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.
Lattard
V
V
Unité de Toxicologie et de Métabolisme Comparés des Xénobiotiques, UMR INRA et DGER, Ecole Nationale Vétérinaire de Lyon, 69280 Marcy l'étoile, France.
Buronfosse
T
T
Lachuer
J
J
Longin-Sauvageon
C
C
Moulin
C
C
Benoit
E
E
D000595:Amino Acid Sequence; D000818:Animals; D013956:Antithyroid Agents / Q000378:metabolism; D001483:Base Sequence; D002384:Catalysis; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000032:analysis; D004926:Escherichia coli; D015870:Gene Expression; D008713:Methimazole / Q000378:metabolism*; D008969:Molecular Sequence Data; D010105:Oxygenases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014018:Tissue Distribution
D013956:Antithyroid Agents; D018076:DNA, Complementary; D012333:RNA, Messenger; D011994:Recombinant Proteins; D008713:Methimazole; D010105:Oxygenases; C020066:dimethylaniline monooxygenase (N-oxide forming)