QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: dmd.106.010827v1 dmd.106.010827v2 34/12/1995    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Borbás, T. Articles by Cashman, J. R. Search for Related Content PubMed PubMed Citation Articles by Borbás, T. Articles by Cashman, J. R.

Investigation of Structure and Function of a Catalytically Efficient Variant of the Human Flavin-Containing Monooxygenase Form 3

Human BioMolecular Research Institute, San Diego, California (J.Z., M.A.C., J.R.C.); and Division of Pharmacology and Drug Safety, Gedeon Richter Ltd., Budapest, Hungary (T.B., I.L.)

(Received April 25, 2006; accepted September 13, 2006)

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
To characterize the contribution of amino acid 360 to the functional activity of the human flavin-containing monooxygenase form 3 (FMO3) and form 1 (FMO1) in the oxygenation of drugs and chemicals, we expressed four FMO3 variants (i.e., Ala³⁶⁰-FMO3, His³⁶⁰-FMO3, Gln³⁶⁰-FMO3, and Pro³⁶⁰-FMO3) and one FMO1 variant (i.e., Pro³⁶⁰-FMO1) and compared them to wild-type enzymes (Leu³⁶⁰-FMO3 and His³⁶⁰-FMO1, respectively). The amino acid substitutions were introduced into wild-type FMO3 or FMO1 cDNA by site-directed mutagenesis. The thermal stability of variants of Leu³⁶⁰ FMO3 was also studied, and the thermal stability was significantly different from that of wild-type FMO3. The influence of different substrates to modulate the catalytic activity of FMO3 variants was also examined. Selective functional substrate activity was determined with mercaptoimidazole, chlorpromazine, and 10-[(N,N-dimethylaminopentyl)-2-(trifluoromethyl)]phenothiazine. Compared with wild-type FMO3, the Ala³⁶⁰-FMO3 and His³⁶⁰-FMO3 variants were less catalytically efficient for mercaptoimidazole S-oxygenation. N-Oxygenation of chlorpromazine was significantly less catalytically efficient for His³⁶⁰-FMO3 compared with wild-type FMO3. Human Pro³⁶⁰-FMO1 was significantly more catalytically efficient at S-oxygenating mercaptoimidazole and chlorpromazine compared with wild-type FMO1. The data support the mechanism that the Pro³⁶⁰ loci affect thermal stability of FMO3. Because different amino acids at position 360 affect substrate oxygenation in a unique fashion compared with that of FMO3 stimulation, we conclude that the mechanism of stimulation of FMO3 is distinct from that of enzyme catalysis. A molecular model of human FMO3 was also constructed to help explain the results. The increase in catalytic efficiency observed for Pro³⁶⁰ in human FMO3 was also observed when the His of FMO1 was replaced by Pro at loci 360.

For FMO3, certain single nucleotide polymorphisms (SNPs) are associated with significant functional differences in enzyme activity (Cashman and Zhang, 2006). Certain amines, including drugs, dietary agents, and xenobiotics metabolized by FMO3 as a prominent metabolic pathway may be significantly altered if an FMO3 SNP is present (Kang et al., 2000; Cashman et al., 2001; Cashman, 2002; Park et al., 2002). For example, humans normally N-oxygenate approximately >96.5% of benzydamine in an FMO3-dependent process. For individuals with defective FMO3, a 2.7- to 3.3-fold decrease in the average percentage of benzydamine N-oxide formation has been observed (Mayatepek et al., 2004). The metabolism of trimethylamine (TMA) illustrates another example of a dramatic effect on FMO3-mediated metabolism in which individuals who possess defective FMO3 decrease TMA N-oxygenation. Because FMO3 is solely responsible for TMA N-oxygenation in vivo, mutations of FMO3 that cause defective TMA N-oxygenation lead to a rare metabolic disorder called trimethylaminuria (Dolphin et al., 1997; Cashman, 2002a; Cashman et al., 2003). In addition to rare mutations that cause trimethylaminuria, common SNPs can also decrease FMO3-mediated metabolism and have clinical consequences (Koukouritaki et al., 2005). In the case of sulindac and chemoprevention of colorectal cancer, for example, FMO3 SNPs may improve the overall clinical efficacy of the drug. The active sulfide metabolite of sulindac (i.e., sulindac sulfide) is S-oxygenated to the S-oxide and then to the sulfone by FMO3 (Hamman et al., 2000). SNPs of FMO3 at the E158K and E308G loci decrease functional activity of FMO3 and decrease the retro-oxygenation of sulindac sulfide to inactive sulfoxide, and increase the efficacy of sulindac. Among sulindac-treated patients who did not develop familial adenomatous polyposis, 33% were homogenous for E158K and 17% were homozygous for E308G variants (Hisamuddin et al., 2004).

Common FMO3 SNPs vary widely across ethnic groups, and it is possible that interethnic variation contributes to therapeutic drug variability for drugs metabolized by FMO3 (Cashman et al., 2001; Furnes et al., 2003). For example, certain ethnic groups have a much greater prevalence of common FMO3 SNPs and possess a greater incidence of abnormal TMA N-oxygenation (Mitchell et al., 1997). A rare Leu³⁶⁰Pro FMO3 SNP has only been observed in individuals of African descent (Lattard et al., 2003). cDNA expression of the enzyme showed that Pro³⁶⁰-FMO3 possessed an extensive metabolizer phenotype in vitro. Thus, mutation of Leu³⁶⁰ of FMO3 to a Pro is an example of a mutation that has been shown to have a higher V_max for several substrates, with little effect on the K_m (Lattard et al., 2003). Our recent discovery of this pharmacologically important polymorphism led us to explore further the effect of other amino acid substitutions at position 360 of FMO3 and to create a molecular model to help understand the physical properties and the affinity, stimulation, and turnover of a number of known FMO3 substrates.

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

 
Chemicals. Mercaptoimidazole (MMI), chlorpromazine HCl, and all other chemicals and reagents were purchased from Aldrich Chemical Co. (Milwaukee, WI) in the highest purity commercially available. The components of the NADPH generating system were obtained from Sigma Chemical Co. (St. Louis, MO). Buffers and other agents were purchased from VWR Scientific, Inc., (San Diego, CA). The phenothiazine analog 10-[(N,N-dimethylaminopentyl)-2-(trifluoromethyl)]phenothiazine (5-DPT) was synthesized by the method described previously (Brunelle et al., 1997).

Cloning and Expression. Wild-type human FMO3 and several variants including Ala³⁶⁰-FMO3, His³⁶⁰-FMO3, Gln³⁶⁰-FMO3, Pro³⁶⁰-FMO3, and wild-type human FMO1 and its variant Pro³⁶⁰-FMO1 were expressed as fusion proteins containing N-terminal maltose-binding protein and C-terminal polyhistidine tags (MBP-FMO3-His₆ and MBP-FMO1-His₆, respectively) as described previously (Brunelle et al., 1997; Lattard et al., 2004). Escherichia coli JM109 cells were transformed with the pMAL-FMO-His₆ plasmid and grown at 37°C in SOC medium (2% Bacto tryptone, 0.5% yeast extract, 8 mM NaCl, 10 mM MgCl₂, 2.5 mM KCl, 20 mM glucose) to an absorbance of 0.5 to 0.6 at 600 nm. Then, 0.2 mM isopropyl ß-thio galactopyranoside, 0.05 mM riboflavin, and 100 µg/ml ampicillin were added. The cells were further incubated overnight at 30°C. Cells were harvested by centrifugation at 6000g for 10 min and resuspended in lysis buffer (50 mM Na₂HPO₄, pH 8.4, 0.5% Triton X-100) containing 0.2% L--phosphatidylcholine, 0.5 mM phenylmethylsulfonyl fluoride, and 100 mM flavin adenine dinucleotide. After incubation for 30 min at 4°C, the resuspended cells were disrupted by sonication (i.e., three 2-min bursts separated by periods of cooling). The solution was centrifuged at 18,000g for 30 min at 4°C. The resulting supernatant was placed onto an amylose column (New England BioLabs, Beverly, MA). The pellets were extracted one more time as described above. The resulting supernatant was loaded onto the same amylose column and eluted as described below.

Purification of MBP-FMO-His₆ Fusion Proteins. All purification procedures were carried out at 4°C. The resulting supernatants were applied (0.5 ml/min) to an amylose column (35 ml) equilibrated with buffer A (50 mM Na₂HPO₄ pH 8.4, 0.5% Triton X-100). The column was washed with 5 volumes of buffer A containing 100 mM flavin adenine dinucleotide. Bound proteins were eluted with 10 mM maltose in buffer A containing 100 mM flavin adenine dinucleotide. Eluted fractions were then applied to a HisTrap Chelating column (HisTrap Kit; GE Healthcare, Little Chalfont, Buckinghamshire, UK) equilibrated with buffer B (20 mM phosphate, pH 7.4, 0.5 M NaCl, 10 mM imidazole, and 0.5% Triton X-100) according to the manufacturer's specifications. After washing with buffer B, bound proteins were eluted with 200 mM imidazole in buffer B. Eluted protein was fractionated by SDS-PAGE (Laemmli, 1970) and visualized with Coomassie Blue staining. The fractions containing the bis-fusion protein were pooled and precipitated by adding PEG 500 to achieve a final concentration of 20% PEG (w/v) and incubated on ice for 30 min. The PEG-precipitated protein was collected by centrifuging at 18,000 rpm for 20 min. The protein was resuspended in buffer A containing 100 mM flavin adenine dinucleotide and 10% glycerol at 4°C. The total protein concentration was measured by bicinchoninic acid assay (Pierce Inc., Rockford, IL) using serum bovine albumin as a standard.

Determination of MBP-FMO-His₆ Concentration by SDS-PAGE and Coomassie Blue Staining. SDS-PAGE was done as described by Laemmli (1970). MBP-FMO1-His₆ and MBP-FMO3-His₆ were quantified by Coomassie Blue staining and compared with a bovine serum albumin standard. In brief, MBP-FMO-His₆ proteins and different quantities of standard bovine serum albumin (i.e., 2, 1.5, 1.0, 0.5, and 0.1 µg per lane) were fractionated by electrophoresis on a 10% polyacrylamide gel under denaturing conditions and stained with Coomassie Blue. After destaining, FMO quantification was done by densitometry analysis using Scion Image software (public domain; http://www.scioncorp.com/). The relationship between the relative intensity of the detected signal and the amount of FMO was linear (r² = 0.9).

Enzyme Assays. The oxygenation of MMI was determined by monitoring the oxidation of NADPH associated with S-oxygenation of this substrate (Dixit and Roche, 1984). The assay contained 50 mM sodium phosphate buffer (pH 8.4), 0.5 mM diethylenetriaminepentaacetic acid (DETAPAC), 0.2 mM NADPH, and 0 to 0.1 mg of MBP-FMO-His₆. Reactions were initiated by the addition of substrate and monitored at 340 nm after obtaining a stable baseline. Kinetic parameters for MMI S-oxygenation were obtained from individual experiments after the addition of decreasing amounts of substrate (1000, 250, 50, 25, 12.5, 6.25, and 3.125 µM final concentration) to the sample cuvette.

The N-oxygenation of chlorpromazine or 5-DPT was also determined as previously described (Lomri et al., 1993; Brunelle et al., 1997). In brief, a typical incubation mixture contained 50 mM potassium phosphate buffer (pH 8.4), 0.4 mM NADP⁺, 0.4 mM glucose 6-phosphate, 1 IU of glucose-6-phosphate dehydrogenase, 0.8 mM DETAPAC, and 0 to 0.9 mg of MBP-FMO-His₆. Reactions were initiated by the addition of 5-DPT or chlorpromazine, incubated at 37°C for 20 min, and stopped by addition of 4 volumes of cold dichloromethane. After addition of 20 mg of Na₂CO₃, the incubations were mixed and centrifuged to partition metabolites and the remaining 5-DPT or chlorpromazine into the organic fraction. The organic fraction was evaporated, dissolved in methanol, mixed thoroughly, centrifuged, and analyzed by HPLC as described previously (Brunelle et al., 1997). N-Oxygenation of 5-DPT or chlorpromazine was determined by quantifying the amount of authentic product formed by HPLC. Chromatography was done on a Hitachi HPLC fitted with an AXXIOM (Richard Scientific, Novato, CA) silica phase column (4.5 µm x 25 cm) with a mobile phase of methanol/isopropanol/60% HClO₄ (55:45:0.01, v/v) and UV detection. This HPLC system separated chlorpromazine, 5-DPT, chlorpromazine N-oxide, and 5-DPT N-oxide that gave retention volumes of 3.1, 2.9, 4.1, and 4.2 ml, respectively. Kinetic constants for the N-oxygenation of chlorpromazine were obtained from individual experiments after the addition of decreasing amounts of chlorpromazine HCl dissolved in water (600, 300, 200, 150, and 127 µM final concentration).

Heat Inactivation Studies. The thermal stability of MBP-FMO3-His₆ variants and wild-type enzyme was examined by monitoring the consumption of NADPH and S-oxygenation of MMI. Enzyme was placed into separate test tubes containing 50 mM phosphate buffer (pH 8.4), DETAPAC and placed on ice. The test tube was taken out of the ice and treated with heat (40°C) for the indicated time and then immediately returned to ice. Shortly thereafter, the heat-treated enzyme was combined with NADPH and transferred to a cuvette for a photometric assay using MMI (100 µM). Non-heat-treated enzyme was used as a control to determine 100% activity. Percentage of remaining activity as a function of time was determined by dividing the observed activity for the heat-treated enzyme by that of the non-heat-treated enzyme.

Molecular Modeling of Human FMO3. The SWISS-MODEL system was used for carrying out homology modeling [SWISS PBD (Protein Data Bank) viewer, Template Selection ExPDB database, and PromodII] (Peitsch, 1995; Guex and Peitsch, 1997; Schwede et al., 2003). Four PDB (Berman et al., 2000) structures were selected for homology-modeling the structure of human FMO3: glutathione reductase (1get) (Mittl et al., 1994), NADPH-peroxidase (1npx) (Stehle et al., 1991), a protein with similarity to flavin-containing monooxygenases (1vqw) (Eswaramoorthy et al., 2006), and phenylacetone monooxygenase (1w4x) (Malito et al., 2004). Several FMO sequences were involved in the analysis (including FMO3 of human, rat, cow, bull, chicken, and mouse; FMO1 of human, rat, and mouse; and rabbit FMO2). Secondary structure was predicted with different methods.

Data Analysis. The estimation of kinetic parameters was achieved by the incubation of at least five different substrate concentrations with MBP-FMO-His₆. Incubations were done in duplicate or triplicate. Data were analyzed with nonlinear regression curve fit tools in GraphPad software using the Michaelis-Menten model. The ordinary least-squares criterion was used to fit the Michaelis-Menten model to the data, taking velocity as the dependent variable. The minimum values of the sum of squared residuals were computed using NAG foundation library routine EO4FDF (Numerical Algorithms Group Ltd., Natick, MA), a combined Gauss-Newton and modified Newton algorithm, using function values only. Data are presented as the mean ± standard deviation from separate experiments. Statistical analysis was done using GraphPad 2.01 (GraphPad Software, Inc., San Diego, CA). Kruskal-Wallis statistical analysis was run to compare values for FMO3 variants with values for wild-type FMO3 enzyme. The Mann-Whitney test was run to compare values for FMO1 variant with wild-type enzyme versus wild-type FMO1 enzyme. Two-way ANOVA with Bonferroni post hoc test was run to determine the effect of isoform and modulators on enzyme activity and the interaction of these factors. The confidence interval selected was 95%.

	Results

Top Abstract Materials and Methods Results Discussion References

 
Previously, the human FMO3 gene variant Pro³⁶⁰-FMO3 was observed only in individuals of African descent, as identified either by MassEXTEND (a high-throughput chip-based genotype variation detection method) with matrix-assisted laser desorption ionization time-of-flight mass spectrometry, described before (Cashman et al., 2001), or by DNA resequencing (Lattard et al., 2003). For the African-American population examined, the Pro³⁶⁰-FMO3 allele frequency was 0.019. cDNA expression of the Pro³⁶⁰-FMO3 showed that the variant was approximately 2- to 4-fold more catalytically efficient in the oxygenation of prototypical substrates than wild-type enzyme (Lattard et al., 2003). For human FMO3, the leucine at loci 360 is not conserved among different species and different FMO isoforms. In mouse and rabbit FMO3, the 360-position is substituted with glutamine. In FMO1, the position is replaced with histidine and is conserved among different species (i.e., human, mouse, rabbit, and pig). To investigate whether the 360-position plays a regulatory role for FMO function, site-directed mutagenesis was used to construct and express four human FMO3 variants and one variant of human FMO1 for comparison with wild-type FMO3 and FMO1, respectively. The four variants were selected based on the idea of a simple alanine replacement, a histidine replacement to mimic FMO1, a glutamine replacement to mimic FMO3 in rodents, and a proline to represent the L360P FMO3 SNP identified in an African-American population.

Several FMO3 variants (i.e., Ala³⁶⁰, His³⁶⁰, Gln³⁶⁰, and Pro³⁶⁰) and a single FMO1 variant (Pro³⁶⁰) were introduced into wild-type FMO3 and FMO1 cDNA, respectively. These mutants, along with wild-type FMOs, were overexpressed as MBP-FMO-His₆ fusion proteins in E. coli JM-109 and purified as described under Materials and Methods. Enzyme fractions were purified to approximately 90% purity and used for kinetic studies.

Preliminary studies showed that human MBP-FMO3 supplemented with NADPH catalyzed the rapid oxygenation of MMI, chlorpromazine, and 5-DPT to their corresponding S-oxide or tertiary amine N-oxides, respectively. The formation of product was a linear function of protein concentration (0–0.16 mg of wild-type FMO3 protein, 0–0.35 mg of Ala³⁶⁰-FMO3, 0–0.9 mg of His³⁶⁰-FMO3, 0–0.5 mg of Gln³⁶⁰-FMO3, and 0–0.15 mg of Pro³⁶⁰-FMO3), with incubation time of at least 10 min. Similarly, formation of oxygenation products by human FMO1 was a linear function of protein concentration (0–0.05 mg of wild-type FMO1 and 0–0.03 mg of Pro³⁶⁰-FMO1), with incubation time of at least 8 min.

View larger version (14K): [in this window] [in a new window]   FIG. 1. Study of the thermal stability of human FMO3 and 360 loci variants. Each enzyme was treated at 40°C for the indicated time. Remaining activity relative to untreated enzyme was plotted against treatment time for wild-type (), L360P (), L360H (), and L360A ().

Detailed kinetic studies were done with the FMO3 variants and compared with wild-type enzyme. The K_m and V_max values obtained for each variant are summarized in Tables 1 and 2. As shown by the kinetic constants listed in Table 1, MMI was an excellent substrate for the wild-type FMO3, having an apparent K_m of 18.1 µM and V_max of 30.3 nmol/min/mg enzyme. To examine the effect of position 360 amino acid variants, the S-oxygenation of MMI was determined by measuring NADPH consumption.

Compared with wild-type FMO3, the catalytic efficiency (i.e., V_max/K_m) of Ala³⁶⁰ -FMO3, His³⁶⁰-FMO3, and Gln³⁶⁰-FMO3 was approximately a third or less of the wild-type value. The catalytic efficiency of Pro³⁶⁰-FMO3 was greater than that of wild-type FMO3. Kruskal-Wallis statistical analysis showed that the Ala³⁶⁰-FMO3 and His³⁶⁰-FMO3 variants were significantly different from wild-type FMO3.

To examine the influence of the Pro³⁶⁰ substitution on a closely related enzyme, wild-type human FMO1 was converted into Pro³⁶⁰-FMO1 by site-directed mutagenesis. As shown in Table 1, the catalytic efficiency of Pro³⁶⁰-FMO1 was greater than 3-fold that of wild-type FMO1. The Mann-Whitney test showed that the Pro³⁶⁰-FMO1 variant was significantly different from wild-type enzyme.

Chlorpromazine was used to investigate the ability of each variant to catalyze the N-oxygenation of a tertiary amine to its N-oxide. Chlorpromazine N-oxide formation was monitored by analysis of organic extracts of incubation mixtures by HPLC. Compared with wild-type FMO3, Ala³⁶⁰-FMO3 and Gln³⁶⁰-FMO3 N-oxygenated chlorpromazine with similar catalytic efficiency (Table 2). His³⁶⁰-FMO3 N-oxygenated chlorpromazine with less catalytic efficiency than wild-type enzyme, and this was confirmed by Kruskal-Wallis statistical analysis. Pro³⁶⁰-FMO3 had twice the catalytic efficiency of wild-type enzyme. The decrease in V_max/K_m for His³⁶⁰-FMO3 was largely due to a lower V_max value, suggesting a decrease in turnover for the tertiary amine. The increase in catalytic efficiency for Pro³⁶⁰-FMO3 compared with wild-type enzyme was largely due to a decrease in K_m, suggesting an increase in the affinity of the enzyme for the tertiary amine.

In agreement with that observed for FMO3, chlorpromazine N-oxygenation was more efficient for Pro³⁶⁰-FMO1. As shown in Table 2, the catalytic efficiency of Pro³⁶⁰-FMO1 was 38% greater than that of wild-type FMO1. As judged by the Mann-Whitney test, the values for Pro³⁶⁰-FMO1 were significantly different from those of wild-type enzyme. The difference in catalytic efficiency apparently was due to changes in both K_m and V_max.

Previously, it was reported that imipramine and chlorpromazine stimulated the FMO3-dependent S-oxygenation of MMI (Wyatt et al., 1998). If the Pro³⁶⁰ amino acid is proximal to the positive regulatory site, then alteration of this amino acid could influence FMO-dependent stimulation of substrate oxygenation. Table 3 shows that wild-type FMO3-catalyzed MMI S-oxygenation was stimulated by chlorpromazine. However, of the Leu³⁶⁰-FMO3 variants examined, compared with nonstimulated enzyme, none showed a statistically significant stimulation of MMI S-oxygenation on the basis of two-way ANOVA and a Bonferroni post hoc test. In all cases examined, the degree of stimulation was less than that observed for wild-type FMO3.

Data of Table 4 show the effect of chlorpromazine on the N-oxygenation of the tertiary amine 5-DPT as well as the effect of 5-DPT on the N-oxygenation of chlorpromazine. Chlorpromazine stimulated the FMO3-mediated N-oxygenation of 5-DPT, but the effect was dependent on the amino acid substitution at position 360, and this effect did not reach statistical significance. The rank order of stimulation of FMO3-mediated 5-DPT N-oxygenation was His³⁶⁰-FMO3 > Gln³⁶⁰-FMO3 > Ala³⁶⁰-FMO3 >> Pro³⁶⁰-FMO3 = wild-type FMO3. In contrast, in the presence of 5-DPT, chlorpromazine N-oxygenation was decreased, and this was judged to be statistically significant on the basis of two-way ANOVA and a Bonferroni post hoc test. The rank order stimulation was as follows: Pro³⁶⁰-FMO3 = Gln³⁶⁰-FMO3 = Ala³⁶⁰-FMO3 = wild-type FMO3 > His³⁶⁰-FMO3.

Molecular modeling of the human FMO3 structure was done in an attempt to understand the possible interaction of the amino acid at position 360 with the NADPH or FAD cofactors. Four PDB structures were selected for homology-modeling the structure of human FMO3. Glutathione reductase (1get) and NADPH-peroxidase (1npx) were used because they had been used in a previous study (Cashman, 2002b). Two other structures were suggested by the SWISS-MODEL Template Selection ExPDB database (Peitsch, 1995; Guex and Peitsch, 1997; Schwede et al., 2003) [i.e., a protein with similarity to flavin-containing monooxygenases (1vqw) and phenylacetone monooxygenase (1w4x)]. The four PDB structures were manually aligned and superimposed. Two conserved domains of the FAD binding domain and the NADPH binding domain could be found in the superimposed structures (Fig. 2). With the exception of these domains, the structures were quite divergent.

View larger version (56K): [in this window] [in a new window]   FIG. 2. Depiction of the conserved FAD binding and NADPH binding domains of the four enzymes used to create a model of human FMO3. The four enzyme structures were modeled together as indicated by different colors (i.e., 1get, green; 1vqw, yellow; 1w4x, red; and 1npx, blue). The NADPH structure is colored with violet and the FAD structure is colored with magenta.

The molecular model derived is shown in Fig. 3. Based on the model, Leu³⁶⁰ lies near, but is not in direct contact with, the FAD adenine moiety. The amino acids of the reference structures corresponding to Leu³⁶⁰ of human FMO3 (i.e., 1get T293, 1vqw Y325, 1W4X T425, 1NPX T271) also showed that the corresponding amino acid was proximal to the FAD (data not shown).

View larger version (58K): [in this window] [in a new window]   FIG. 3. Molecular model of human FMO3. The color scheme (blue–turquoise–green–yellow) indicates the deviation from reference structures (i.e., blue shows the best fit), whereas every structural element obtained by loop search was colored red. The coordinates of the FAD (magenta) and NADPH (violet) were imported from the 1get structure. This crude structure was acceptable for localization of amino acid 360. The atoms of Leu360 are represented by van der Waals spheres (i.e., the yellow amino acid slightly below the FAD).

	Discussion

Top Abstract Materials and Methods Results Discussion References

In a previous study, a few individuals (i.e., 1%) were identified as possessing the Pro³⁶⁰-FMO3 variant, and we reported that Pro³⁶⁰-FMO3 was more catalytically efficient than wild-type FMO3 (Lattard et al., 2003). Pro³⁶⁰-FMO3 oxygenated MMI, TMA, and 5-DPT approximately 3-, 5-, and 2-fold more efficiently, respectively, than wild-type FMO3. For these substrates, the K_m values for Pro³⁶⁰-FMO3 and wild-type enzyme were similar, but the V_max values were greater. It is possible that Pro³⁶⁰-FMO3 could facilitate desorption of NADP⁺ or dehydration of FAD pseudobase water and, thus, speed up the FMO reaction. Evidence that one or the other of these processes is the rate-limiting step in FMO catalysis has been reported previously (Poulsen and Ziegler, 1979; Beaty and Ballou, 1980). Another possibility is that Pro³⁶⁰-FMO3 has an effect on the structure of FMO3 because it is known that substitution of a Pro into an enzyme can change protein structure. We did observe a significant difference in the effect of 360-amino acid substitution on the thermal stability and half-life of Leu³⁶⁰-FMO3 variants (Fig. 1), and we suspect that the effect of the 360-loci substitution has a key effect on function. Accordingly, detailed kinetic studies were undertaken.

We examined variants at the 360 loci to gain insight into structural features that may contribute to increasing the catalytic efficiency of Pro³⁶⁰-FMO3. Compared with wild-type human FMO3, Pro³⁶⁰-FMO3 S-oxygenated MMI with greater catalytic efficiency. Ala³⁶⁰-FMO3, His³⁶⁰-FMO3, and Gln³⁶⁰-FMO3 variants all showed less catalytic efficiency compared with wild-type enzyme (Table 1). N-Oxygenation of chlorpromazine with the Pro³⁶⁰ variant was more catalytically efficient than that of the wild-type enzyme. His³⁶⁰-FMO3, but not Ala³⁶⁰-FMO3, and Gln³⁶⁰-FMO3 were less catalytically efficient than wild-type enzyme at chlorpromazine N-oxygenation (Table 2).

We also examined variants of human FMO1 at the 360 loci to determine whether the observations about FMO3 extended to human FMO1. The Pro³⁶⁰-FMO1 variant catalyzed the S-oxygenation of MMI with greater catalytic efficiency than wild-type enzyme (Table 1). Likewise, Pro³⁶⁰-FMO1 was more catalytically efficient at N-oxygenating chlorpromazine than was wild-type enzyme (Table 2). Thus, in a manner similar to that of human FMO3, replacement of the His³⁶⁰ of human FMO1 with Pro³⁶⁰ affords an enzyme with greater catalytic efficiency. Because human FMO1 and FMO3 share 53% sequence identity (Lawton et al., 1994), it is not surprising that the factors at loci 360 that work to increase catalytic efficiency in FMO3 are also at work for FMO1.

It has been reported that tertiary amines such as chlorpromazine and imipramine stimulate oxygenation of other FMO3 substrates such as MMI (Wyatt et al., 1998). The data pointed to a regulatory site for FMO3 that facilitated substrate turnover (Cashman, 1995). In the study herein, we observed significant stimulation by chlorpromazine of MMI S-oxygenation in the presence of wild-type FMO3, but Ala³⁶⁰-FMO3, His³⁶⁰-FMO3, Gln³⁶⁰-FMO3, and Pro³⁶⁰-FMO3 variants did not show a statistically significant effect (Table 3). In the presence of chlorpromazine, wild-type FMO3-mediated MMI S-oxygenation was stimulated 24%. By a simple substitution of Leu³⁶⁰ in wild-type FMO3 for Ala³⁶⁰-, His³⁶⁰-, Gln³⁶⁰-, or Pro³⁶⁰-FMO3, the enzyme lost its responsiveness to stimulation. In summary, for MMI S-oxygenation, the 360 loci of FMO3 can contribute to modulation of stimulation in the presence of chlorpromazine, but the effect is not a simple matter of hydrophobic or basic interactions. The mechanism responsible for an increase in catalytic efficacy by substitution of Pro at loci 360 appears to be distinct from that of enzyme stimulation.

We also examined the influence of chlorpromazine to stimulate 5-DPT N-oxygenation as well as the influence of 5-DPT to stimulate chlorpromazine N-oxygenation. In the case of variants Ala³⁶⁰-FMO3, His³⁶⁰-FMO3, and Gln³⁶⁰-FMO3, the N-oxygenation of 5-DPT was stimulated in the presence of chlorpromazine, but this did not reach statistical significance (Table 4). The fact that both aliphatic and basic amino acid substitution at loci 360 cause a similar percentage of stimulation of FMO3 suggests that the mechanism of enzyme stimulation of FMO3 may involve multiple structural effects. The effect of amino acid substitution on stimulation of substrate oxygenation as a consequence of changes at loci 360 is quite distinct for MMI and 5-DPT, and suggests that these substrates are handled differently by FMO3.

In the presence of 5-DPT, the 360 loci variants and wild-type FMO3 enzymes decreased chlorpromazine N-oxygenation (Table 4). We interpret this as alternate substrate competitive inhibition of 5-DPT on chlorpromazine N-oxygenation. Previously, we showed that, compared with 5-DPT, chlorpromazine was an inferior substrate for FMO3, probably because of the shorter aliphatic side chain associated with this tertiary amine (Lomri et al., 1993). In contrast, 5-DPT possesses a more optimal aliphatic side chain length to place the tertiary amine further into the substrate-binding channel of FMO3 and afford more efficient N-oxygenation. That no stimulation of N-oxygenation of chlorpromazine by 5-DPT was observed and, in fact, alternate competitive substrate inhibition was observed suggests that the mechanism of alternate substrate stimulation is distinct from that of alternative tertiary amine N-oxygenation (or alternative substrate competitive inhibition).

Two FMO models were previously reported (Cashman, 2002b; Ziegler, 2002) using glutathione reductase (Mittl et al., 1994) and NADPH peroxidase (Stehle et al., 1991) structures as templates. The previously developed human FMO3 models were based on threading the FMO3 sequence onto the X-ray structure of the flavoproteins, whereas the model developed herein was based on homology modeling using the cofactor binding domains as key linchpins. The recently published structures of phenylacetone monooxygenase (i.e., a Baeyer-Villiger monooxygenase) (Malito et al., 2004) and structures of the FMO from Schizosaccharomyces pombe (Eswaramoorthy et al., 2006) encouraged us to construct a new human FMO3 model. Comparison of the FMO3 sequences with other monooxygenases of known structures revealed well defined FAD and NADP⁺ binding domains that were readily modeled. In our model, as well as the earlier models, these cofactor domains have typical dinucleotide-binding folds and other known conserved structural motifs as part of these domains (Krueger and Williams, 2005). Based on the prior model, we proposed that FMO3 amino acid 360 is near the FAD cofactor region (Fig. 3). Leucine 360 is proximal but not in direct contact with the FAD. This amino acid may be involved in domain rotation thought to be occurring during catalysis (Malito et al., 2004; Ballou et al., 2005). We interpret the amino acid substitution at position 360 as having an influence on dehydration of the FAD pseudo base. If amino acid 360 has a determining role in the flexibility of FMO3, then this amino acid may have an influence on NADP⁺ desorption.

Beyond the conserved domains, there are insertions that are unique in mammalian FMOs compared with other monooxygenases. A 40-residue polypeptide chain segment insertion in the FAD binding domain occurs in the interface between the two domains. The C terminus of the FAD binding domain contains mostly -helical polypeptide chains that extend from the FAD domain to the NADPH domain. In addition, the NADPH binding domain exhibits an insertion of 66 amino acids right after the ßß NADPH binding unit. The position of this insertion is the same as in the Baeyer-Villiger monooxygenase (Malito et al., 2004), but the insertion is shorter in the FMO and contains only two -helixes. These insertions might play a role in substrate binding and the catalytic mechanism of human FMOs, but delineating this will require further refinement of the model structures.

In summary, for human FMO3, interindividual variation of enzymatic activity can contribute to discernible differences in drug or chemical metabolism (Cashman and Zhang, 2002). Genetic polymorphisms of genes encoding FMO enzymes resulting in "fast metabolizer" phenotype may lead to rapid drug metabolism and potentially a decrease in therapeutic efficacy of drugs. Based on the kinetics and the molecular model, the change in catalytic efficacy as a function of modulation of amino acid position 360 is likely due to changes in the function of FAD, but further refinement is required for more detailed structural analysis (i.e., substrate binding or influence of mutations on structures). In contrast to MMI, which moves into the active site domain, it is possible that, because the phenothiazine ring system of 5-DPT is precluded from entering the putative substrate binding domain, amino acid interactions in this region of the protein modulate the extent of substrate stimulation. The results for effects of amino acid substitution on FMO3 catalytic efficiency were distinct from those observed for stimulation of MMI S-oxygenation by chlorpromazine. We interpret these findings to suggest that the contribution of the 360 loci for FMO3 substrate stimulation is different from the influence of the 360 loci on FMO catalysis.

Because the 360 loci variant has only been found thus far in individuals of African descent, the functional variation of Leu³⁶⁰-FMO3 described herein may contribute to ethnic group-dependent metabolism of chemicals and dietary materials that are FMO3 substrates. The relevance of the Pro³⁶⁰-FMO3 SNP to human drug metabolism needs to be investigated further.

	Acknowledgments

 
We thank Vignesh Raman for technical assistance in enzyme preparation.

	Footnotes

 
The financial support of the National Institute of Health (DK59618) is gratefully acknowledged. Vignesh Raman was supported by a grant from the San Diego Foundation and the James Copley Foundation.

Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.

doi:10.1124/dmd.106.010827.

ABBREVIATIONS: FMO, flavin-containing monooxygenase; MBP, maltose-binding fusion protein; MBP-FMO3-His₆, C-terminal poly-histidine (His₆) fusion protein; SNP, single nucleotide polymorphism, PAGE, polyacrylamide gel electrophoresis; PEG, polyethylene glycol; TMA, trimethylamine; TMA N-oxide, trimethylamine N-oxide; MMI, mercaptoimidazole; 5-DPT, 10-[(N,N-dimethylaminopentyl)-2-(trifluoromethyl)]phenothiazine, DETAPAC, diethylenetriaminepentaacetic acid; HPLC, high-performance liquid chromatography; PDB, Protein Data Bank; ANOVA, analysis of variance; 1get, glutathione reductase; 1npx, NADPH-peroxidase; 1vqw, a protein with similarity to flavin-containing monooxygenases; 1w4x, phenylacetone monooxygenase.

Address correspondence to: John R. Cashman, Human BioMolecular Research Institute, 5310 Eastgate Mall, San Diego, CA 92121. E-mail: jcashman{at}hbri.org

	References

Top Abstract Materials and Methods Results Discussion References

 


Akerman BR, Forrest S, Chow L, Youil R, Knight M, and Treacy EP (1999) Two mutations of the FMO3 gene in a proband with trimethylaminuria. Hum Mutat 13: 376–379.[CrossRef][Medline]

Ballou DP, Entsch B, and Cole LJ (2005) Dynamics involved in catalysis by single-component and two-component flavin-dependent aromatic hydroxylases. Biochem Biophys Res Commun 338: 590–598.[CrossRef][Medline]

Beaty N and Ballou D (1980) A kinetic investigation of liver microsomal mixed function amine oxidase, in Microsomes, Drug Oxidation and Chemical Carcinogenesis, (Coon MJ, Conney AH, Estabrook RW, Gelboin HV, Gillette JR, and O'Brien PJ eds) pp 209–302, Academic Press, New York.

Berman HM, Westbrook J, Feng Z, Gilliland G, Bhat TN, Weissig H, Shindyalov IN, and Bourne PE (2000) The Protein Data Bank. Nucleic Acids Res 28: 235–242.[Abstract/Free Full Text]

Brunelle A, Bi YA, Lin J, Russell B, Luy L, Berkman C, and Cashman J (1997) Characterization of two human flavin-containing monooxygenase (form 3) enzymes expressed in Escherichia coli as maltose binding protein fusions. Drug Metab Dispos 25: 1001–1007.[Abstract/Free Full Text]

Cashman JR (1995) Structural and catalytic properties of the mammalian flavin-containing monooxygenase. Chem Res Toxicol 8: 165–181.

Cashman JR (2002a) Human flavin-containing monooxygenase (form 3): polymorphisms and variations in chemical metabolism. Pharmacogenomics 3: 325–339.[CrossRef][Medline]

Cashman JR (2002b) Flavin monooxygenases, in Enzyme Systems that Metabolize Drugs and Other Xenobiotics (Ioannides C ed) pp 67–94, John Wiley and Sons, Ltd., London.

Cashman JR, Camp K, Fakharzadeh SS, Fennessey PV, Hines RN, Mamer OA, Mitchell SC, Schlenk D, Smith RL, Tjoa SS, et al. (2003) Biochemical and clinical aspects of the human flavin-containing monooxygenase form 3 (FMO3) related to trimethylaminuria. Curr Drug Metab 4: 151–170.[CrossRef][Medline]

Cashman JR and Zhang J (2002) Interindividual differences of human flavin-containing monooxygenase 3: genetic polymorphisms and functional variation. Drug Metab Dispos 30: 1043–1052.[Abstract/Free Full Text]

Cashman JR and Zhang J (2006) Human flavin-containing monooxygenases. Annu Rev Pharmacol Toxicol 46: 65–100.[CrossRef][Medline]

Cashman JR, Zhang J, Leushner J, and Braun A (2001) Population distribution of human flavin-containing monooxygenase form 3: gene polymorphisms. Drug Metab Dispos 29: 1629–1637.[Abstract/Free Full Text]

Dixit A and Roche TE (1984) Spectrophotometric assay of the flavin-containing monooxygenase and changes in its activity in female mouse liver with nutritional and diurnal conditions. Arch Biochem Biophys 233: 50–63.[CrossRef][Medline]

Dolphin CT, Janmohamed A, Smith RL, Shephard EA, and Phillips IR (1997) Missense mutation in flavin-containing monooxygenase 3 gene, underlies fish-odour syndrome. Nat Genet 17: 491–494.[CrossRef][Medline]

Dolphin CT, Janmohamed A, Smith RL, Shephard EA, and Phillips IR (2000) Compound heterozygosity for missense mutations in the flavin-containing monooxygenase 3 (FMO3) gene in patients with fish-odour syndrome. Pharmacogenetics 20: 799–807.

Eswaramoorthy S, Bonanno JB, Burley SK, and Swaminathan S (2006) Mechanism of action of a flavin-containing monooxygenase. Proc Natl Acad Sci USA 103: 9832–9837.[Abstract/Free Full Text]

Furnes B, Feng J, Sommer S, and Schlenk D (2003) Identification of novel variants of the flavin-containing monooxygenase gene family in African Americans. Drug Metab Dispos 31: 187–193.[Abstract/Free Full Text]

Guex N and Peitsch MC (1997) SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling. Electrophoresis 18: 2714–2723.[CrossRef][Medline]

Hamman MA, Haehner-Daniels BD, Wrighton SA, Rettie AE, and Hall SD (2000) Stereoselective sulfoxidation of sulindac sulfide by flavin-containing monooxygenases. Comparison of human liver and kidney microsomes and mammalian enzymes. Biochem Pharmacol 60: 7–17.[CrossRef][Medline]

Hernandez D, Janmohamed A, Chandan P, Phillips IR, and Shephard EA (2004) Organization and evolution of the flavin-containing monooxygenase genes of human and mouse; identification of novel gene pseudogene clusters. Pharmacogenetics 14: 117–130.[CrossRef][Medline]

Hines RN, Hopp KA, Fanco J, Saeian K, and Begun FP (2002) Alternative processing of the human FMO6 gene renders transcripts incapable of encoding a functional flavin-containing monooxygenase. Mol Pharmacol 62: 320–325.[Abstract/Free Full Text]

Hisamuddin IM, Whebi MA, Chao A, Wyre HW, Hylind LM, Giardiello FM, and Yang VW (2004) Genetic polymorphisms of human flavin-containing monooxygenase 3 in sulindac-mediated primary chemoprevention of familial adenomatous polyposis. Clin Cancer Res 10: 8357–8362.[Abstract/Free Full Text]

Kang JH, Chung WG, Lee KH, Park CS, Kang JS, Shin IC, Roh HK, Dong MS, Baek HM, and Cha YN (2000) Phenotypes of flavin-containing monooxygenase activity determined by ranitidine N-oxidation are positively correlated with genotypes of linked FM03 gene mutations in a Korean population. Pharmacogenetics 10: 67–78.[CrossRef][Medline]

Koukouritaki SB and Hines RN (2005) Flavin-containing monooxygenase genetic polymorphism: impact on chemical metabolism and drug development. Pharmacogenomics 8: 807–822.

Koukouritaki SB, Poch MT, Cabacungan ET, McCarver DG, and Hines RN (2005) Discovery of novel flavin-containing monooxygenase 3 (FMO3) single nucleotide polymorphisms and functional analysis of upstream haplotype variants. Mol Pharmacol 68: 383–392.[Abstract/Free Full Text]

Krueger SK and Williams DE (2005) Mammalian flavin-containing monooxygenases: structure/function, genetic polymorphisms, and role in drug metabolism. Pharmacol Ther 106: 357–387.[CrossRef][Medline]

Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (Lond) 227: 680–685.[CrossRef][Medline]

Lattard V, Zhang J, and Cashman JR (2004) Alternative processing events in human FMO genes. Mol Pharmacol 65: 1517–1525.[Abstract/Free Full Text]

Lattard V, Zhang J, Tran Q, Furnes B, Schlenk D, and Cashman JR (2003) Two new polymorphisms of the FMO3 gene in Caucasian and African-American populations: comparative genetic and functional studies. Drug Metab Dispos 31: 854–860.[Abstract/Free Full Text]

Lawton M, Cashman J, Cresteil T, Dolphin C, Elfarra A, Hines RN, Hodgson E, Kimura T, Ozols J, Phillips IR, et al. (1994) A nomenclature for the mammalian flavin-containing monooxygenase gene family based on amino acid sequence identity. Arch Biochem Biophys 308: 254–257.[CrossRef][Medline]

Lomri N, Gu Q, and Cashman JR (1992) Molecular cloning of flavin-containing monooxygenase (form II) cDNA from adult human liver. Proc Natl Acad Sci USA 89: 1685–1689.[Abstract/Free Full Text]

Lomri N, Yang Z, and Cashman JR (1993) Expression in Escherichia coli of the flavin-containing monooxygenase D (form II) from adult human liver: determination of a distinct tertiary amine substrate specificity. Chem Res Toxicol 6: 425–429.[CrossRef][Medline]

Malito E, Alfieri A, Fraaije MW, and Mattevi A (2004) Crystal structure of a Baeyer-Villiger monooxygenase. Proc Natl Acad Sci USA 101: 13157–13162.[Abstract/Free Full Text]

Mayatepek E, Flock B, and Zschocke J (2004) Benzydamine metabolism in vivo is impaired in patients with deficiency of flavin-containing monooxygenase 3. Pharmacogenetics 14: 775–777.[CrossRef][Medline]

Mitchell SC, Zhang AQ, Barrett T, Ayesh R, and Smith RL (1997) Studies on the discontinuous N-oxidation of trimethylamine among Jordanian, Ecuadorian and New Guinean populations. Pharmacogenetics 7: 45–50.[CrossRef][Medline]

Mittl PRE, Berry A, Scrutton NS, Perham RN, and Schulz GE (1994) Anatomy of an engineered NAD-binding site. Protein Sci 9: 1504–1514.

Park CS, Kang JH, Chung WG, Yi HG, Pie JE, Park DK, Hines RN, McCarver DG, and Cha YN (2002) Ethnic differences in allelic frequency of two flavin-containing monooxygenase 3 (FMO3) polymorphisms: linkage and effects on in vivo and in vitro FMO activities. Pharmacogenetics 12: 77–80.[CrossRef][Medline]

Peitsch MC (1995) Protein modeling by email. Bio/Technology 13: 658–660.[CrossRef]

Poulsen LL and Ziegler DM (1979) The liver microsomal FAD-containing monooxygenase: spectral characterization and kinetic studies. J Biol Chem 254: 6449–6455.[Abstract/Free Full Text]

Schwede T, Kopp J, Guex N, and Peitsch MC (2003) SWISS-MODEL: an automated protein homology-modeling server. Nucleic Acids Res 31: 3381–3385.[Abstract/Free Full Text]

Stehle T, Ahmed SA, Claiborne A, and Schulz GE (1991) Structure of NADH peroxidase from Streptococcus faecalis 10C1 refined at 2.16 angstroms resolution. J Mol Biol 221: 1325–1330.[Medline]

Treacy EP, Akerman BR, Chow LM, Youil R, Bibeau C, Lin J, Bruce AG, Knight M, Danks DM, Cashman JR, et al. (1998) Mutations of the flavin-containing monooxygenase gene (FMO3) cause trimethylaminuria, a defect in detoxication. Hum Mol Genet 7: 839–845.[Abstract/Free Full Text]

Wyatt MK, Overby LH, Lawton MP, and Philpot RM (1998) Identification of amino acid residues associated with modulation of flavin-containing monooxygenase (FMO) activity by imipramine: structure/function studies with FMO1 from pig and rabbit. Biochemistry 37: 5930–5938.[CrossRef][Medline]

Zhang J and Cashman JR (2006) Quantitative analysis of FMO gene mRNA levels in human tissues. Drug Metab Dispos 34: 19–26.[Abstract/Free Full Text]

Ziegler DM (1988) Flavin-containing monooxygenases: catalytic mechanism and substrate specificities. Drug Metab Rev 19: 1–32.[Medline]

Ziegler DM (2002) An overview of the mechanism, substrate specificities, and structure of FMOs. Drug Metab Rev 34: 503–511.[CrossRef][Medline]

This Article Abstract Full Text (PDF) All Versions of this Article: dmd.106.010827v1 dmd.106.010827v2 34/12/1995    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Borbás, T. Articles by Cashman, J. R. Search for Related Content PubMed PubMed Citation Articles by Borbás, T. Articles by Cashman, J. R.