title: Influence of FAD structure on its binding and activity with the C406A mutant of recombinant human liver monoamine oxidase A.
abstract: The FAD binding site of human liver monoamine oxidase A (MAO A) has been investigated by mutagenesis of the amino acid site of covalent FAD attachment (Cys-406) to an alanyl residue. Expression of the C406A mutant in Saccharomyces cerevisiae results in the formation of an active enzyme, as found previously with the rat liver enzyme. The activity of this mutant enzyme is labile to solubilization, thus requiring all experiments to be done with membrane preparations. C406A MAO A was expressed in a rib 5(-) strain of S. cerevisiae in the presence of 16 different riboflavin analogues. Inactive apoC406A MAO A is formed by induction of the enzyme in the absence of riboflavin. FAD but not FMN or riboflavin restores catalytic activity with an apparent K(d) of 62 +/- 5 nm. The results from both in vivo and in vitro reconstitution experiments show increased activity levels (up to approximately 7-fold higher) with those analogues exhibiting higher oxidation-reduction potentials than normal flavin and decreased activity levels with analogues exhibiting lower potentials. Analogues with substituents on the pyrimidine ring bind to C406A MAO A more weakly than normal FAD, suggesting specific interactions with the N(3) and N(1) positions. Analogues with substituents in the 7 and 8 positions bind to C406A MAO A with affinities comparable with that of normal FAD. These results are discussed in regard to functional significance of 8alpha-covalent binding of flavins to proteins.
authors.affiliation: Departments of Biochemistry and Chemistry, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
authors.forename: R K
authors.initials: RK
authors.lastname: Nandigama
authors.forename: D E
authors.initials: DE
authors.lastname: Edmondson
mesh_terms: D001665:Binding Sites; D004795:Enzyme Stability; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008995:Monoamine Oxidase / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D010084:Oxidation-Reduction; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D012256:Riboflavin / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D013329:Structure-Activity Relationship
chemical_list: D011994:Recombinant Proteins; D005182:Flavin-Adenine Dinucleotide; D008995:Monoamine Oxidase; D012256:Riboflavin