Drug Metabolism and Disposition Fast Forward
First published on December 1, 2006; DOI: 10.1124/dmd.106.013094
0090-9556/07/3503-328-330$20.00
DMD 35:328-330, 2007
SHORT COMMUNICATION
Effect of Genetic Variants of the Human Flavin-Containing Monooxygenase 3 on N- and S-Oxygenation Activities
Makiko Shimizu,
Hiroshi Yano,
Satomi Nagashima,
Norie Murayama,
Jun Zhang,
John R. Cashman, and
Hiroshi Yamazaki
Laboratory of Drug Metabolism and Pharmacokinetics, Showa Pharmaceutical University, Tokyo, Japan (M.S., H.Y., S.N., N.M., H.Y.); and Human BioMolecular Research Institute, San Diego, California (J.Z., J.R.C.)
(Received September 22, 2006;
accepted November 29, 2006)
 |
Abstract
|
|---|
The decreased capacity of the flavin-containing monooxygenase 3 (FMO3) to oxygenate xenobiotics including trimethylamine is believed to contribute to metabolic disorders. The aim of this study was to functionally characterize FMO3 variants recently found in a Japanese population and compare them with selective functional activity of other FMO3 variants. Recombinant Glu158Lys and Glu158Lys-Glu308Gly FMO3 expressed in Escherichia coli membranes showed slightly decreased N-oxygenation of benzydamine and trimethylamine. Selective functional S-oxygenation of these variants by methyl p-tolyl sulfide or sulindac sulfide was comparable to that of wild-type FMO3. The Glu158Lys-Thr201Lys-Glu308Gly and Val257Met-Met260Val variants showed significantly decreased oxygenation of typical FMO3 substrates (i.e., approximately one-tenth of the Vmax/Km values). Val257Met FMO3 had a lower catalytic efficiency for methyl p-tolyl sulfide and sulindac sulfide S-oxygenation. However, compared with wild-type FMO3, Val257Met FMO3 showed a similar catalytic efficiency for N-oxygenation of benzydamine and trimethylamine. The catalytic efficiency for benzydamine and trimethylamine N-oxygenation by Arg205Cys FMO3 was only moderately decreased, but it possessed decreased sulindac sulfide S-oxygenation activity. Kinetic analysis showed that Arg205Cys FMO3 was inhibited by sulindac in a substrate-dependent manner, presumably because of selective interaction between the variant enzyme and the substrate. The results suggest that the effects of genetic variation of human FMO3 could operate at the functional level for N- and S-oxygenation for typical FMO3 substrates. Genetic polymorphism in the human FMO3 gene might lead to unexpected changes of catalytic efficiency for N- and S-oxygenation of xenobiotics and endogenous materials.
The flavin-containing monooxygenase (FMO; EC 1.14.13.8
[EC]
) is an NADPH-dependent enzyme that catalyzes the oxygenation of a wide variety of compounds containing nitrogen, sulfur, or other heteroatoms (Krueger and Williams, 2005
; Cashman and Zhang, 2006). FMO3 is considered a prominent form expressed in adult human liver (Lomri et al., 1992) and plays a role in processing nucleophilic drugs such as the anticancer drug tamoxifen, the pain medication codeine, the antifungal drug ketoconazole, the addictive chemical nicotine found in tobacco, and the diet-derived chemical trimethylamine (Cashman et al., 2000; Ziegler, 2002).
Because of its strong linkage with the genetic disorder trimethylaminuria, or fish-like odor syndrome, considerable work has been done to relate coding region polymorphisms of the FMO3 gene to interindividual differences in FMO3 phenotype (Dolphin et al., 1997a; Cashman, 2002, 2004). According to the list of FMO3 gene mutations summarized in a systematic and trivial name web database, Glu158Lys, Val257Met, and Glu158Lys-Glu308Gly FMO3 forms are common genetic FMO3 polymorphisms (Hernandez et al., 2003). There is considerable genetic variation reported among different ethnic groups (Cashman et al., 2001). In the course of identification of novel mutations of FMO3 and/or haplotypes of the FMO3 gene in Japanese individuals suffering from trimethylaminuria (Yamazaki et al., 2004), we recently reported some variants of the FMO3 gene using genomic DNA from individuals that showed low FMO3 metabolic capacity (Yamazaki et al., 2005, 2007). In our preliminary reports (Fujieda et al., 2003; Shimizu et al., 2006), there were some FMO3 variants such as Thr201Lys, Arg205Cys, or Met260Val that were thus far only observed in a Japanese population. Interestingly, Thr201Lys and Met260Val variants also existed together with the well known Glu158Lys-Glu308Gly and Val257Met mutations, respectively, in the FMO3 gene (Shimizu et al., 2006). However, the complete characterization of these FMO3 variants has not been examined in detail.
Herein we report data showing low catalytic efficiency of three novel FMO3 variants expressed in bacteria that are causative of abnormal N- and S-oxygenation of typical FMO3 substrates including benzydamine, trimethylamine, methyl p-tolyl sulfide, and sulindac sulfide (Fig. 1) and compare the data from wild-type enzyme with FMO3 mutants including Glu158Lys, Val257Met, and Glu158Lys-Glu308Gly. The effect of Arg205Cys and Val257Met FMO3 mutations on the rates of N- and S-oxygenation of model substrates are also reported.
View larger version (16K):
[in this window]
[in a new window]
|
FIG. 1. Chemical structures of FMO3 substrates used in this study. Arrows indicate the positions of N-or S-oxygenation.
|
|
|
Materials and Methods
|
|---|
Chemicals. Benzydamine hydrochloride, trimethylamine N-oxide, methyl p-tolyl sulfide, methyl p-tolyl sulfoxide, sulindac sulfoxide (Z-5-fluoro-2-methyl-1-[p-(methylsulfinyl)benzyldiene]indene-3-acetic acid), and sulindac sulfide were purchased from Sigma-Aldrich (St. Louis, MO). Trimethylamine hydrochloride was obtained from Wako Pure Chemicals (Osaka, Japan). Benzydamine N-oxide (>99.0% purity; Yeung and Rettie, 2006) was a generous gift from Dr. A. E. Rettie (University of Washington, Seattle, WA). The other chemicals and reagents used were obtained in the highest grade commercially available.
Recombinant FMO3 Protein Preparation. The procedure for preparation of wild-type FMO3 and Arg205Cys FMO3 cDNA was reported previously (Yamazaki et al., 2007). To produce Glu158Lys, Glu158Lys-Glu308Gly, Glu158Lys-Thr201Lys-Glu308Gly, Val257Met, and Val257Met-Met260Val FMO3, site-directed mutagenesis was done using the QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA). The wild-type and modified FMO3 cDNAs were introduced into the pTrc99A expression vector (GE Healthcare, Little Chalfont, Buckinghamshire, UK) and then transformed into Escherichia coli strain JM109 as described previously (Yamazaki et al., 2007). The entire coding regions of the wild-type and mutagenized FMO3 cDNAs including the mutated sites were verified by resequencing both strands. From bacterial pellets, membrane FMO3 fractions were prepared by a series of fractionations and high-speed centrifugation steps. The amount of recombinant FMO3 (0.025–0.10 nmol FMO3/mg bacterial protein) was determined by immunoquantification using an anti-FMO3 antibody (BD Gentest, Woburn, MA) by comparison with a standard of recombinant human FMO3 (BD Gentest) according to the manufacturer's data sheet (Yamazaki et al., 2007).
Enzyme Assays. Rates of N-oxygenation of benzydamine (Yeung and Rettie, 2006) and S-oxygenation of methyl p-tolyl sulfide (Stevens et al., 2003) and sulindac sulfide (Hamman et al., 2000) were determined using HPLC methods described previously with minor modifications. In brief, a typical incubation mixture consisted of 50 mM potassium phosphate buffer (pH 8.4), an NADPH-generating system (0.25 mM NADP+, 2.5 mM glucose 6-phosphate, and 0.25 unit/ml glucose-6-phosphate dehydrogenase), a substrate, and bacterial membranes (5.0 pmol of FMO3) in a final volume of 0.20 ml. Incubations were carried out at 37°C for 10 min: the linearity of product formation was confirmed with recombinant FMO3 preparations for 30 min. The incubation was terminated by adding 0.20 ml of ice-cold acetonitrile for benzydamine and methyl p-tolyl sulfide and by adding 50 µl of 50% phosphoric acid and 1.0 ml of ethyl acetate for sulindac sulfide. For sulindac sulfide, after centrifugation at 2000g for 10 min, the organic phase was transferred to a clean tube and evaporated to dryness at 40°C. The residue was dissolved in 0.20 ml of mobile phase (50% acetonitrile in 25 mM potassium phosphate buffer, pH 3.0) and introduced onto the HPLC apparatus. For benzydamine and methyl p-tolyl sulfide, the aqueous supernatant was centrifuged at 2000g for 10 min and was subjected to HPLC equipped with an analytical C18 column (4.6 x 150 mm, 5 µm). The formation of methyl p-tolyl sulfoxide and sulindac sulfoxide was monitored at wavelengths of 237 nm (Stevens et al., 2003) and 360 nm (Hamman et al., 2000), respectively. Benzydamine N-oxide was determined fluorometrically (Yeung and Rettie, 2006).
Rates of N-oxygenation of trimethylamine were determined by gas chromatography as described previously (Yamazaki et al., 2007). The kinetic analysis of N- or S-oxygenation was done using a nonlinear regression analysis program (KaleidaGraph, Synergy Software, Reading, PA). When substrate inhibition was observed, an equation of v = Vmax · [S]/(Km + [S] + [S]2/Ks) was used instead of the Michaelis-Menten equation, and [S] and Ks were defined as substrate concentration and substrate inhibition constant, respectively.
|
Results and Discussion
|
|---|
To investigate the effects of novel FMO3 variants found in Japanese and to compare them with other FMO3 variants, recombinant wild-type, Glu158Lys, Glu158Lys-Glu308Gly, Glu158Lys-Thr201Lys-Glu308Gly, Val257Met, Val257Met-Met260Val, and Arg205Cys FMO3 were expressed in E. coli membranes. Kinetic parameters for N-oxygenation of benzydamine and trimethylamine activities were determined by nonlinear regression analysis for six variant FMO3s and were compared with those of wild-type FMO3 expressed in bacterial membranes (Table 1). Apparent Vmax values of Glu158Lys-Thr201Lys-Glu308Gly and Val257Met-Met260Val FMO3 for benzydamine N-oxygenation were approximately 1/10 and 1/20, respectively, of that of wild-type enzyme, but apparent Km values were not different. Apparent catalytic efficiency (Vmax/Km) of Glu158Lys, Glu158Lys-Glu308Gly, and Arg205Cys FMO3 were slightly decreased compared with wild-type FMO3; however, catalytic function of Val257Met was similar to that of the wild-type FMO3. A similar series of results was obtained using trimethylamine N-oxygenation as a marker reaction catalyzed by wild-type and variant FMO3 enzymes in terms of effects of amino acid substitutions (Table 1).
View this table:
[in this window]
[in a new window]
|
TABLE 1 N- and S-Oxygenation of wild-type and variant FMO3 expressed in E. coli membranes
Each substrate (0–1000 µM benzydamine, 0–500 µM trimethylamine, 0–1000 µM methyl p-tolyl sulfide, and 0–300 µM sulindac sulfide) was incubated with recombinant FMO3 (5–50 pmol Eq) at 37°C for 10 to 30 min in the presence of an NADPH-generating system. Kinetic parameters were calculated from a fitted curve by nonlinear regression (mean ± S.E.).
|
|
Kinetic parameters for S-oxygenation of methyl p-tolyl sulfide and sulindac sulfide activities were also determined and compared with those of wild-type FMO3 expressed in bacterial membranes (Table 1). Apparent Vmax/Km values for methyl p-tolyl sulfide and sulindac sulfide S-oxygenation of Val257Met FMO3 as well as Glu158Lys, Glu158Lys-Glu308Gly, Glu158Lys-Thr201Lys-Glu308Gly, Val257-Met-Met260Val, and Arg205Cys FMO3 were decreased compared with wild-type FMO3. In contrast to the N-oxygenation described above, apparent Km and Vmax values of Glu158Lys-Thr201Lys-Glu308Gly and Val257Met-Met260Val FMO3 for methyl p-tolyl sulfide S-oxygenation were increased and decreased, respectively, compared with wild-type FMO3. A similar series of results was obtained using sulindac sulfide S-oxygenation catalyzed by these same variant FMO3s (Table 1), except for the case of Arg205Cys FMO3, which showed a unique substrate-velocity curve that resembled substrate inhibition, as shown in Fig. 2. Because wild-type FMO3 has no free cysteine residues in the native form, this inhibition might be caused by selective interaction between Arg205Cys FMO3 and sulindac sulfide or the S-oxide, sulindac. However, because most of the reported FMO3 inhibition is generally considered to be due to competition with NADP/H (Cashman and Zhang, 2006), further studies are needed to interpret this interesting phenomenon. Apparent instability of the enzyme or substrate inhibition by methyl p-tolyl sulfide on its S-oxygenation was not observed under the present conditions (data not shown).
The structural organization of the human FMO3 gene has been reported (Dolphin et al., 1997b), and because the functional FAD- and NADP+-binding domains of the human FMO3 are encoded by exon 2 and exon 5, respectively (Dolphin et al., 1997b), it is possible that the mutation(s) located near the N terminus of FMO3 could be responsible for decreased expression of the gene and/or abnormal functional activity or instability of the encoded protein. Previously, inactive variants of FMO3 at positions in exon 2 and exon 5 have been reported (Hernandez et al., 2003; Zhang et al., 2003). The present results collectively suggest that novel Glu158Lys-Thr201Lys-Glu308Gly and Val257Met-Met260Val FMO3 possessed little functional activity for N- and S-oxygenation, suggesting that additional changes in specific regions of exon 5 and exon 6 of the FMO3 gene were required for functional activity. Consistently, individuals harboring Val257Met-Met260Val/Arg500stop and Glu158Lys-Thr201Lys-Glu308Gly/Arg205Cys FMO3 genotypes showed low FMO3 metabolic capacity (<40%) as phenotyped by measuring trimethylamine and trimethylamine N-oxide excreted in urine (Shimizu et al., 2006; Yamazaki et al., 2007).
In genotype-phenotype studies of individuals with trimethylaminuria, the Val257Met FMO3 mutation showed no significant changes in trimethylamine N-oxygenation (Cashman, 2002, 2004; Dolphin et al., 1997a). However, the Val257Met amino acid substitution in FMO3 could decrease the S-oxygenation of some xenobiotics while not changing the extent of N-oxygenation (Table 1). In the present study, the Arg205Cys substitution of FMO3, which was found with low allele frequency in a Japanese self-reported cohort of trimethylaminuria (Yamazaki et al., 2007), was the most representative example showing different effects between FMO3 substrate N- and S-oxygenation (Fig. 2; Table 1). In conclusion, effects of genetic variation of the human FMO3 could lead to unexpected changes in catalytic efficiency of N- and S-oxygenation for typical FMO3 substrates, and this may have clinical consequences.
|
Acknowledgments
|
|---|
We thank Sachiko Tomioka, Maki Karino, and Yumiko Wakiya for their help.
|
Footnotes
|
|---|
This work was supported in part by the Ministry of Education, Culture, Sports, Science and Technology of Japan and Japan Research Foundation for Clinical Pharmacology. J.R.C. was supported financially by a grant from the National Institutes of Health (Grant DK 59618).
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
doi:10.1124/dmd.106.013094.
ABBREVIATIONS: FMO, flavin-containing monooxygenase; HPLC, high-performance liquid chromatography.
Address correspondence to: Prof. Dr. Hiroshi Yamazaki, Showa Pharmaceutical University, 3-3165 Higashi-tamagawa Gakuen, Machida, Tokyo 194-8543, Japan. E-mail: hyamazak{at}ac.shoyaku.ac.jp
|
References
|
|---|
Cashman JR (2002) Human flavin-containing monooxygenase (form 3): polymorphisms and variations in chemical metabolism. Pharmacogenomics 3: 325–339.[CrossRef][Medline]
Cashman JR (2004) The implications of polymorphisms in mammalian flavin-containing monooxygenases in drug discovery and development. Drug Discov Today 9: 574–581.[CrossRef][Medline]
Cashman JR, Akerman BR, Forrest SM, and Treacy EP (2000) Population-specific polymorphisms of the human FMO3 gene: significance for detoxication. Drug Metab Dispos 28: 169–173.[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]
Dolphin CT, Janmohamed A, Smith RL, Shephard EA, and Phillips IR (1997a) Missense mutation in flavin-containing mono-oxygenase 3 gene, FMO3, underlies fish-odour syndrome. Nat Genet 17: 491–494.[CrossRef][Medline]
Dolphin CT, Riley JH, Smith RL, Shephard EA, and Phillips IR (1997b) Structural organization of the human flavin-containing monooxygenase 3 gene (FMO3), the favored candidate for fish-odor syndrome, determined directly from genomic DNA. Genomics 46: 260–267.[CrossRef][Medline]
Fujieda M, Yamazaki H, Togashi M, Saito T, and Kamataki T (2003) Two novel single nucleotide polymorphisms (SNPs) of the FMO3 gene in Japanese. Drug Metab Pharmacokinet 18: 333–335.[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, Addou S, Lee D, Orengo C, Shephard EA, and Phillips IR (2003) Trimethylaminuria and a human FMO3 mutation database. Hum Mutat 22: 209–213.[CrossRef][Medline]
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]
Lomri N, Gu Q, and Cashman JR (1992) Molecular cloning of the flavin-containing monooxygenase (form II) cDNA from adult human liver. Proc Natl Acad Sci USA 89: 1685–1689.[Abstract/Free Full Text]
Shimizu M, Fujita H, Aoyama T, and Yamazaki H (2006) Three novel single nucleotide polymorphisms of the FMO3 gene in a Japanese population. Drug Metab Pharmacokinet 21: 245–247.[CrossRef][Medline]
Stevens JC, Melton RJ, Zaya MJ, and Engel LC (2003) Expression and characterization of functional dog flavin-containing monooxygenase 1. Mol Pharmacol 63: 271–275.[Abstract/Free Full Text]
Yamazaki H, Fujieda M, Cashman JR, and Kamataki T (2005) Mild trimethylaminuria observed in a Japanese cohort with liver damage. Am J Med 118: 803–805.[CrossRef][Medline]
Yamazaki H, Fujieda M, Togashi M, Saito T, Preti G, Cashman JR, and Kamataki T (2004) Effects of the dietary supplements, activated charcoal and copper chlorophyllin, on urinary excretion of trimethylamine in Japanese trimethylaminuria patients. Life Sci 74: 2739–2747.[CrossRef][Medline]
Yamazaki H, Fujita H, Gunji T, Zhang J, Kamataki T, Cashman JR, and Shimizu M (2007) Stop codon mutations in the flavin-containing monooxygenase 3 (FMO3) gene responsible for trimethylaminuria in a Japanese population. Mol Genet Metab 90: 58–63.[CrossRef][Medline]
Yeung CK and Rettie AE (2006) Benzydamine N-oxygenation as a measure of flavin-containing monooxygenase activity. Methods Mol Biol 320: 157–162.[Medline]
Zhang J, Tran Q, Lattard V, and Cashman JR (2003) Deleterious mutations in the flavin-containing monooxygenase 3 (FMO3) gene causing trimethylaminuria. Pharmacogenetics 13: 495–500.[CrossRef][Medline]
Ziegler DM (2002) An overview of the mechanism, substrate specificities, and structure of FMOs. Drug Metab Rev 34: 503–511.[CrossRef][Medline]
![[Feedback]](/icons/banner/feedbackACT.gif)
![[For Subscribers]](/icons/banner/subscriberACT.gif)
![[Archive]](/icons/banner/archiveACT.gif)
![[Search]](/icons/banner/searchACT.gif)
![[Contents]](/icons/banner/tocACT.gif)
Top
Abstract
Materials and Methods
) and Arg205Cys (
) FMO3 cDNA expressed in bacteria. Kinetic analysis was done using nonlinear regression analysis employing the Michaelis-Menten equation, v = Vmax · [S]/(Km + [S]), or v = Vmax · [S]/(Km + [S] + [S]2/Ks) for substrate inhibition.