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 V max /K m 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) 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, 2002Cashman, , 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...

Human flavin-containing monooxygenase 3 (FMO3)-mediated microsomal oxygenation activities, levels of FMO3 protein and FMO3 mRNA, and its modification were investigated in Japanese livers genotyped for the FMO3 gene. Significant correlations were observed for benzydamine N-oxygenation or methyl p-tolyl sulfide S-oxygenation activities (in the range of ~20- to ~40-fold) and the FMO3 levels determined immunochemically in liver microsomes (r2 = 0.73 – 0.75, p < 0.0001, n = 16). Preincubation with the reducing agent ascorbate revealed that FMO3 activities in some liver samples were suppressed. Microsomal FMO3 protein contents (~40-fold) were correlated with FMO3 mRNA levels (r2 = 0.55, p = 0.0010, n = 16), but the FMO3 haplotypes did not affect FMO3 mRNA expression (~100-fold) under the present conditions. On the contrary, FMO3 mRNA levels were multivariately correlated with trans-acting factors, i.e. hepatic nuclear factor 4 (HNF-4) mRNA and nuclear factor Y box-binding protein (NF-Y) mRNA (r2 = 0.31, p = 0.0017, n = 37). These results suggest that considerable individual differences in FMO3 levels may exist in Japanese livers. The liver-enriched transcription factor HNF-4 appears to be a determinant of FMO3 expression in livers, as well as the ubiquitous factor NF-Y.

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We reported the human flavin-containing monooxygenase 3 (FMO3) haplotypes (Pharmacogenet. Genomics: 17, 827, 2007). The objective was to gain the insight into transcriptional regulation in a Japanese population. The wild-type FMO3 reporter plasmids carrying 5'-flanking sequence from the transcriptional initiation site of the FMO3 haplotype 1 (prepared from three individuals) showed higher luciferase activities in HepG2 cells than those from the FMO3 haplotypes 2 and 3, with the wild-type coding region. Several deletion mutants of the FMO3 haplotype 1 (extending from -5,167 to -1,764, numbered relative to the A of the ATG translational initiation codon) revealed that the region of -2,064 to -1,804 contained an important cis-acting element(s) for activation of the FMO3 gene expression. Putative hepatocyte nuclear factor-4 (HNF-4) binding site and CCAAT box, but not Yin Yang 1 element, could be responsible cis-acting elements of the FMO3 gene, by site-directed mutagenesis analysis. The unknown suppressive cis-element(s) at the 5'-upstream region from -2,064 might show genetic polymorphism, because the FMO3 haplotypes 2 and 3 had three and ten mutations, respectively. These results suggest that the putative HNF-4 binding site and CCAAT box could be responsible cis-acting elements of the FMO3 gene in Japanese.

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