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Determination of FAD-binding domain in flavin-containing monooxygenase 1 (FMO1)
1Division of Drug Metabolism, Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan.
Abstract:
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.
Insights
Flavin-containing monooxygenases (FMOs) require specific glycines in their FAD-binding domain for catalytic activity. Mutations in this domain reduce FAD content and alter enzyme kinetics, highlighting the importance of glycine residues for FMO function.
Area of Science:
- Biochemistry
- Enzymology
Background:
- Flavin-containing monooxygenases (FMOs) are crucial flavoenzymes involved in various metabolic processes.
- FMOs contain a flavin adenine dinucleotide (FAD) molecule per monomer, essential for their catalytic activity.
Purpose of the Study:
- To investigate the interaction site between FMO and FAD within the FMO1 protein.
- To elucidate the role of specific glycine residues in the FAD-binding domain of FMO1 for enzyme function and catalysis.
Main Methods:
- Expression and characterization of four mutant rat liver FMO1 proteins in yeast.
- Immunochemical analysis to assess protein similarity to the unmodified form.
- Quantification of FAD content in wild-type and mutant enzymes.
- Enzyme kinetic studies (Km and Vmax) for imipramine N-oxidation.
- Investigation of the effects of n-octylamine on enzyme activity.
Main Results:
- All four FMO1 mutants exhibited reduced FAD content compared to the wild-type.
- A mutant (RFMOm1) with the first glycine of the GxGxxG motif changed to alanine retained catalytic activity but showed altered kinetic parameters (increased Km, decreased Vmax) for imipramine N-oxidation.
- Three other mutants (RFMOm2, RFMOm3, RFMOm4) were catalytically inactive.
- n-Octylamine enhanced the activity of both wild-type and RFMOm1 enzymes, lowering Km and raising Vmax for imipramine N-oxidation.
Conclusions:
- The study suggests that three glycine residues, particularly the second and third, within the proposed FAD-binding domain are critical for FMO1 catalytic activity.
- The results highlight the structural and functional importance of the GxGxxG motif for FAD binding and enzymatic function in FMOs.
- n-Octylamine may act as a modulator of FMO1 activity, influencing substrate binding and turnover rate.