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 extension. Complete cDNA was amplified, cloned, and sequenced. The cDNA encodes a protein of 531 amino acids which contains the NADPH- and FAD-binding sites and 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 Escherichia 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 immunoblotting. Recombinant rat FMO3 showed activities of methimazole S-oxidation, and 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 corresponding to the 2.3-kb FMO3 transcript was detected in RNA samples from rat liver and kidney while a weak signal was observed with lung RNA samples. In contrast, the probe did not hybridize with any RNA from brain, adipose tissue, or muscle.

Expression of two different FMOs in sheep liver.

Five different members of the flavin-dependent monooxygenase gene family from different animal species have been described to date. We report the purification and characterization of two FMOs from sheep liver. The predominant isoform was purified 240-fold and was homogenous in SDS PAGE. Its molecular weight (MW) was 58 KDa. The N-terminal sequence, the cross-reactivity with anti-rat FMO3 antibodies, and the catalytic properties such the ability to metabolize trimethylamine (TMA) and to stereoselectively produce L-methionine-d-sulfoxide from L-methionine, are all consistent with this protein being an FMO3. The minor form has a MW of 59 KDa and cross-reacts with anti-rat FMO1 antibodies. The methimazole S-oxidase activity catalyzed by this form was not inhibited by TMA but was inhibited by imipramine. These facts imply that this protein is an FMO1. The expression profile of FMO in sheep liver is similar to that in the human or the mouse but differs from the rat profile.

The flavin-containing monooxygenases in rat liver: evidence for the expression of a second form different from FMO1.

A second form of rat liver FMO, FMO-A, was separated and purified by chromatography on Blue Sepharose Fast flow 6. This FMO-A is different from FMO1 by antigenic properties (anti-FMO-A did not cross-react with FMO1, and reciprocally) and by catalytic properties (the Km for trimethylamine was 3.8 microM and 141.4 microM for FMO-A and FMO1, respectively; the Km for imipramine was 536 microM and 17.4 microM for FMO-A and FMO1, respectively). Furthermore, N-terminal amino sequencing revealed differences in the primary structure of these two FMOs although they both contained the highly conserved FAD-binding domain (Gly-X-Gly-X-X-Gly).

Chiral sulfoxidation of albendazole by the flavin adenine dinucleotide-containing and cytochrome P450-dependent monooxygenases from rat liver microsomes.

The enantioselectivity of the in vitro sulfoxidation of the prochiral drug albendazole was investigated in rat liver microsomes. When biological material obtained from control rats and phenobarbital-, 3-methylcholanthrene-, or dexamethazone-pretreated rats was subjected to specific immunological and chemical inhibitors, it was shown that two main enzymatic systems--cytochrome P450s and flavin-containing monooxygenase (FMO)--were responsible for the sulfoxidation. Purified FMO from rat liver was used to study the enantioselectivity of this enzyme in the sulfoxidation of albendazole. The enantiospecificity of FMO is the reverse of that of the P450s. Nevertheless, each P450 isoenzyme involved in this reaction presents its own individual stereoselectivity.