Characterization of a dinucleotide-binding site in monoamine oxidase B by site-directed mutagenesis.
Monoamine oxidase B (MAO B), an integral protein of the outer mitochondrial membrane, catalyzes the oxidative deamination of neuroactive and vasoactive amines. The oxidation step is coupled to the reduction of an obligatory FAD cofactor. In this study, we have examined the role of one amino acid (Glu34) in human MAO B that is thought to play a crucial role in binding to the 2'-hydroxy group of ribose in the AMP moiety of FAD. Glu34 is located within a region of the MAO B molecule of high sequence identity to the dinucleotide-binding site in other flavoproteins. In MAO B, this region is postulated to consist of a beta 1-sheet-alpha-helix-beta 2-sheet motif which culminates with a Glu at the C-terminal end of the second beta-sheet. We used site-directed mutagenesis to convert Glu at position 34 to Asp, Gln, and Ala. The wild-type and mutant cDNAs were then transiently transfected into COS-7 cells and assayed for MAO B activity. All three variants exhibited a dramatic decrease in the enzymatic activity as compared to wild-type MAO B, and only the Asp variant retained any detectable activity. Our studies indicate that the Glu34 residue in human MAO B is essential for catalysis. Whether Glu34 is responsible only for alignment of the FAD for participation in the oxidation/reduction cycle or also for the initial binding of FAD to the apoenzyme remains to be determined.
Kwan
S W
SW
Division of Medicinal Chemistry, College of Pharmacy, University of Texas at Austin 78712-1074.
Lewis
D A
DA
Zhou
B P
BP
Abell
C W
CW
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002478:Cells, Cultured; D004252:DNA Mutational Analysis; D018076:DNA, Complementary / Q000235:genetics; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism*; D018698:Glutamic Acid / Q000235:genetics; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008995:Monoamine Oxidase / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D014162:Transfection
D018076:DNA, Complementary; D011994:Recombinant Proteins; D005182:Flavin-Adenine Dinucleotide; D018698:Glutamic Acid; D008995:Monoamine Oxidase