Determination of FAD-binding domain in flavin-containing monooxygenase 1 (FMO1).
The flavin-containing monooxygenases (FMOs) are a family of flavoenzymes and contain one molecule of FAD per monomer. In order to demonstrate where FMO interacts with FAD, four mutants for the rat liver FMO1 protein were expressed in yeast and characterized. All four mutants were immunochemically similar to the unmodified form, although the contents of FAD in all four mutants were much lower than that in the unmodified form. Interestingly, the mutant generated by changing the first glycine of the proposed FAD-binding domain (GxGxxG) to alanine revealed catalytic activities, but was lower than those seen with the unmodified form. The conversion of the first glycine to alanine markedly increased and decreased the Km and Vmax values for imipramine N-oxidation, respectively. The other three mutants (RFMOm2, RFMOm3, and RFMOm4) were catalytically inactive. Our results suggest that three glycines, especially the second and third glycines, in the proposed FAD-binding domain were necessary for FMO to show catalytic activities. Using RFMOm1 and the unmodified form, the effects of n-octylamine on the activity of FMO1 were investigated. The activities of both wild-type and RFMOm1 enzymes for all of the compounds examined were enhanced by n-octylamine. The Km and Vmax values of both RFMOm1 and the unmodified form for imipramine N-oxidation were lowered and raised by n-octylamine, respectively.
Kubo
A
A
Division of Drug Metabolism, Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan.
Itoh
S
S
Itoh
K
K
Kamataki
T
T
D000818:Animals; D001665:Binding Sites; D005182:Flavin-Adenine Dinucleotide; D005998:Glycine / Q000235:genetics; D007099:Imipramine / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008861:Microsomes / Q000201:enzymology; D016297:Mutagenesis, Site-Directed; D009249:NADP; D010105:Oxygenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013854:Thioamides / Q000378:metabolism
D011994:Recombinant Proteins; D013854:Thioamides; D005182:Flavin-Adenine Dinucleotide; D009249:NADP; C015539:thiobenzamide; D010105:Oxygenases; C020066:dimethylaniline monooxygenase (N-oxide forming); D007099:Imipramine; D005998:Glycine