Monoamine oxidase B (MAO B)The major amine-degrading enzymes in the central nervous system and peripheral tissues of mammals are monoamine oxidase A and B (MAO 1 A and B, amine:oxygen, oxidoreductase (deaminating, flavin-containing), EC 1.4.3.4). These isozymes are integral proteins of the outer mitochondrial membrane (1) and can be distinguished by differences in substrate preference (2), inhibitory specificity (3), tissue and cell distribution (4 -6), and immunological properties (7-9). Furthermore, comparison of their nucleotide and deduced amino acid sequences show that human MAO A and B are two distinct proteins encoded by different genes (10).Oxidation of amines by MAO is coupled to the reduction of an obligatory cofactor, FAD, which is covalently linked to the enzyme. Five types of bonds are generally found in the covalent linkage of flavins to their respective apoproteins (11). These include a histidine residue which can be attached through its N-1 or N-3 atom to the 8␣-methyl group of the isoalloxazine ring to form a tertiary amine; a cysteine residue which forms a thioether linkage with either the 8␣-methyl group or the C-6 of the xylene ring of the flavin molecule; or a tyrosine residue can become linked to the 8␣-methyl group to form an (O)-8␣-flavin bond. In MAO A and B, the 8␣-methyl group of FAD is bound covalently to cysteine through a thioether linkage in the pentapeptide SGGCY (12, 13). Comparison of this segment with the complete deduced amino acid sequences of MAO A and B indicated that FAD is covalently bound to Cys 406 in MAO A and Cys 397 in MAO B, respectively (10). In addition, site-directed mutagenesis studies of MAO B, where Cys 397 was substituted with serine or histidine, showed that this cysteine residue is essential for catalytic activity (14,15).Although the amino acid sequences surrounding the FAD covalent attachment site in different flavoproteins bear little homology, a distinct non-covalent FAD-binding site displays high sequence identity in many FAD-containing enzymes of diverse function (16,17). This non-covalent FAD binding region is commonly referred to as the dinucleotide-binding site or motif due to its interaction with the AMP moiety of FAD. This motif consists of a  1 -sheet-␣-helix- 2 -sheet beginning with a highly conserved Gly-X-Gly-X-X-Gly sequence between the first -sheet and the ␣-helix. The second -sheet usually ends with a glutamate residue in which the ␥-carboxylate group is thought to interact through a hydrogen bond with the 2Ј-hydroxyl group of ribose in the AMP moiety of FAD. In MAO A and B, this motif is located at the N terminus of MAO A (residues 15-43) and MAO B (residues 6 -34) and ends in Glu 43 and Glu 34 , respectively. Site-directed mutagenesis studies, where Glu 34 was replaced with aspartate, glutamine, or alanine, resulted in near complete or total loss of catalytic activity in MAO B (18).A fundamental process in the intracellular generation of functional flavoenzymes is the molecular mechanism which generates holoenzyme from apoenzyme a...
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)
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