The Flavin-Containing Monooxygenase (FMO) is arguably the most important understudied mammalian enzyme known today. FMO is a major component of the defenses that protect humans against potentially toxic chemicals in their environment, including xenobiotics, drugs and pesticides. A compound containing a tertiary amine or a nucleophilic sulfur nucleus is efficiently N- or S-oxygenated by FMO and converted to a form more readily excreted. Even though FMO is present in high levels in every secretory cell thus far examined, a physiological basis for its action remains unclear. Knowledge of the amino acid sequence of FMO deduced from cDNA sequencing will enable an understanding of: (1) tissue or species selective FMO drug or xenobiotic metabolism, and (2) tissue specific enzyme mechanism. We have cloned human liver and human placenta FMO employing a lambda gtll antibody screening procedure. The data strongly suggest that at least for placenta FMO we have a full length cDNA. In order to confirm this we obtained FMO amino acid sequence, deduced nucleotide sequence, constructed a probe and demonstrated that this probe selectively hybridizes to FMO cDNA. In this research proposal we will accomplish the following: (1) clone human liver and placenta and hog liver FMO cDNA using a lambda gtlO oligonucleotide selection procedure. This will provide a set of cDNA in which to confirm our original clones, (2) confirm the identity of FMO cDNA clones with (a) hybridization selection, and (b) antibody fingerprinting, (3) Verify the FMO cDNA by comparing nucleotide sequence with amino acid sequence deduced from (a) gas phase peptide sequencing and (b) mass spectrometry. (4) Express the human liver and placenta FMO cDNA. These studies will provide detailed information which will explain isozyme or tissue specific FMO drug and xenobiotic metabolism. The information gained will be of therapeutic value in achieving an understanding of tissue selective targeting of drugs or drug metabolites.
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