MOLECULAR-CLONING, SEQUENCING, AND EXPRESSION IN ESCHERICHIA-COLI OF MOUSE FLAVIN-CONTAINING-MONOOXYGENASE-3 (FMO3) - COMPARISON WITH THE HUMAN ISOFORM

The sequence of mouse flavin-containing monooxygenase 3 (FMO3) was obt ained from several clones isolated from a mouse liver cDNA library, Th e nucleotide sequence of mouse FMO3 was 2020 bases in length containin g 37 bases in the 5' flanking region, 1602 in the coding region, and 3 81 in the 3' flanking region, The derived protein sequence consisted o f 534 amino acids including the putative flavin adenine dinucleotide a nd NADP(+) pyrophosphate binding sites (characteristic of mammalian FM Os) starting at positions 9 and 191, respectively. The mouse FMO3 prot ein sequence was 79 and 82% identical to the human and rabbit FMO3 seq uences, respectively, Mouse FMO3 was expressed in Escherichia coli and compared to E. coli expressed human FMO3. The FMO3 proteins migrated with the same mobility (similar to 58 kDa) as determined by sodium dod ecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. Th e expressed FMO3 enzymes (mouse and human forms) were sensitive to hea t and reacted in a similar manner toward metal ions and detergent, Cat alytic activities of mouse and human FMO3 were high toward the substra te methimazole; however, in the presence of trimethylamine and thioace tamide, FMO-dependent methimazole oxidation by both enzymes was reduce d by greater than 85%. Other substrates which inhibited methimazole ox idation were thiourea and thiobenzamide and to a lesser degree N,N-dim ethylaniline. When probed with mouse FMO3 cDNA, FMO3 transcripts were detected in hepatic mRNA samples from female mice, but not in samples from males. FMO3 was detected in mRNA samples from male and female mou se lung, but FMO3 message was not detected in mouse kidney sample from either gender, Results of immunoblotting confirmed the tissue-and gen der-dependent expression of mouse FMO3. (C) 1997 Academic Press.