Functional characterization of flavin-containing monooxygenase 1B1 expressed in Saccharomyces cerevisiae and Escherichia coli and analysis of proposed FAD- and membrane-binding domains.

A cDNA encoding the flavin-containing monooxygenase of rabbit lung (FMO 1B1) was expressed in yeast and Escherichia coli and the recombinant enzymes characterized. A high copy, isopropyl-1-thio-beta-D-galactopyranoside (IPTG)-inducible E. coli expression vector, pKKHC, was used for expression in E. coli strain JM109, and a galactose-inducible vector, YEp53, was used for expression in yeast strain 334. Following transcriptional induction with IPTG or galactose, subcellular fractions were prepared and analyzed immunochemically and catalytically. Antibodies to rabbit FMO 1B1 were used to detect the recombinant proteins in the 100,000 x g pellet prepared from the 10,000 x g supernatant fraction of yeast homogenates and the 2,000 x g supernatant fraction of E. coli homogenates. No FMO 1B1 was detected in cytosol. Mobilities of the recombinant proteins in SDS-polyacrylamide gel electrophoresis appeared identical to that of the native microsomal enzyme. Catalytic similarity to the native FMO 1B1 was demonstrated by the ability of the expressed enzymes to metabolize methimazole, thiourea, dimethylaniline, and cysteamine, but not chlorpromazine or imipramine. In addition, the recombinant enzymes exhibited a number of the unique physical properties associated with FMO 1B1, including stability to elevated temperature and activation by sodium cholate and magnesium chloride. Based on the specific content of FAD, the level of expression was estimated to be approximately 2% of the total protein in the E. coli 100,000 x g particulate fraction and 1% in the fraction from yeast. To demonstrate the utility of the E. coli expression system for studying structure/function relationships of the flavin-containing monooxygenase, two mutant FMOs were expressed and characterized. One mutant, formed by deletion of a putative membrane-anchoring peptide (the 26 carboxyl-terminal amino acids) was tested for membrane association. No difference in the subcellular distribution was found between the truncated and unmodified proteins, suggesting that the 26-residue COOH-terminal peptide is not important in membrane association. Catalytic analysis of the truncated FMO 1B1 established its functional similarity to the full-length protein, indicating that the COOH terminus does not contribute to any of the unique properties of the lung enzyme.(ABSTRACT TRUNCATED AT 400 WORDS)