Monoamine oxidase (MAO) is responsible for the oxidation of biogenic and dietary amines. It exists as two isoforms, A and B, which have a 70% amino acid identity and different substrate and inhibitor specificities. This study reports the identification of residues responsible for conferring this specificity in human MAO A and B. Using site-directed mutagenesis we reciprocally interchanged three pairs of corresponding nonconserved amino acids within the central portion of human MAO. Mutant MAO A-I335Y became like MAO B, which exhibits a higher preference for ␤-phenylethylamine than for the MAO A preferred substrate serotonin (5-hydroxytryptamine), and became more sensitive to deprenyl (MAO B-specific inhibitor) than to clorgyline (MAO A-specific inhibitor). The reciprocal mutant MAO B-Y326I exhibited an increased preference for 5-hydroxytryptamine, a decreased preference for ␤-phenylethylamine, and, similar to MAO A, was more sensitive to clorgyline than to deprenyl. These mutants also Monoamine oxidase (MAO 1 ; amine:oxygen oxidoreductase (deaminating) (flavin-containing), EC 1.4.3.4) catalyzes the oxidative deamination of biogenic and xenobiotic amines and plays an important role in regulating their levels. MAO is a flavin-adenine dinucleotide-containing enzyme located on the mitochondrial outer membrane (1-3). It exists in two forms, A and B. MAO A preferentially oxidizes serotonin (5-hydroxytryptamine, 5-HT) and is inhibited by low concentrations of clorgyline (4) and Ro 41-1049 (5), whereas MAO B preferentially oxidizes ␤-phenylethylamine (PEA) and is inhibited by low concentrations of (Ϫ)-deprenyl (6) and Ro 16 -6491 (7). Dopamine is a common substrate (8). MAO A and B are composed of 527 and 520 amino acids, respectively, and have a 70% amino acid identity (9). They are closely linked on the X-chromosome (10) and have an identical intron-exon organization, indicating that they are derived from a common ancestral gene (11). Higher 5-HT and norepinephrine levels and a phenotype characterized by increased aggressive behavior is observed when the MAO A gene is deficient in humans (12) and in mice (13). Disruption of the MAO B gene in mice results in increased PEA but not 5-HT, norepinephrine, or dopmanie and confers a resistance to the Parkinsonism-inducing toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (14). Because MAO is an integral membrane protein, it is difficult to crystallize, and its three-dimensional structure has not been reported.To determine the region(s) responsible for the substrate and inhibitor specificities of the two isoenzymes, we and other groups have made point mutations and chimeric MAO A/B enzymes (15-21). It has been shown that reciprocally interchanging amino acids Phe-208 in MAO A and its corresponding residue in MAO B, Ile-199, was sufficient to partially reverse the substrate and inhibitor specificities of rat MAOs (22) but not human MAOs (21). This indicated that different amino acid residues may determine specificity in human and rat MAOs. We also found that the residues that...

Monoamine oxidase (MAO) is a key enzyme responsible for the degradation of serotonin, norepinephrine, dopamine, and phenylethylamine. It is an outer membrane mitochondrial enzyme existing in two isoforms, A and B. We have recently generated 14 site-directed mutants of human MAO A and B, and we found that four key amino acids, Lys-305, Trp-397, Tyr-407, and Tyr-444, in MAO A and their corresponding amino acids in MAO B, Lys-296, Trp-388, Tyr-398, and Tyr-435, play important roles in MAO catalytic activity. Based on the polyamine oxidase three-dimensional crystal structure, it is suggested that Lys-305, Trp-397, and Tyr-407 in MAO A and Lys-296, Trp-388, and Tyr-398 in MAO B may be involved in the non-covalent binding to FAD. Tyr-407 and Tyr-444 in MAO A (Tyr-398 and Tyr-435 in MAO B) may form an aromatic sandwich that stabilizes the substrate binding. Asp-132 in MAO A (Asp-123 in MAO B) located at the entrance of the U-shaped substrate-binding site has no effect on MAO A nor MAO B catalytic activity. The similar impact of analogous mutants in MAO A and MAO B suggests that these amino acids have the same function in both isoenzymes. Three-dimensional modeling of MAO A and B using polyamine oxidase as template suggests that the overall tertiary structure and the active sites of MAO A and B may be similar.Monoamine oxidase (MAO, 1 EC 1.4.3.4; amine:oxygen oxidoreductase (deaminating, flavin-containing)) is a flavoprotein located at the outer membranes of mitochondria in neuronal, glial, and other cells. It catalyzes the oxidative deamination of monoamine neurotransmitters such as serotonin, norepinephrine, and dopamine and appears to play important roles in several psychiatric and neurological disorders (for review see Refs. 1 and 2). In addition, it is also responsible for the biotransformation of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine into 1-methyl-4-phenylpyridinium, a Parkinsonian producing neurotoxin (3-5). Recently, it has been shown that MAO may contribute to the apoptotic process because inhibition of MAO activity suppressed cell death (6).MAO exists in two forms, namely MAO A and MAO B. MAO A preferentially oxidizes serotonin (5-hydroxytryptamine) and is irreversibly inhibited by low concentrations of clorgyline (7). MAO B preferentially oxidizes phenylethylamine (PEA) and benzylamine, and it is irreversibly inactivated by low concentrations of pargyline and deprenyl (8). Dopamine, tyramine, and tryptamine are common substrates for both MAOs. MAO A and B consist of 527 and 520 amino acids, respectively, and have a 70% identity (9). Each isoenzyme has a FAD covalently linked to a cysteine residue, Cys-406 in MAO A and Cys-397 in MAO B, through an 8␣-(cysteinyl)-riboflavin (10 -13). They exhibit identical exon-intron organization, and they are probably derived from the duplication of a common ancestral gene (14 Recently, the x-ray crystal structure of a related enzyme namely polyamine oxidase (PAO) has been obtained (25). PAO catalyzes the oxidation of the secondary amino group of polyamines, such as sp...

Monoamine oxidase is an outer mitochondrial membrane protein that catalyzes the deamination of a number of neurotransmitters and dietary amines. To determine the roles of the carboxyl-terminal amino acids on the activity and solubility of human monoamine oxidase (MAO B), 10 sequential mutants were made with stop codons at amino acid positions 511, 504, 498, 492, 486, 481, 476, 467, 417, and 397, respectively. All truncated mutants were expressed in Sf21 insect cells using baculovirus, and the enzyme kinetic parameters were determined. Truncations at amino acid positions 511, 504, and 498 slightly decreased MAO B catalytic activity and had no significant changes on deprenyl inhibition. Further deletions up to amino acid 417 decreased the specific activity 10 -100-fold without significant changes of the K m for phenylethylamine or dopamine or the IC 50 for deprenyl and clorgyline. The truncation mutant C397, which lacks covalently attached FAD, was inactive. Progressive carboxylterminal truncations up to position 481 were correlated with increased solubility of MAO B mutants. 47% of the activity of the truncated C481 was found in the 105,000 ؋ g supernatant in the absence of detergent. However, further truncated mutants, C476, C467, and C417, remained associated with the membrane fraction. In contrast to crude homogenate, the water-soluble C481 mutant was rapidly inactivated at 4°C and 37°C, which indicates that the membrane environment is required for the stability of MAO B. Expression of the green fluorescent protein-MAO B C481 fusion protein revealed that this mutant was located in the cytoplasm, whereas its counterpart in MAO A, truncated mutant C490, was located on the mitochondria. These results suggest that the carboxyl-terminal amino acid residues 417-520 of MAO B are not directly involved in the active site but are required for maintaining the appropriate conformation and interaction with the outer mitochondrial membrane. The different solubilities of the various carboxyl-terminal truncation mutants indicate that the interaction of MAO B with mitochondrial membrane is not simply anchoring through the carboxylterminal hydrophobic tail. Further, our results suggest that the carboxyl-terminal of MAO A and B plays different roles in mitochondrial attachment.Monoamine oxidase A and B (MAO 1 A and MAO B, EC 1.4.3.4) are flavoisoenzymes, which catalyze the oxidative deamination of neuroactive, vasoactive, and dietary amines (1). MAO A and B are encoded by different genes (2) and have different substrate and inhibitor specificities (3, 4). MAO A preferentially oxidizes serotonin (5-hydroxytryptamine) and is irreversibly inhibited by low concentrations of clorgyline. MAO B preferentially oxidizes phenylethylamine (PEA) and benzylamine, and it is irreversibly inactivated by low concentrations of pargyline and deprenyl. Dopamine, tyramine, and tryptamine are common substrates for both MAOs. MAO A and B consist of 527 and 520 amino acids, respectively, and have a 70% identity (2). Each type of enzyme has a FAD covale...

It has been reported previously that reciprocally switching Phe 208 and Ile 199 in rat monoamine oxidase (MAO) A and B, respectively, was sufficient to switch their substrate and inhibitor preferences. In this study, the same mutants were made in the human forms of MAO. When compared with MAO A, MAO A-F208I showed a sixfold decrease in the specificity constant k cat /K m for both the MAO A-and the MAO B-preferring substrates 5-hydroxytryptamine and ␤-phenylethylamine, respectively. The reciprocal point mutant MAO B-I199F had no effect on substrate affinity. To investigate if the region neighboring these two residues is responsible for conferring preferences, we have also made chimeric constructs by reciprocally switching the corresponding amino acid segments 159 -214 in 1304 -1309 (2000).Monoamine oxidase [MAO; amine:oxygen oxidoreductase (deaminating) (flavin-containing), EC 1.4.3.4] catalyzes the oxidative deamination of biogenic and xenobiotic amines and plays an important role in regulating their levels. It is a flavin-adenine dinucleotidecontaining enzyme located on the mitochondrial outer membrane (Nara et al., 1966;Greenawalt and Schnaitman, 1970;Shih et al., 1999). It exists as two isoenzymes, A and B. MAO A preferentially oxidizes serotonin [5-hydroxytryptamine (5-HT)] and is inhibited by low concentrations of clorgyline, whereas MAO B preferentially oxidizes low concentrations of ␤-phenylethylamine (PEA) and is inhibited by low concentrations of deprenyl. Common substrates include tyramine (Johnston, 1968;Knoll and Magyar, 1972) and dopamine. MAO A and B are composed of 527 and 520 amino acids, respectively, and have a 70% amino acid identity (Bach et al., 1988). They are closely linked on the X chromosome (Lan et al., 1989) and have an identical intron-exon organization, indicating that they are derived from a common ancestral gene (Grimsby et al., 1991). Knocking out the MAO A gene resulted in aggressive behavior in mice (Cases et al., 1995), whereas knocking out the MAO B gene resulted in mice resistant to the parkinsonism-inducing toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (Grimsby et al., 1997).To determine the region(s) responsible for the distinct substrate and inhibitor preferences of the two isoenzymes, we and other groups have made point mutants and chimeric MAO constructs exchanging corresponding portions of the isoenzymes (Gottowik et al., 1993(Gottowik et al., , 1995Wu et al., 1993;Tsugeno et al., 1995;Cesura et al., 1996;Chen et al., 1996). It has been shown that amino acid segments 161-375 in human MAO A and 152-366 in human MAO B contain part of the domain responsible for determining preference (Grimsby et al., 1996). It was later discovered that reciprocally switching amino acids F208 in rat MAO A and its corresponding residue in rat MAO B, I199, was sufficient to switch the substrate and inhibitor preferences of rat MAO A and B (Tsugeno and Ito, 1997). To determine if the human MAO equivalents of these two residues, which are located within the segments in our ea...

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