nitrocellulose, and horseradish peroxidase-conjugated secondary antibodies for Western blotting were purchased from Bio-Rad (Richmond, CA). Geneclean II was from BIO 101 Inc. (La Jolla, CA). Hyperfilm MP and enhanced chemiluminescence kit were from Amersham Pharmacia Biotech (Arlington Heights, IL). Oligonucleotides for PCR were synthesized by the Center for Gene Research and Biotechnology (Oregon State University). PCR was performed in a 96-well Robocycler (Stratagene, La Jolla, CA). FAD determinations were made on a PerSeptive Biosystems (Framingham, MA) CytoFluor Series 4000 fluorescent plate reader.

Cloning of mFMO2. The Bac-to-Bac baculovirus expression system was used to express mFMO2-535 cDNA obtained from plasmid pBK-CMV-FMO2, a positive clone from cDNA library screening (Yueh et al., 1997). Plasmid DNA was digested with EcoRI/XbaI to yield a 1.6-kbp full-length cDNA, which was subsequently ligated to EcoRI/XbaI cut pFastBac1, placing the mFMO2-535 cDNA downstream of a baculovirus promoter derived from the polyhedrin gene. Recombinant plasmid was isolated and used to transform DH10Bac Escherichia coli cells, which contain the baculovirus shuttle vector (bacmid) with a mini-att TN7 target site, as well as the helper plasmid that transposes the gene insert from pFastBac1 together with flanking baculovirus sequences into the bacmid. White recombinant bacmid colonies were visualized on LB agar plates containing the chromogenic substrate bluo-gal. High molecular weight DNA was prepared from recombinant bacmid clones. PCR was performed with PUC/M13 primers using DNA from recombinant colonies as template, and PCR products were visualized by UV light subsequent to electrophoresis through a 1% agarose gel (0.5 µg/ml of ethidium bromide, 40 mM Tris acetate, 2 mM EDTA), to confirm transposition of the clone into the bacmid.

Cloning of rFMO2. We worked with a rabbit cDNA clone (provided by R. N. Hines, The Medical College of Wisconsin, Milwaukee, WI) initially cloned in pUC19. We originally re-engineered this clone so that the ATG start site was embedded in an NcoI restriction site, for use in an E. coli expression vector, pET-30a. This was accomplished by PCR-directed mutagenesis with a forward primer (5'-GGT ACC CCC ATG GCA AAG AAG GTG GCA GT) that created the NcoI site and a reverse primer (5'-CGG ATC CTT AGA ACC ATT GC) that created a BamHI site downstream of the stop codon. PCR conditions were as follows: 99°C denaturation for 13 s, 54°C annealing for 45 s, and 68°C extension for 3, 5, and 7 min for cycles 1 to 10, 11 to 20, and 21 to 30, respectively. Reactions contained Expand High Fidelity polymerase (0.26 units/10 µl), magnesium (1.5 mM), dNTPs (10 mM, each), primers (0.3 µM), and template DNA (2.5 ng/10 µl) in 1× High Fidelity buffer. The PCR fragment was digested with NcoI/BamHI and was cloned into pCR2.1 (Invitrogen, San Diego, CA) in DH5, and was subsequently sequenced. For baculovirus expression, the clone was digested with NcoI/HindIII and was gel purified.

Viral Preparation and Protein Expression. Sf9 insect cells were routinely maintained in Sf-900II serum-free medium containing 10 mg/liter gentamicin in a 500-ml sterile shaker flask at 27°C on an orbital shaker (125 rpm). Sf9 cells (1 × 10⁶) were seeded into 35-mm wells of a 6-well plate with 2 ml of medium/well. Then they were transfected with recombinant bacmid (mFMO2-535, mFMO2-471, and rFMO2-535) via carrier cellfectin reagent according to the manufacturer's protocol (Life Technologies). Recombinant baculovirus was harvested at 72 h post-transfection and was amplified (1-2 ml of primary virus/100 ml) in Sf9 cells in log phase (2 × 10⁶ cells/ml) to prepare high titer virus stocks. After 3 to 7 days, cells were pelleted to remove debris, and the viral supernatant was collected. Sixty milliliters of secondary virus was used to infect 600 ml of Sf9 cells in suspension cultures (2 × 10⁶ cells/ml). FAD was supplemented (10 µg/ml) for expression. Insect cells were harvested at 96 h postinfection, washed with buffer containing 20 mM potassium phosphate (pH 7.4) and 137 mM sodium chloride, repelleted, and stored at 80°C until further use.

Protein Isolation and Western Analysis of Expressed Proteins. Microsomes were prepared from the insect cell pellets (Guengerich, 1989). Pellets were resuspended in 10 mM potassium phosphate (pH 7.6), 20% glycerol, 1 mM EDTA, and 0.4 mM PMSF (buffer A) and homogenized with a motor-driven Teflon-tipped pestle on ice. The suspension was centrifuged at 1000g for 10 min at 4°C to remove large cellular debris. The balance of the supernatant was centrifuged at 100,000g for 80 min at 4°C to pellet the microsomes. The cytosol was removed and an aliquot saved, while the pellets were thoroughly resuspended in approximately 3 pellet-volumes of buffer A.

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Generation of Polyclonal Antibody. Custom-made polyclonal rabbit anti-monkey FMO2 antibody was produced (HTI Bio-Products, Ramone, CA) in two rabbits from purified expressed mFMO2-535. The standard 70-day protocol includes one preimmunization serum collection, four immunizations, and three serum collections per rabbit. The last two collections of antiserum were shipped for testing. The antiserum was used without purification in hybridization buffer (1:10,000) consisting of 20 mM potassium phosphate (pH 7.4), 137 mM sodium chloride, 0.05% Tween 20, and 2.0% bovine serum albumin to detect expressed mFMO2-535, mFMO2-471, and rFMO2-535.

FMO Catalytic Activity. N-Oxidation was determined for expressed FMO2 monkey protein (one batch) and for purified rabbit lung FMO2 with [¹⁴C]DMA (Lee et al., 1993) and [³H](S)-nicotine (Williams et al., 1990) as substrates. N-Oxygenation of [¹⁴C]DMA was determined by HPLC following incubation of microsomes (0.1 mg) or purified FMO (20 µg) in buffer with [¹⁴C]DMA and an NADPH-generating system in a total volume of 0.1 ml. An aliquot of supernatant from terminated reactions was injected onto an HPLC column (Whatman RAC II partisil 5 ODS, 3 µm, 4.6 × 100 mm; Whatman, Clifton, NJ). Products were eluted with 62% methanol at a flow rate of 0.5 ml/min, which resolved the DMA-N-oxide and N-methylaniline metabolites from DMA. Eight assays were run for each sample with DMA concentrations of 0.03 to 0.3 mM. The actual DMA concentration was determined for each reaction by liquid scintillation counting (LS6500, Beckman Coulter, Fullerton, CA) an aliquot of the supernatant. These concentrations were used to determine the apparent Michaelis-Menten constant (K_m) and maximal rate of metabolism (V_max) from Lineweaver-Burke plots.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Identification of Expressed FMO2. On average we recovered approximately 7.5 mg of microsomal protein from 100 ml of infected insect cells (range was from 3-14 mg per 100 ml). Western analysis of cytosolic and microsomal proteins demonstrated that expressed mFMO2-535, mFMO2-471, and rFMO2-535 were correctly targeted to the membrane fraction; mFMO2-535 and rFMO2-535 migrate with the same apparent molecular weight under denaturing polyacrylamide electrophoresis conditions (data not shown). Each of our FMO2 antibodies (anti-monkey and anti-rabbit) was able to detect FMO2 proteins from the other species. Western blots of the microsomal proteins demonstrated efficient expression of FMO2 for all clones (Fig. 1; Table 1) with FMO content of some preparations as high as 2 nmol of FMO/mg of microsomal protein (12%). A control vector-bacmid viral infection did not produce any cross-reacting protein (data not shown).

View larger version (27K): [in this window] [in a new window]   Fig. 1.   Western blots of microsomes from expressed monkey and rabbit baculovirus clones. A, lanes 1 to 4 were loaded with 1.9, 1.1, 0.56, and 0.28 pmol of expressed purified mFMO2-535, respectively; lanes 5 and 6 were loaded with 0.5 µg of microsomes from two separate batches of expressed mFMO2-535; lanes 7 and 8 contained 2.0 µg of microsomes from two separate batches of expressed mFMO2-471. B, lanes 1 to 4 were loaded with 5.0, 3.0, 1.5, and 0.75 pmol of purified rFMO2-535 from rabbit lung, respectively; lanes 5 and 6 contained 1.0 µg of microsomes from two separate batches of expressed rFMO2-535.

Enzyme Activity. As was previously observed (Williams et al., 1990) (S)-nicotine is a relatively poor substrate for rFMO2-535 with an apparent V_max 100-fold lower than FMO1 from porcine liver (K_m, 380 µM; V_max, 57 min¹). Nicotine was also a relatively poor substrate for mFMO2-535, with an apparent K_m of 1700 µM (ca. 3-fold higher than the K_m for rFMO2-535); however, the apparent V_max with mFMO2-535 was an order of magnitude greater than the rabbit ortholog (Table 2). The net result is that V_max/K_m with nicotine as the substrate is 3.4 times higher for the monkey compared with the rabbit FMO2.

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Alteration of Enzyme Activity by Effectors of FMO. Further characterization of S-oxygenation, under conditions known to alter catalytic activity of FMO (Devereux and Fouts, 1974; Williams et al., 1985), identified additional differences between expressed rFMO2-535 and mFMO2-535 orthologs. In all cases the response observed for the rFMO2-535 samples was similar to that reported for purified rabbit lung FMO (Williams et al., 1985; Nikbakht et al., 1992). The rate of activity (compared with standard conditions) at lower pH values and the pH optima of mFMO2-535 and rFMO2-535 were almost identical (Fig. 2). However, at pH 10.5 no activity was detectable (calculated velocity  0) from mFMO2-535, while rFMO2-535 retained from 84 to 131% of the activity measured at pH 8.5. The two clones differed significantly in their response to pH (clone by pH interaction effect: p = 0.0012, 4 df); however, the entire difference was due to their response at pH 10.5 (p = 0.0003, 1 df).

View larger version (29K): [in this window] [in a new window]   Fig. 2.   Effect of pH on methimazole-dependent S-oxygenation by expressed mFMO2-535 and rFMO2-535 microsomal protein. The assay was performed with microsomes from two separate batches from each clone as described under Materials and Methods using Tricine buffer at the indicated pH values. Velocities calculated from assays performed under standard conditions were designated as 100% for each batch of each clone, and the results from the test conditions were normalized accordingly. Results shown are the mean normalized results from the separate batches. The error bars show the maximal observed activity. Velocities for 100% were 27.5 and 29.2 min1 for mFMO2-535 batches and 29.5 and 37.1 min1 for rFMO2-535 batches using estimates of FMO content derived from the flavin assay (Table 1).

View larger version (29K): [in this window] [in a new window]   Fig. 3.   Effect of protein concentration and buffer composition during heat treatment on methimazole-dependent S-oxygenation by expressed mFMO2-535 and rFMO2-535 microsomal protein. Assays were performed as described (see Materials and Methods) with standard assay conditions. Protein to be assayed was diluted to a concentration of 0.2 µg/µl (dilute) or 3.0 µg/µl (concentrated) in either Tricine buffer (100 mM Tricine, pH 8.5, 1 mM EDTA) or storage buffer (10 mM potassium phosphate pH 7.4, 20% glycerol, 0.1 mM EDTA, 0.4 mM PMSF) before heating (5 min at 45°C then iced). Velocities were normalized as described in Fig. 2, with normalization to the unheated treatment. The error bars show the maximal observed activity. Velocities for 100% with unheated, undiluted microsomal samples were 26.7 and 24.2 min1 for mFMO2-535 batches and 28.0 and 34.5 min1 for rFMO2-535 batches.

View larger version (33K): [in this window] [in a new window]   Fig. 4.   Alteration of methimazole-dependent S-oxygenation achieved with expressed mFMO2-535 and rFMO2-535 microsomal protein by effectors of FMO activity. Assays were run under standard conditions (see Materials and Methods) with effectors added to the final indicated concentration after DTNB addition, but preceding the 3-min incubation at 37°C. Velocities were normalized to the results obtained under standard conditions, without additions as described in Fig. 2. The error bars show the maximal observed activity. Velocities for 100% were 23.9 and 23.0 min1 for mFMO2-535 batches and 24.2 and 29.9 min1 for rFMO2-535 batches.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

We report here the efficient expression of FMO2 in a baculovirus-mediated system that makes it possible to produce quantities of protein for our future studies including preliminary assessment of the properties of human FMO2.1, determination of gene structure-function relationships, and the study of specific mutations. Expressed mFMO2-535 possessed similar catalytic activity to that of expressed and native rFMO2-535 purified from lung microsomes with respect to S-oxygenation of methimazole, and it was actually somewhat more efficient at N-oxygenation of the two substrates (DMA and nicotine) examined, as measured by V_max/K_m.

The amino acid sequence predicted from the cloned FMO2 cDNA (Yueh et al., 1997) has 97% identity with the reported human FMO2*1 allele (Dolphin et al., 1998). mFMO2-471, which lacks the 64-amino acid residues of the C terminus, was produced in baculovirus to mimic human FMO2.2A, and it was compared with expressed mFMO2-535 protein. No difference in subcellular distribution was found, suggesting that this C-terminal peptide is not required for membrane association. This further confirms the findings from an expressed rabbit FMO2 mutant missing the final 26 residues of the C terminus (Lawton and Philpot, 1993). However, while this mutant maintained its membrane association like mFMO2-471, it also retained enzyme activity, whereas mFMO2-471 did not.

Comparison of the FMO content predicted from Western blotting with that from FAD determination led us to believe that the truncated protein was not effectively binding FAD. Supplementation of FAD to the assay for S-oxidation did not alter the activity of either the active mFMO2-535 or the mFMO2-471 truncated proteins; however, we lacked a positive control for this assay variation (e.g., an expressed FMO known to lack FAD and activity, whose activity could be restored with supplementation). Nonetheless, our finding that N-oxidation of DMA and S-oxidation of methimazole is reduced to undetectable levels by the truncation is in complete agreement with published work on expressed human FMO2.2A (Dolphin et al., 1998). They also demonstrated that FMO2.2A was membrane associated and lacked the ability to catalyze S-oxidation with methimazole as substrate. Our studies indicate that if residual capacity for S-oxidation is retained by mFMO2-471, it has been reduced to less than 0.1% of the activity observed with mFMO2-535.

The FMO2 isoform has often been characterized as stable at high pH, thermal stable, and stimulated by detergents and magnesium. Our results with the monkey FMO2 ortholog demonstrate that these generalizations have their limitations. We measured a sharp drop in activity by mFMO2-535 at pH values that do not appreciably decrease the activity of rFMO2-535, a nearly complete loss of activity by mFMO2-535 in response to thermal conditions that eliminate only about 40% of rFMO2-535 activity, and some effector-specific ortholog differences.

We have demonstrated that there are significant ortholog differences in activity in response to 1% cholate when methimazole is used as the test substrate. Venkatesh and coworkers (1991) demonstrated that the increase or decrease in relative activity caused by the presence of detergent was dependent on the detergent used, the mouse FMO isoform under study, and the specific substrate under study. Thus, ortholog differences between rFMO2-535 and mFMO2-535 may well show a different pattern if other substrates are paired with the detergents we studied.

That more ortholog differences have not previously been identified may be due to the generally small spectrum of conditions investigated, the choice of parameters tested (most investigators test for response to magnesium), the orthologs under study, or a failure to carefully control test parameters when comparing orthologs (i.e., concentration and buffer composition during heat treatment). In addition, as our experiments with CHAPS demonstrate, slightly different chemical preparations can produce very different results.

Active protein produced from chimeric constructs could be useful in assigning protein properties to specific regions of the amino acid sequence. Our efforts to produce active rabbit FMO1 × FMO2 protein (data not shown) and work by others (D. M. Ziegler and L. L. Poulsen, personal communication) indicate a general inability to produce active protein from interisoform chimeras involving large sequence stretches. By contrast, ortholog chimeras can be produced that are active (Wyatt et al., 1998). Thus, it should be possible to produce active protein from mFMO2-535 × rFMO2-535 chimeras; we have demonstrated here that such chimeras could be assayed for numerous differences.

Genotyping experiments have demonstrated that 26% (Whetstine et al., 2000) of people of African American descent carry the FMO2*1 allele, and these individuals are in theory capable of expressing catalytically active FMO2.1 enzyme. Identification of full-length protein in some human samples by Western blotting indicates that there are at least two phenotypes of hFMO2 (full-length and undetectable). The genotyping results, produced in cooperation with Dr. Hines' laboratory, showed that only individuals who expressed FMO2.1 protein had the FMO2*1 allele encoding full-length protein. The DMA assay revealed no catalytically active (at least at the present detection limit) FMO2 in lung microsomes from any phenotype. There are at least two explanations for this observation. We have demonstrated here that mFMO2-535 is more thermolabile than the rabbit ortholog. If human FMO2.1 is also thermolabile, post-mortem degradation before freezing of lung tissue may significantly reduce or eliminate catalytic activity but leave the primary structure intact. We also find in the present study that, based on K_m, DMA is not a particularly good substrate for mFMO2. Again, if the primate orthologs are similar in this respect, the lack of DMA-N-oxygenation previously reported in human microsomes known to contain FMO2.1 (Whetstine et al., 2000) may not be definitive. Therefore, potential substrates for N- or S-oxygenation other than those examined here should be tested for human FMO2 metabolism to determine whether this polymorphism affects drug metabolism in humans.