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...