DETERMINATION OF FAD-BINDING DOMAIN IN FLAVIN-CONTAINING MONOOXYGENASE-1 (FMO1)

The flavin-containing monooxygenases (FMOs) are a family of flavoenzym es and contain one molecule of FAD per monomer, In order to demonstrat e where FMO interacts with FAD, four mutants for the rat liver FMO1 pr otein were expressed in yeast and characterized. All four mutants were immunochemically similar to the unmodified form, although the content s of FAD in all four mutants were much lower than that in the unmodifi ed form, Interestingly, the mutant generated by changing the first gly cine of the proposed FAD-binding domain (GxGxxG) to alanine revealed c atalytic activities, but was lower than those seen with the unmodified form, The conversion of the first glycine to alanine markedly increas ed and decreased the K-m and V-max values for imipramine N-oxidation, respectively. The other three mutants (RFMOm2, RFMOm3, and RFMOm4) wer e catalytically inactive. Our results suggest that three glycines, esp ecially the second and third glycines, in the proposed FAD-binding dom ain were necessary for FMO to show catalytic activities. Using RFMOm1 and the unmodified form, the effects of n-octylamine on the activity o f FMO1 were investigated. The activities of both wild-type and RFMOm1 enzymes for all of the compounds examined were enhanced by n-octylamin e, The K-m and V-max values of both RFMOm1 and the unmodified form for imipramine N-oxidation were lowered and raised by n-octylamine, respe ctively. (C) 1997 Academic Press.