Regulation of Flavin-Containing Monooxygenase

The flavin-containing monooxygenases (FMOs) are important for the oxidation of a variety of environmental toxicants, natural products, and therapeutics. Consisting of six family members (FMO1–5), these enzymes exhibit distinct but broad and overlapping substrate specificity and are expressed in a highly tissueand species-selective manner. Corresponding to previously identified regulatory domains, a YY1 binding site was identified at the major rabbit FMO1 promoter, position 8 to 2, two overlapping HNF1 sites, position 132 to 105, and two HNF4 sites, position 467 to 454 and 195 to 182. Cotransfection studies with HNF1 and HNF4 expression vectors demonstrated a major role for each of these factors in enhancing FMO1 promoter activity. In contrast, YY1 was shown by site-directed mutagenesis to be dispensable for basal promoter activity but suppressed the ability of the upstream domains to enhance transcription. Finally, comparisons between rabbit and human FMO1 demonstrated conservation of each of these regulatory elements. With the exception of the most distal HNF4 site, each of the orthologous human sequences also was able to compete with rabbit FMO1 ciselements for specific protein binding. These data are consistent with these same elements being important for regulating human FMO1 developmentaland tissue-specific expression. The flavin-containing monooxygenases (FMOs) (EC 1.14.13.8) are a family of microsomal enzymes important for the oxidative metabolism of a wide range of compounds that possess soft nitrogen, sulfur, selenium, and phosphorous nucleophilic centers. Substrates include dietary components such as trimethylamine and methionine, pesticides such as fonfos and phorate, therapeutic agents such as imipramine, cimetidine, and ketoconazole, and plant alkaloids such as nicotine (for review, see Rettie and Fisher, 1999). Five mammalian FMO isoforms have been identified (FMO1–5), each exhibiting a distinct but unusually broad and overlapping substrate specificity that is partly attributable to the unique catalytic mechanism of the FMO (Poulsen and Ziegler, 1995). These enzymes are expressed at high levels in several tissues and in all animal species examined (Poulsen, 1991). In the human, up to a 10-fold range in interindividual FMO activity has been reported (Overby et al., 1997). However, unlike the cytochrome P450-dependent monooxygenase family, such interindividual variation is unlikely to be caused by differential environmental exposure; with rare exception, FMO expression is not affected by exogenous agents. Expression of the different FMO isoforms is highly tissueand species-selective, can be affected by endogenous steroids (Rettie and Fisher, 1999), and also is influenced by genetic variability (Cashman et al., 2000; Whetstine et al., 2000). An extensive number of reports have appeared on FMO protein chemistry and overall expression pattern (for review, see Rettie and Fisher, 1999). In contrast and despite the impact differential expression must have on targetand species-selective therapeutic efficacy and toxicant susceptibility, little is known regarding specific molecular mechanisms regulating FMO