Cloning and sequencing of flavin-containing monooxygenases FMO3 and FMO4 from rabbit and characterization of FMO3.

Abstract

The flavin-containing monooxygenases (FMO) are a family of enzymes that contain putative FAD- and NADPH-binding domains within the first 200 residues of their N termini. The cDNAs encoding these enzymes contain an area of relatively high identity over the 5' half of the coding region. Rabbit genomic DNA was probed under low stringency conditions, with a mixture of 5' cDNA fragments encoding rabbit FMO1, FMO2, or FMO5. Bands associated specifically with FMO1, FMO2, or FMO5 were resolved by analysis at high stringency with individual probes. Several bands were detected that could not be assigned to FMO1, FMO2, or FMO5. The behavior of the 5' probes at low versus high stringency was used to facilitate the isolation of cDNAs corresponding to the unknown DNA bands. A cDNA library was constructed from rabbit liver mRNA and screened under low stringency hybridization conditions (30 degrees C, 50% formamide, 1 x SSC, 0.1% SDS) with the mixture of 5' FMO1, FMO2, and FMO5 cDNA probes. A total of 157 clones was detected. Of these, 117 clones remained under high stringency hybridization conditions (65 degrees C, 50% formamide, 0.1 x SSC, 0.1% SDS) and were identified as FMO1 (95 clones) or FMO5 (22 clones). Of the 40 remaining clones, 36 were characterized by sequence analysis as encoding FMO3, previously identified at the protein level by Ozols (Ozols, J. (1991) Arch. Biochem. Biophys. 290, 103-115) as a second rabbit liver FMO. Four clones were shown to encode an FMO not previously described for the rabbit, FMO4. No clones encoding FMO2 were isolated from the liver library. Sequence analysis revealed that FMO3 and FMO4 are 56% identical, and analysis of genomic DNA indicated that each is encoded by a single gene. Message distribution was tissue-, species-, and form-specific. The properties of FMO3 cDNA expressed in Escherichia coli were found to be more similar to those of FMO1 than FMO2, but to differ significantly from both. Rabbit genomic DNA was probed under conditions of low stringency with a mixture of 5' cDNA fragments encoding all five FMO forms and produced results consistent with the possibility of one additional FMO.