A Key Amino Acid Responsible for Substrate Selectivity of Monoamine Oxidase A and B*

Monoamine oxidase (MAO) oxidizes biologically important amines including neurotransmitters and plays a central role in the regulation of intracellular level of these amines. Two distinct forms of MAO (MAO A and MAO B) were defined based on differences in substrate and inhibitor specificities. We earlier reported that the region between about residues 120 and 220 of rat MAO is responsible for determination of the substrate selectivity of MAO A and B (Tsugeno, Y. Hirashiki, I., Ogata, F., and Ito, A. (1995) J. Biochem. (Tokyo) 118, 974–980). To determine the essential amino acids in this region that participate in substrate recognition, a series of mutant enzymes in which amino acid residues that are conserved among various species but are different between the two forms of the enzyme were replaced with the corresponding amino acids of the counterpart and were engineered from the cDNAs of rat liver MAO A and B, and affinities for several substrates were examined. A single mutation in which Phe-208 in MAO A was substituted by the corresponding residue of Ile in MAO B was sufficient to convert the A-type substrate selectivity, and the reverse was exactly the case. Phe at this position was replaceable with Tyr for the A-type specificity and Ile was replaceable with Val and Ala for the B-type. Thus, aromatic and aliphatic residues seem to contribute to render substrate selectivity of MAO A and MAO B, respectively.

Monoamine oxidase (MAO) 1 oxidizes biologically important amines, including neurotransmitters, trace amines, and neurotoxins, and plays a central role in regulating intracellular levels of these amines (1)(2)(3)(4)(5). Two distinct forms of MAO, termed MAO A and MAO B, were defined based on differences in substrate and inhibitor specificities and tissue distributions (6 -9). Human placenta tissue contains predominantly MAO A, whereas platelets, lymphocytes, and chromaffin cells express primarily MAO B (10 -13). MAO A preferentially oxidizes serotonin and is irreversibly inactivated by low concentrations of the acetylenic inhibitor clorgyline. MAO B oxidizes phenylethylamine and is irreversibly inactivated by pargyline and deprenyl. Dopamine, tyramine, and tryptamine are substrates for both forms of MAO.
The extent of MAO activity is associated with various behavioral aberrations. MAO A deficiency was found to be associated with impulsive aggressive behavior (14). Reduction of MAO B activity may synergize with nicotine to produce diverse behavioral and epidemiological effects of smoking tobacco (15). Inhibitors of MAO A are widely used to treat affective disorders, whereas MAO B inhibitors are of benefit for subjects with neurological disorders such as Parkinson's disease. MAO B inhibition is associated with the enhanced activity of dopamine, as well as with the decreased production of hydrogen peroxide, a reactive oxygen species. One needs to know which region or residues in the molecule confer MAO A or MAO B enzymatic activity to develop highly selective inhibitors as well as to elucidate recognition mechanisms of related enzymes.
cDNAs of the two isozymes of MAO have been cloned from human, bovine, and rat tissues (16 -20). Primary structures deduced from the cDNAs revealed that rat liver MAO A and B consist of 526 and 520 amino acid residues, respectively, and that the amino acid sequences of the two enzymes are approximately 70% identical and show more than 90% sequence similarity when conservative amino acid substitutions are included (19,20). We used chimeric enzymes constructed from MAO A and MAO B and found that the region between about residues 120 -220 is responsible for determination of the substrate specificity of MAOs, whereas the middle portion, about residues 220 -400, may relate to the relative catalytic activity toward substrates (21). Essentially the same results were reported by Gottowik et al. (22); Shih et al. described the importance of the C-terminal and middle portions of MAO B to maintain MAO B in an active form (23,24). In the present work, we attempted to elucidate amino acids responsible for substrate recognition of MAOs. We obtained evidence that a single amino acid, Phe-208 in MAO A and Ile-199 at the corresponding position in MAO B, has a vital role determining the substrate selectivity of MAOs.

EXPERIMENTAL PROCEDURES
Materials-Oligonucleotides were purchased from Biologica Co. (Nagoya, Japan). Restriction endonucleases and DNA-modifying enzymes were purchased from Nippon Gene (Toyama, Japan), Toyobo (Osaka, Japan), Takara Shuzo (Kyoto, Japan), and New England Bio  (26). The original and chimeric cDNAs constructed above were then inserted into pYcDE2, a yeast expression vector.
Expression of Mutant MAOs in Yeast Cells and Preparation of Mito-* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The transformed cells were cultured to the mid-logarithmic phase in YPD medium with vigorous shaking, and then the cells were harvested by centrifugation at 2,200 ϫ g for 10 min. Mitochondrial fraction was prepared as described (21). Briefly, after treatment of the cells with Zymolyase 100 T (2 mg/g wet cells) for 60 min at 30°C, the resultant spheroplasts were lysed in 10 mM HEPES-KOH buffer, pH 7.4, containing 0.65 M sorbitol, 1 mM EDTA, and a protease inhibitor mixture (0.1 mg/ml chymostatin, 2 mg/ml aprotinin, 1 mg/ml pepstatin A, 1.1 mg/ml phosphoramidon, 7.2 mg/ml E-64, 0.5 mg/ml leupeptin, 2.5 mg/ml antipain, 0.1 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride) with a Dounce homogenizer. The lysate was centrifuged at 1,000 ϫ g for 10 min, and the supernatant was further centrifuged at 10,000 ϫ g for 10 min to obtain the mitochondrial fraction. The mitochondrial pellet was finally suspended in 10 mM HEPES-KOH buffer, pH 7.4, containing 15% glycerol, 0.65 M sorbitol, and 1 mM EDTA and stored at Ϫ80°C until use.
Assay of MAO Activity-Because each mutant enzyme had a different stability after solubilization of the mitochondrial membranes, we used fresh mitochondrial fractions for MAO activity. The activity was determined by the radiometric procedure described by Wurtmann and Axelrod (28), using as substrates serotonin, tryptamine, tyramine, and PEA. The assay mixture contained the mitochondrial fraction (50 g of protein) in a total volume of 60 l of 50 mM phosphate buffer, pH 7.4. The reaction, initiated by adding the substrate, was run for 20 min at 37°C and was stopped by the addition of 40 l of 2 M HCl. When tryptamine and PEA were used as substrate, the reaction time was set within the period (2 min) for which the reaction progressed linearly. The product was extracted with water-saturated ethyl acetate-toluene (1:1 v/v), and the radioactivity that was transferred into the organic solvent phase was estimated in a Packard Tri-Carb liquid scintillation spectrometer.
In inhibition experiments, mitochondria were preincubated with various concentrations of clorgyline or deprenyl in 50 mM phosphate buffer, pH 7.4, at 37°C for 30 min, and the remaining activity was determined as described above.

RESULTS
Comparison of the primary structures of the two forms of rat MAO with those from other species revealed a greater degree of similarity between the same form from different species than between different forms from the same species. The amino acid residues that are identical in different species in each form can be inferred to have been conserved during evolution because of their functional importance. The residues that are conserved among various species but different between the two forms might be those responsible for substrate selectivity of MAO. In a region between residues 120 and 220 that has been determined to be important in conferring substrate selectivity of MAO A and MAO B (21), only nine amino acid residues are identical in each form of MAO but are different between the two forms (amino acid residues boxed in Fig. 1). Five nonconservative amino acid residues in MAO B (shaded in Fig. 1) were replaced with the corresponding amino acids in MAO A, by site-directed mutation using oligonucleotides. The cDNAs encoding the wild-type and mutated MAOs were introduced into yeast cells, and the mitochondrial fractions were isolated from the cells expressing the enzymes, as described under "Experimental Procedures." Monoamine oxidase activities in the mitochondrial fractions were measured using as substrates serotonin, tryptamine, tyramine, and PEA.
As described in the previous report (21), the specific activities of the mutant proteins based on mitochondrial protein varied, and the mutants derived from MAO B, even the parent MAO B, usually had very low activity in yeast cells with every substrate used. Thus, a comparison of the V max values among the mutant enzymes would be invalid. In the present study, affinity for four substrates (K m ) served as a criterion for ascertaining which type of the substrate selectivity was rendered to the mutant enzymes (Table I). The MAO B variants carrying a His residue instead of Leu at position 139, MAO B(L139H), has substantially the same affinity profile for the substrates as that of the wild-type MAO B, that is, high affinity to PEA and no detectable activity toward serotonin. Affinity for the substrates of the mutant with triple substitutions at positions 172, 175, and 177, MAO B(C172N/A175S/T177P), was also unaffected. However, the mutant B(I199F), in which Ile-199 was converted to Phe, exhibited a dramatic increase in affinity for serotonin and tyramine, and their K m values were close to those of MAO A, although practically no change was observed in the affinity for tryptamine but with some decrease in that for PEA. The result indicates that the amino acid residue at position 199 in MAO B is important for selective binding of some substrates such as serotonin and tyramine. Because the above data pointed to Ile-199 as the single most important determinant of substrate selectivity of MAO B, we asked whether the corresponding amino acid in MAO A plays the same role in the substrate recognition. Phe at position 208 in MAO A was mutated to Ile to make the counter mutant of B(I199F), A(F208I). As shown in Table I, the mutation caused a marked reduction in affinity for serotonin and tyramine, to the same level of that in MAO B with practically no change in the affinity to tryptamine and PEA. This confirmed that an amino acid residue at the position 208 in MAO A and the residue at the corresponding position in MAO B plays a vital role in determination of substrate selectivity of MAO A and MAO B.
To determine whether structural differences between Ile and Phe, a hydrophobic branched chain and an aromatic ring, respectively, contribute to different affinities for the substrates, Phe-208 in MAO A was replaced with Tyr, Val, and Ala. Mutation of Phe to Tyr had no effect on affinity for all the substrates used. However, mutants in which Phe was converted to the residues with aliphatic side chains, A(F208V) and A(F208A), exhibited exactly the same affinity profile as that of the mutant A(F208I) discussed above. Thus, substrate selectivity of MAO can be explained by the different aromatic and aliphatic side chains of amino acids at this position.
Effects of the type-specific inhibitors on the mutation at position 208 in MAO A were examined using clorgyline and deprenyl as inhibitors for MAO A and MAO B, respectively. PEA was used as the substrate because all the enzymes examined oxidized this substrate. IC 50 values of clorgyline and deprenyl for MAO A were reported to be 0.025 M and 5.0 M, respectively, and those for MAO B were reported to be 79 M and 0.13 M, respectively (21). 0.5 and 1 M of clorgyline and deprenyl, respectively, were then used to examine the different sensitivity of the mutants to the two inhibitors (Fig. 2). At these concentrations of inhibitors, MAO A activity was abolished by clorgyline, whereas the full activity remained even in the presence of deprenyl. The reverse was true for MAO B. As expected from data on the substrate selectivity of B-type, B(L139H) and B(C172N/A175S/T177P) were inhibited only with deprenyl. However, B(I199F) had substantially the same inhibition pattern as those mutants and MAO B, although the enzyme did exhibit an A-type nature at least with regard to the affinity for serotonin and tyramine. On the contrary, patterns of sensitivity to inhibitors of A(F208I), A(F208V), and A(F208A) converted to that of the B-type enzymes with the single amino acid mutation, as in the case for substrate selectivity. A(F208Y) showed the same inhibitor sensitivity as the parent enzyme. Thus, except for B(I199F), all mutant enzymes had inhibitor sensitivity expected from their substrate selectivity. DISCUSSION We obtained evidence that an amino acid residue at position 208 in MAO A and the residue at the corresponding position 199 in MAO B plays an important role in determination of substrate selectivity of MAO A and MAO B. An enzyme with aromatic amino acids, such as Phe and Tyr, at this position has a similar affinity for all substrates examined, whereas ones with amino acids with an aliphatic side chain, such as Ile, Val, and Ala, have little affinity for serotonin and tyramine and a high affinity for tryptamine and PEA, although these amino acids are usually classified as hydrophobic amino acids. A good correlation between aromatic amino acid at this position and MAO A-type substrate specificity was seen in trout MAO (29), which has Phe at this position and properties in substrate specificity and inhibitor sensitivity more like those of mammalian MAO A, although it does share a similar extent of homology (about 70%) with both mammalian MAO A and MAO B.
MAO A has a similar affinity for most substrates with aromatic rings, yet over a 1000-fold difference in the affinity among substrates was observed for MAO B. The finding of participation of the aromatic side chain in substrate recognition of MAO A suggests thatinteraction between aromatic rings of substrates and the enzyme plays a major part in their interaction and could explain why MAO A has a similar affinity for aromatic substrates. This was further confirmed by inhibition experiments using aromatic and nonaromatic compounds. Serotonin oxidizing activity of MAO A was competitively inhibited by PEA and 3-phenyl-1-propylamine with K i values of about 150 M, whereas the value of cyclohexane methylamine was over 10 times higher (about 2 mM). On the other hand, the presence of an amino acid with an aliphatic side chain at the position responsible for substrate selectivity of MAO B as well as large differences in its affinity to aromatic substrates indicate involvement of different interactions from theinteraction between substrates and the enzyme. PEA and tryptamine were good substrates for MAO B, whereas the enzyme showed practically no activity toward serotonin and tyramine, which have an extra phenolic hydroxyl group to the same carbon skeleton as tryptamine and PEA, respectively. Affinity of the enzymes for serotonin and tyramine was markedly reduced with a single mutation of residue 208 of MAO A from aromatic to aliphatic amino acids. It has been shown that C 5 -C 10 linear aliphatic amines are preferentially oxidized by MAO B (30). Taken together, these results indicate that a hydrophobic van der Waals' interaction, instead of an aromatic one, seems to be involved in recognition of substrates by MAO B and the phenolic hydroxyl group may strongly interfere such an interaction between substrates and the enzyme. When Ile at position 199 in MAO B was replaced with Phe, aromatic interaction may take place of hydrophobic one in substrate recognition of the mutant enzymes, and it acquired reasonable affinity to compounds with phenolic hydroxyl group. Although most mutant enzymes have the sensitivity to type-specific inhibitors expected from their substrate selectivity, one mutant,  B(I199F), exhibited the B-type sensitivity, in contrast to its substrate preference of the A-type. Polarity of the two chloride groups in clorgyline may be too large and strong to be accepted in the substrate-binding pocket of MAO B, even after substitution of Ile-199 by an aromatic amino acid. The present study provides the first experimental evidence for identification of a key amino acid participating the substrate selectivity of MAO. Our observations provide important information for molecular design of highly selective inhibitors.