Molecular cloning and characterization of a full-length flavin-dependent monooxygenase from yeast.
Eucaryotes contain a class of enzymes called flavin-dependent monooxygenases (FMOs). Unlike mammals, yeast have only a single isoform-yFMO. Deletion mutants suggested that yFMO may play a role in folding proteins which contain disulfide bonds. Recently we detected two nucleotide errors in the GenBank sequences attributed to the yFMO gene. This previously led us to express and characterize a 373-residue catalytically active protein instead of the correct 432-residue enzyme. Here we report the sequencing, expression, and enzyme characterization of the full-length form of yFMO. Comparison of the two forms of yFMO showed similar pH profiles and K(m), K(cat), and V(max) values using glutathione as a substrate. These results indicate that the full-length yeast FMO has biochemical and catalytic properties similar to those of the truncated protein. Therefore, it is likely that the hypotheses concerning the enzyme's function proposed earlier are still valid.
Zhang
Man
M
Department of Chemistry and Biochemistry, Institute of Cellular and Molecular Biology, University of Texas, Austin, TX 78712, USA.
Robertus
Jon D
JD
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D030561:Databases, Nucleic Acid; D004926:Escherichia coli / Q000235:genetics; D005978:Glutathione / Q000378:metabolism; D006639:Histidine / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D010105:Oxygenases / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D006639:Histidine; D010105:Oxygenases; C020066:dimethylaniline monooxygenase (N-oxide forming); D005978:Glutathione