title: Mutagenesis at a highly conserved tyrosine in monoamine oxidase B affects FAD incorporation and catalytic activity.
abstract: Monoamine oxidase B (MAO B), an integral protein of the outer mitochondrial membrane, catalyzes the oxidative deamination of various neuroactive and vasoactive amines. A covalently bound FAD cofactor at Cys-397 of human MAO B is required for the oxidation of the amine substrates. In addition to the covalent binding site, MAO B also contains a noncovalent FAD binding region (residues 6-34) known as the dinucleotide binding motif. Previously, we have shown that Glu-34 is required for catalytic activity, presumably by forming a hydrogen bond between the carboxylate group of glutamate and the 2'-hydroxyl group of ribose in the AMP moiety of FAD. In this work, we have identified a third FAD binding site in MAO B (residues 39-46) by sequence comparisons to other flavoenzymes. The conserved sequence contains a tyrosine residue (Tyr-44) which, based on the X-ray crystal structure of ferredoxin-NADP+ reductase, is postulated to participate in FAD binding through van der Waals contact with the isoalloxazine ring and a hydrogen bond to the 3'-hydroxy of the ribityl moiety. To test the postulated role of this tyrosine residue, site-directed mutants that encode substitutions at Tyr-44 were prepared and expressed in mammalian COS-7 cells. Variant MAO B enzymes were then characterized with respect to enzymatic activity and [14C]FAD incorporation. Substitution of tyrosine with phenylalanine had no effect on MAO B activity or the level of [14C]FAD incorporation compared to the wild-type enzyme, indicating that the hydroxyl group of the tyrosine residue was not essential at residue 44.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Division of Medicinal Chemistry, College of Pharmacy, University of Texas at Austin 78712-1074, USA.
authors.forename: B P
authors.initials: BP
authors.lastname: Zhou
authors.forename: D A
authors.initials: DA
authors.lastname: Lewis
authors.forename: S W
authors.initials: SW
authors.lastname: Kwan
authors.forename: T J
authors.initials: TJ
authors.lastname: Kirksey
authors.forename: C W
authors.initials: CW
authors.lastname: Abell
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D017360:Arabidopsis / Q000201:enzymology; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D002522:Chlorocebus aethiops; D017124:Conserved Sequence; D004926:Escherichia coli / Q000201:enzymology; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D006801:Humans; D007668:Kidney; D007700:Kinetics; D008961:Models, Structural; D008969:Molecular Sequence Data; D008995:Monoamine Oxidase / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D017354:Point Mutation; D011487:Protein Conformation; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D018724:Spinacia oleracea / Q000201:enzymology; D014162:Transfection; D014443:Tyrosine
chemical_list: D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D005182:Flavin-Adenine Dinucleotide; D014443:Tyrosine; D008995:Monoamine Oxidase