Flavinylation of monoamine oxidase B.
Monoamine oxidase B (MAO B) catalyzes the oxidative deamination of biogenic and xenobiotic amines. The oxidative step is coupled to the reduction of an obligatory cofactor, FAD, which is covalently linked to the enzyme at Cys397. In this study, we developed a novel riboflavin-depleted (Rib-) COS-7 cell line to study the flavinylation of MAO B. ApoMAO B can be obtained by expressing MAO B cDNA in these cells. We found that MAO B is expressed equally in the presence or absence of FAD and that apoMAO B can be inserted into the outer mitochondrial membrane. Flavinylation of MAO B was achieved by introducing MAO B cDNA and different flavin derivatives simultaneously into Rib- COS-7 cells via electroporation. Since the addition of riboflavin, FMN, or FAD resulted in equal levels of MAO B activity, we conclude that the flavin which initially binds to apoMAO B is FAD. In our previous work, we used site-directed mutagenesis to show that Glu34 in the dinucleotide-binding motif of MAO B is essential for MAO B activity, and we postulated that this residue is involved in FAD binding. In this study, we tested the role of residue 34 in flavin binding by expressing wild-type or mutant MAO B cDNA in Rib- COS-7 cells with the addition of [14C]FAD. We found that Glu34 is essential for both FAD binding and catalytic activity. Thus, FAD binds to MAO B in a dual manner at Glu34 noncovalently and Cys397 covalently. We conclude that Glu34 is critical for the initial non-covalent binding of FAD and is instrumental in delivering FAD to the covalent attachment site at Cys397.
Zhou
B P
BP
Division of Medicinal Chemistry, College of Pharmacy, University of Texas, Austin 78712-1074, USA.
Lewis
D A
DA
Kwan
S W
SW
Abell
C W
CW
D000818:Animals; D001051:Apoenzymes / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D002460:Cell Line; D018076:DNA, Complementary / Q000235:genetics; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D005415:Flavins / Q000378:metabolism*; D018698:Glutamic Acid / Q000235:genetics / Q000378:metabolism; D066298:In Vitro Techniques; D008928:Mitochondria / Q000201:enzymology; D008995:Monoamine Oxidase / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed
D001051:Apoenzymes; D018076:DNA, Complementary; D005415:Flavins; D005182:Flavin-Adenine Dinucleotide; D018698:Glutamic Acid; D008995:Monoamine Oxidase