Cloning, sequencing, tissue distribution, and heterologous expression of rat flavin-containing monooxygenase 3

The sequence of rat FMO3 was obtained by RT-PCR and 5'/3' terminal extensio n. Complete cDNA was amplified, cloned, and sequenced. The cDNA encodes a p rotein of 531 amino acids which contains the NADPH- and FAD-binding sites a nd a hydrophobic carboxyl terminus characteristic of FMOs. This sequence is 81, 81., and 91% identical to sequences of human, rabbit, and mouse FMO3, respectively, and 60% identical to rat FMO1. Rat FMO3 was expressed in Esch erichia coli. The recombinant protein and the native protein purified from rat liver microsomes migrated with the same mobility (56 kDa) as determined in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblo tting. Recombinant rat FMO3 showed activities of methimazole S-oxidation, a nd NADPH oxidation associated with the N- or S-oxidation of trimethylamine and thioacetamide, in good concordance with those reported for human FMO3. When probed with rat FMO3 cDNA (bases 201 to 768), a strong signal correspo nding to the 2.3-kb FMO3 transcript was detected in RNA samples from rat li ver and kidney while a weak signal was observed with lung RNA samples. In c ontrast, the probe did not hybridize with any RNA from brain, adipose tissu e, or muscle. (C) 2001 Academic Press.