Flavinylation of Monoamine Oxidase B

Abstract

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...

ADP Competes with FAD Binding in Putrescine Oxidase

“…In general, several roles can exist for the covalent linkage(s) between a protein and its flavin cofactor (22)(23)(24). For MAO-A and MAO-B, the role of the covalent flavin linkage has been extensively studied (25)(26)(27)(28)(29), and the effect of replacing the linking cysteine residue (Cys-406 in MAO-A and Cys-397 in MAO-B) has been investigated (30,31). These mutagenesis studies revealed that, while replacement of the cysteine prevents covalent FAD incorporation, the covalent flavin linkage is not essential for catalytic activity, neither for targeting and insertion of the protein in the mitochondrial membrane.…”

Monoamine Oxidase B Isolated from Bovine Liver Exists as Large Oligomeric Complexes in Vitro

“…Site-directed mutagenesis has been used to identify specific amino acid residues (glutamate and tyrosine) in a region of M A 0 B located near the amino terminus that participate in the non-covalent binding of FAD [14, 1.51. Furthermore, changes of glutamate to aspartate, glutamine or alanine can affect covalent flavinylation of M A 0 B [16]. Also, hybrids of M A 0 A and M A 0 B have been examined in attempts to identify which amino acids or regions of the molecule might confer isozyme specificity [17].…”

Characterization of a Highly Conserved FAD-binding Site in Human Monoamine Oxidase B

“…14 C]FAD was synthesized by a modified method of Manstein and Pai (22) as described previously (18).…”

“…This dinucleotide-binding site is observed in many flavoproteins with diverse functions and is thought to consist of a ␤ 1 -␣-␤ 2 motif (19), in which the terminal glutamate (Glu 34 ) interacts with the 2Ј-hydroxyl group of the ribose moiety of FAD. Site-directed mutagenesis studies, which replaced Glu 34 with alanine, aspartate, or glutamine, resulted in a dramatic loss of FAD coupling and, consequently, a corresponding loss of MAO B enzymatic activity (1,18). A second FAD-binding region was found adjacent to the dinucleotide-binding motif in MAO B (residues 39 -46 in human MAO B).…”

“…Detergent-extracted bovine liver MAO B appears to operate as an oligomeric complex (17). Although the precise steps required for flavinylation of MAO B remain unknown, it appears that FAD initially binds noncovalently to the consensus sequences near the amino terminus (1,2) and subsequently is covalently linked to Cys 397 (18). Since the MAOs are integral proteins in the outer mitochondrial membrane, it has been difficult to obtain a crystal structure to identify the precise residues that constitute the active site and those that interact with FAD.…”