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

Binhua P. ZhouDagger §, Bo WuDagger , Sau-Wah Kwan, and Creed W. Abellparallel

From the Division of Medicinal Chemistry, College of Pharmacy, and the Institute for Neuroscience and the Institute for Cellular and Molecular Biology, University of Texas, Austin, Texas 78712-1074

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Monoamine oxidase B (MAO B) catalyzes the oxidative deamination of biogenic and xenobiotic amines. The oxidative step is coupled to the reduction of an obligatory cofactor, FAD, which is covalently linked to the apoenzyme at Cys397. Our previous studies identified two noncovalent flavin-binding regions in MAO B (residues 6-34 and 39-46) (Kwan, S.-W., Lewis, D. A., Zhou, B. P., and Abell, C. W. (1995) Arch. Biochem. Biophys. 316, 385-391; Zhou, B. P., Lewis, D. A., Kwan, S.-W., Kirksey, T. J., and Abell, C. W. (1995) Biochemistry 34, 9526-9531). In these regions, Glu34 and Tyr44 were found to be required for the initial binding of FAD. By comparing sequences with enzymes in the oxidoreductase family, we now have found an additional FAD-binding site in MAO B (residues 222-227), which is highly conserved across species (human, bovine, and rat). This conserved sequence contains adjacent glycine and aspartate residues (Gly226 and Asp227). Based on the x-ray crystal structures of several oxidoreductases (Eggink, G., Engel, H., Vriend, G., Terpstra, P., and Witholt, B. (1990) J. Mol. Biol. 212, 135-142; Van Driessche, G., Kol, M., Chen, Z.-W., Mathews, F. S., Meyer, T. E., Bartsch, R. G., Cusanovich, M. A., and Van Beeumen, J. J. (1996) Protein Sci. 5, 1753-1764), the Gly residue at the end of a beta -strand facilitates a sharp turn and extends the beta -carbonyl group of Asp to interact with the 3'-hydroxyl group of the ribityl chain of FAD. To assess the hypothesis that Gly226 and Asp227 are involved in FAD binding in MAO B, site-specific mutants that encode substitutions at these positions were prepared and expressed in mammalian COS-7 cells. Our results indicate that Gly226 and the beta -carbonyl group of Asp227 are required for covalent flavinylation and catalytic activity of MAO B, but not for noncovalent binding of FAD. Our studies also reveal that mutagenesis at Glu34 and Tyr44 not only interferes with covalent flavinylation and catalytic activity of MAO B, but also with noncovalent binding of FAD. Based on these collective results, we propose that the coupling of FAD to the MAO B apoenzyme is a multistep process.

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Monoamine oxidases A and B (MAOs1 A and B; EC 1.4.3.4) are the major enzymes in the central nervous system and peripheral tissues of mammals that catalyze the oxidative deamination of neuroactive and vasoactive amines (5). These isozymes are integral proteins of the outer mitochondrial membrane (6) and can be distinguished by differences in substrate and inhibitor preferences and in cell and tissue distribution (7-11). Furthermore, comparison of their nucleotide and deduced amino acid sequences shows that MAOs A and B are distinct proteins with a high degree of sequence identity (12, 13). Recent studies (14) have shown that the substrate selectivity of MAOs A and B appears to be determined by a single amino acid residue (Phe208 in MAO A and Ile199 in MAO B). Oxidation of amines by MAO A or B is coupled to the reduction of FAD, an obligatory cofactor. MAO B contains one FAD molecule/subunit (15), which is covalently linked at Cys397 (12, 16). 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 Cys397 (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. However, we identified two regions in MAO B that are crucial for noncovalent FAD binding in our previous studies (1, 2). One is a dinucleotide-binding site, which is located in the N-terminal region of MAO B (residues 6-34). This dinucleotide-binding site is observed in many flavoproteins with diverse functions and is thought to consist of a beta 1-alpha -beta 2 motif (19), in which the terminal glutamate (Glu34) interacts with the 2'-hydroxyl group of the ribose moiety of FAD. Site-directed mutagenesis studies, which replaced Glu34 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). This new region was recognized by comparing sequences in several reductase flavoproteins, including ferredoxin-NADP+ reductase, whose three-dimensional structure has been solved (20). The consensus sequence contains a tyrosine residue (Tyr44), which is postulated to participate in FAD binding through Van der Waals contact with the isoalloxazine ring. We found that the aromatic ring of the tyrosine residue is essential for FAD binding and catalytic activity of MAO B (2).

In addition to the two adjacent FAD-binding regions on the N terminus and the covalent binding site near the C terminus, we found that MAO B contains a fourth FAD-binding site, which was recognized by sequence similarities in other flavoproteins (Fig. 1) (3). This fingerprint site was first found in the oxidoreductase family of flavoproteins by Eggink et al. (3) and has been recently described in several flavoproteins (4). In those flavoproteins whose structure is known (Fig. 1), this fingerprint region consists of a beta -strand followed by an invariant glycine residue that folds into a sharp turn to extend the next invariant aspartate residue into close proximity to the ribityl chain of FAD. The beta -carbonyl group of this highly conserved aspartate forms a hydrogen bond with the 3'-hydroxyl group of the ribityl chain of FAD (21). This region is highly conserved in MAOs A and B across species (human, bovine, and rat), but the function of this region in MAOs A and B remains unknown.


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Fig. 1.   Sequence comparison of a putative FAD-binding site in monoamine oxidases and in oxidoreductase flavoproteins. Two residues, glycine and aspartate (boxed), corresponding to Gly226 and Asp227 in MAO B, are conserved in all monoamine oxidases and oxidoreductases, preceded by a beta -strand. The beta -carbonyl of the conserved aspartate forms a hydrogen bond with the 3'-hydroxyl group of the ribityl moiety of FAD after a sharp turn at the conserved glycine that terminates the beta -strand. This fingerprint site has been verified in all oxidoreductases whose three-dimensional structure is known (3). The oxidoreductases with identified x-ray crystallographic structure are indicated (*).

To test the hypothesis that these two residues are involved in FAD binding in human MAO B, we changed Gly226 to alanine (G226A) and Asp227 to glutamate (D227E), asparagine (D227N), or alanine (D227A) by site-directed mutagenesis and transiently expressed the cDNAs in COS-7 cells. The effects of these substitutions were assessed by enzymatic activity assays, quantitation of expression, covalent [14C]FAD coupling, and noncovalent [14C]FAD binding. Our results indicate that the putative sharp turn at Gly226 and the carbonyl group of Asp227 are required for covalent flavinylation and catalytic activity of MAO B, but not for noncovalent FAD binding. We also analyzed the ability of MAO B variants at Glu34 and Tyr44 to bind FAD noncovalently. This work, in conjunction with our previous studies (1, 2, 18), reveals that the MAO B variants at Glu34 and Tyr44, which lose MAO B catalytic activity and the ability to couple FAD covalently, also cannot bind FAD noncovalently in dot-blot assays. Thus, it appears that the participation of Gly226 and Asp227 in MAO B flavinylation takes place after the initial noncovalent binding of FAD to the consensus sequences of MAO B near the amino terminus, but prior to covalent linkage to Cys397. In addition, we observed that changes at position 227 abolished recognition by the monoclonal antibody MAO B-1C2, indicating that Asp227 may constitute part of the epitope recognition site.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Synthesis of [14C]FAD-- [14C]FAD was synthesized by a modified method of Manstein and Pai (22) as described previously (18).

Site-directed Mutagenesis-- Mutagenesis was performed by the method of Deng and Nickoloff (23) using a Transformer Site-directed Mutagenesis kit (CLONTECH) as described previously (1). Human MAO B cDNA cloned into the EcoRI site of the expression vector pSVK3 (Amersham Pharmacia Biotech) was used to construct MAO B mutants. The mutagenic primers and the corresponding amino acid changes are shown in Fig. 2. Gly at position 226 was replaced with Ala in the G226A variant, and Asp at position 227 was replaced with Glu (D227E), Asn (D227N), or Ala (D227A). For the purpose of screening, all mutagenic primers were designed to create a new restriction site without altering the coding sequence for any other amino acids. An HpaI restriction site was introduced into a 29-mer selection primer to replace the only KpnI site in the vector. The mutant clones were screened for the presence of the new restriction site created by the mutagenic primer. The presence of the correct mutations in all mutant cDNAs was confirmed by double-stranded dideoxy-DNA sequencing (24). Both wild-type and mutant plasmid DNAs were purified through CsC1 gradients prior to transfection studies.


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Fig. 2.   Nucleotide sequences of mutagenic primers used in site-directed mutagenesis. Lowercase letters indicate base substitutions. The codons for the wild-type and mutant cDNAs at positions 226 and 227 are indicated by a single line above the nucleotides. Base substitutions that do not alter the amino acid coding sequence were also included in each mutagenic primer to create a new restriction site (double underline) for the purpose of screening. Side chains corresponding to amino acid substitutions are also shown.

Expression of Wild-type or Mutant MAO B cDNAs-- Mammalian COS-7 cells used for MAO B expression were grown in Dulbecco's modified Eagle's medium with 10% fetal bovine serum and 5% CO2 at 37 °C. Riboflavin-depleted COS-7 cells were generated by maintaining these cells in riboflavin-free Dulbecco's modified Eagle's medium/dialyzed fetal bovine serum (Life Technologies, Inc.) for >100 days (18). Transient transfection by electroporation (25) of wild-type or mutant MAO B cDNAs into COS-7 cells was carried out as described previously (1). In experiments where covalent flavinylation or noncovalent FAD incorporation of wild-type or variant MAO B was studied, 20 µl of 0.8 mM [14C]FAD and 15 µg of MAO B cDNA were simultaneously electroporated into riboflavin-depleted COS-7 cells in riboflavin-free medium (2.5 × 106 cells/0.8 ml). Transfected cells were resuspended in 15 ml of Dulbecco's modified Eagle's medium/dialyzed fetal bovine serum (or riboflavin-free Dulbecco's modified Eagle's medium/fetal bovine serum) and incubated at 37 °C with 5% CO2. Cells were harvested at 48 h and homogenized in a lysis solution (500 µl) containing 20 mM Tris-HCl, 1.0 mM EDTA, and 0.5 mM phenylmethanesulfonyl fluoride, pH 8.0. Extraction of MAO B from each sample was carried out by adding Triton X-100 to a concentration of 0.25% and stirring for 50 min at 4 °C.

Enzyme-linked Immunosorbent Assay (ELISA)-- Protein concentrations of samples containing wild-type or variant MAO B were determined using a Micro-BCA kit (Pierce). All samples were then adjusted to equal protein concentration. MAO B concentration was quantitated by ELISA with a goat polyclonal antibody to MAO B using a modification of the method of Yeomanson and Billett (26) as described previously (1). Expression levels of wild-type or variant MAO B were determined in duplicate for three separate experiments.

Enzymatic Activity Determination-- MAO B activity was measured by a modification of the method of Wurtman and Axelrod (27) as described previously (1). The activities of wild-type and MAO variants were determined in duplicate in three separate experiments. MAO B activity was expressed both as specific activity (nmol of benzylamine/min/mg of total protein) and as enzymatic activity (µmol of benzylamine/min/mg of MAO B).

Immunoprecipitation of Wild-type and Variant MAO B Enzymes-- COS-7 cells transfected with either wild-type or mutant MAO B were homogenized in 300 µl of 20 mM Tris-HCl, 1 mM EDTA, and 0.5 mM phenylmethanesulfonyl fluoride, pH 8.0. MAO B was extracted from the homogenate with 0.25% Triton X-100 for 50 min at 4 °C. The supernatant from each sample was collected after centrifugation at 1300 × g for 5 min, and all supernatants were adjusted to equal MAO B concentrations using ELISA. Supernatants (300 µl) were incubated with 10 µg of a goat anti-MAO B polyclonal antibody overnight at 4 °C, followed by further incubation with 50 µl of immobilized protein G-Sepharose beads for 3 h. The immunocomplexes composed of immobilized protein G-Sepharose, goat antibody, and wild-type MAO B or its variants were collected by centrifugation at 10,000 × g for 20 s and washed six times with 20 mM Tris buffer, pH 8.0.

For Western blotting and fluorography, the wild-type or variant MAO B proteins in the immunocomplexes were solubilized with SDS-PAGE sample buffer and subsequently analyzed. For dot-blot assay, the wild-type or variant MAO B proteins were eluted from the immunocomplexes with 150 µl of 50 mM glycine, pH 3.0. After a brief spin for 20 s at room temperature, the supernatant was immediately neutralized by mixing with 20 µl of 1.0 M Tris-HCl, pH 8.0. Elution and neutralization were repeated twice to ensure complete extraction of wild-type or variant MAO B proteins from the immunocomplexes. The eluents were combined for dot-blot assays.

Western Blot Analysis-- Immunoprecipitated proteins were subjected to 10% SDS-PAGE and analyzed by Western blotting with the MAO B-specific monoclonal antibody, MAO B-1C2 (10), as described previously (1).

Fluorography-- Immunoprecipitated wild-type and variant MAO B enzymes were subjected to electrophoresis on a 10% SDS-polyacrylamide gel. The gel was fixed (7% acetic acid and 10% methanol) for 1 h and processed for fluorography according to Bonner and Laskey (28) with modification as described previously (18). The dried gel was exposed to Kodak X-Omat AR film at -80 °C for 3 weeks.

Dot-blot Assay-- Immunoprecipitated wild-type and variant MAO B enzymes were analyzed in a dot-blot apparatus (Bio-Rad) according to the manufacturer's instructions. Briefly, the nitrocellulose membrane (Schleicher & Schuell) was prewetted with 150 ml of 20 mM Tris and1 mM EDTA, pH 8.0, and assembled into the dot-blot apparatus. Each sample, containing immunoprecipitated wild-type or variant MAO B, was loaded into an individual well before the membrane dried out and filtered through the nitrocellulose membrane under a gentle vacuum pressure, immobilizing the protein onto the membrane surface. The wells were washed twice with 150 µl of 20 mM Tris and 1 mM EDTA, pH 8.0. The nitrocellulose membrane was dried in an oven at 50 °C and exposed to Kodak X-Omat AR film at -80 °C for 3 weeks.

Densitometry-- The fluorogram of SDS-PAGE and the autoradiogram of dot-blot analysis were digitized and quantitated using the program NIH Image 1.62 (47).

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Wild-type and variant MAO B enzymes were expressed in mammalian COS-7 cells since these cells do not contain any detectable endogenous MAO B (1). Expression levels of wild-type and variant MAO B enzymes are shown in Table I. The amount of MAO B expressed for all variants (0.67-0.72 µg of MAO B/mg of protein) was similar to that of wild-type MAO B (0.69 µg/mg of protein). Substitution of Asp227 with glutamate (D227E) or asparagine (D227N) led to a slight decrease (91%) or moderate decrease (70%) in enzymatic activity, respectively (Table I). However, substitution of Asp227 with alanine (D227A) resulted in a substantial loss of enzymatic activity (35%). A fourth mutant, G226A, in which the invariant glycine was replaced with alanine at position 226, also exhibited a marked loss of enzymatic activity (33%). Likewise, the specific activities of all the variants closely correlated with their enzymatic activities.

                              
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Table I
Comparison of expression levels and activities of wild-type and variant human MAO B enzymes
An equal amount of wild-type or variant MAO B cDNA (15 µg) was transiently expressed in COS-7 cells, and the cells were then incubated at 37 °C with 5% CO2 for 48 h. The transfected cells were harvested and homogenized. Wild-type and variant MAO B proteins were then extracted from the cell lysates with Triton X-100. After the total protein concentration was equalized in all samples, MAO B quantitation (by ELISA) and activity measurements were performed. Samples were run in duplicates in each experiment. Each value represents the mean ± S.E. from three separate experiments.

To determine the expression of MAO B by Western blotting, wild-type and variant MAO B enzymes were quantitatively immunoprecipitated with goat anti-MAO B polyclonal antibodies, subjected to SDS-PAGE, and immunoblotted using MAO B-1C2 as primary antibody (10). As shown in Fig. 3 (lanes 2 and 6), wild-type MAO B and G226A have bands of approximately equal intensity corresponding to 59 kDa, whereas the sample prepared from untransfected COS-7 cells (lane 7) showed no MAO B band. Surprisingly, all variants at position 227 (lanes 3-5) did not exhibit a visible band.


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Fig. 3.   Western blot analysis. Wild-type and mutant MAO B cDNAs were transfected in COS-7 cells. Before immunoprecipitation with a goat anti-MAO B polyclonal antibody, expressed wild-type and variant MAO B enzymes were adjusted to equal concentrations (based on ELISA). Immunoprecipitated enzymes were then separated on 10% SDS-polyacrylamide gel, transferred to a nitrocellulose membrane, and analyzed by Western blotting using the MAO B-specific monoclonal antibody, MAO B-1C2. Lane 1, prestained molecular mass markers; lane 2, wild-type MAO B; lane 3, D227E MAO B; lane 4, D227N MAO B; lane 5, D227A MAO B; lane 6, G226A MAO B; lane 7, untransfected COS-7 cells; lane 8, biotinylated molecular mass markers.

Flavinylation of wild-type and variant MAO B enzymes with substitutions at Asp227 and Gly226 was studied by simultaneous electroporation of [14C]FAD and MAO B cDNA. The quantitatively immunoprecipitated wild-type and variant MAO B enzymes were subjected to SDS-PAGE and then fluorography (Fig. 4a). Quantitation of the fluorogram showed that covalent incorporation of [14C]FAD into D227E, D227N, D227A, and G226A was 91, 67, 29, and 28% of that of the wild-type enzyme, respectively (Fig. 4c). These results indicate that wild-type MAO B and the variant D227E can covalently bind to [14C]FAD to approximately equal extents. D227N, D227A, and G226A, however, showed decreased ability to bind [14C]FAD covalently (67, 29, and 28%, respectively).


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Fig. 4.   Effect of site-directed mutagenesis at Gly226 and Asp227 on FAD binding of MAO B. a, fluorography of SDS-PAGE. Wild-type and mutant cDNAs were transfected in riboflavin-depleted COS-7 cells with the addition of exogenous [14C]FAD during electroporation. Expressed wild-type and variant MAO B enzymes were adjusted to equal concentrations based on ELISA before immunoprecipitation. The immunoprecipitated enzymes were separated by 10% SDS-PAGE and subjected to fluorography. Lanes 1 and 8, 14C-methylated molecular mass markers; lane 2, wild-type MAO B; lane 3, D227E MAO B; lane 4, D227N MAO B; lane 5, D227A MAO B; lane 6, G226A MAO B; lane 7, untransfected COS-7 cells. b, autoradiography of dot blotting. Expressed wild-type and variant MAO B enzymes at equal concentrations (based on ELISA) were immunoprecipitated, eluted with glycine buffer, and used in dot-blot analysis. The dried nitrocellulose membrane of dot blotting was then exposed to x-ray film at -80 °C. Dot 1, wild-type MAO B; dot 2, D227E MAO B; dot 3, D227N MAO B; dot 4, D227A MAO B; dot 5, G226A MAO B; dot 6, untransfected COS-7 cells. c, densitometry. The fluorogram and the autoradiogram were digitized and quantitated using the program NIH Image 1.62 (47). Enzymatic activity data are taken from Table I. All data are expressed as percent of the corresponding value of wild-type MAO B.

The total amount of [14C]FAD incorporated into MAO B variants includes both covalently linked FAD and noncovalently bound FAD. The presence of the MAO B band on the fluorograms of SDS-PAGE reflects only FAD covalently bound to the enzyme (Figs. 4a and 5a) since noncovalently bound FAD will be released from the enzyme due to denaturation of proteins prior to and during SDS-PAGE. Consequently, we developed a dot-blot assay to determine the total amount of [14C]FAD that is both covalently and noncovalently incorporated into the wild-type or variant MAO B enzymes. As seen in Fig. 4b, the wild-type and variant enzymes exhibited approximately equal intensities in the dot-blot assay. Densitometric examination of the autoradiogram showed that the total amounts of [14C]FAD bound to D227E, D227N, D227A, and G226A were 92, 89, 88, and 89% of that of wild-type MAO B, respectively (Fig. 4c), indicating that the total amount of [14C]FAD bound to the apoenzyme (covalent and noncovalent binding) is approximately the same in the wild-type and variant enzymes with substitutions at positions 226 and 227. 


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Fig. 5.   Effect of site-directed mutagenesis at Glu34 and Tyr44 on FAD binding of MAO B. The procedures used for SDS-PAGE and the dot-blot assay were the same as described in the legend to Fig. 4. a, fluorogram of SDS-PAGE. Lanes 1 and 8, 14C-methylated molecular mass markers; lane 2, wild-type MAO B; lane 3, E34A MAO B; lane 4, E34D MAO B; lane 5, Y44F MAO B; lane 6, Y44A MAO B; lane 7, untransfected COS-7 cells. b, autoradiography of dot blotting. Dot 1, wild-type MAO B; dot 2, E34A MAO B; dot 3, E34D MAO B; dot 4, Y44F MAO B; dot 5, Y44A MAO B; dot 6, untransfected COS-7 cells. c, densitometry. The fluorogram and the autoradiogram were digitized and analyzed with NIH Image 1.62 (47). Enzymatic activity data were taken from our previous reports (1, 2). All data are expressed as percent of the corresponding value of wild-type MAO B.

In our previous studies (1, 2, 18), we found that Glu34 in the dinucleotide-binding site and Tyr44 in the adjacent FAD-binding region are critical for initial FAD binding. Substitution of Glu34 with aspartate (E34D), or alanine (E34A) resulted in an almost complete loss of enzymatic activity and covalent FAD coupling (Fig. 5a) (1, 18). Substitution of Tyr44 with phenylalanine (Y44F) had no significant effect on enzymatic activity or covalent FAD binding. However, substitution of Tyr44 with alanine (Y44A) resulted in a complete loss of enzymatic activity and covalent FAD binding (Fig. 5a) (2). Whether these variants could still bind FAD noncovalently was not previously known. In this paper, we used dot-blot assays (Fig. 5b) to assess the extent of total FAD binding for these variants. The fluorograms of SDS-PAGE and the autoradiograms of the dot-blot assay were quantitated and compared with the respective enzymatic activities (Fig. 5c). The results of this study revealed that the total FAD binding for these variants correlated well with their covalent binding and enzymatic activities. Y44F had 88% of total FAD binding, 108% of covalent binding of FAD, and 93% enzymatic activity as compared with wild-type MAO B. However, E34D had 15% total FAD binding, 12% covalent binding of FAD, and 7% enzymatic activity compared with wild-type MAO B. E34A and Y44A appeared to lose their ability to bind FAD not only covalently but also noncovalently since no more than 5% of the values for wild-type MAO B were found. The dot-blot patterns of these MAO variants correlated well with their fluorograms and enzymatic activities, indicating that Glu34 and Tyr44 are critical residues that participate in the activation of MAO B through the covalent flavinylation process.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

We have examined the role of a fingerprint site in MAO B that is highly conserved among numerous oxidoreductases that require FAD as a cofactor (3, 4, 21). Several flavoproteins containing this fingerprint site have been crystallized, and the three-dimensional structure of this FAD-binding region is known (3, 4). This new FAD-binding motif consists of a beta -strand that ends with an invariant glycine residue that undergoes a sharp turn, extending the next invariant aspartate residue in close proximity to FAD. The beta -carbonyl group of this highly conserved aspartate forms a hydrogen bond with the 3'-hydroxyl group of the ribityl moiety of FAD.

To examine the role of the analogous residues (Gly226 and Asp227) in MAO B, the glycine was replaced with alanine (G226A), and aspartate was substituted with glutamate (D227E), asparagine (D227N), or alanine (D227A). As expected, the D227E variant retained almost full enzymatic activity (91%) since a carboxyl group is still present in the glutamate residue. Apparently, the length of the side chain is not critical as long as a negatively charged carboxyl group is present at this position. A loss of enzymatic activity was seen for D227N (70%), which was also expected because the replacement of aspartate by asparagine leads to a weaker hydrogen-bonding potential between the beta -carbonyl group of asparagine and the 3'-hydroxyl group of the ribityl moiety of FAD. For the D227A variant, replacing the side chain possessing a beta -carboxyl with a methyl group resulted in a substantial reduction, but not total loss, of enzymatic activity (35%). A reduction of enzymatic activity in the G226A variant (33%) was detected; this is presumably caused by the bulkier side chain of alanine that sterically hindered this residue from undergoing a sharp turn at position 226. The extent of this reduction was virtually the same as in D227A, indicating that both the sharp turn facilitated by Gly226 and the hydrogen bonding between the beta -carbonyl group of Asp227 and the 3'-hydroxyl group of the ribityl chain are critical for the enzymatic activity of MAO B.

The concentration of wild-type and variant MAO B enzymes was determined by ELISA using an anti-MAO B polyclonal antibody. Since quantitation is dependent upon recognition by polyclonal antibodies at multiple epitopes under nondenaturing conditions, major conformational changes could lead to a substantial difference between the apparent concentrations of a variant MAO B and its real value. Our results demonstrated that the concentrations of all variant MAO B enzymes determined by ELISA are very close to the value of wild-type MAO B (Table I), indicating that activity losses of the variant MAO B enzymes were unlikely to be due to defective expression or aberrant conformational changes in the proteins.

Studies on covalent flavinylation of these variant enzymes using quantitative fluorography (Figs. 4 and 5) demonstrated that the MAO B variant D227E incorporated covalently bound [14C]FAD to approximately the same extent (91%) as wild-type MAO B. D227N incorporated a moderate amount of covalently bound [14C]FAD (67% of the wild-type enzyme), but D227A and G226A incorporated much less covalently bound [14C]FAD (29 and 28% of wild-type MAO B, respectively). These results are in agreement with the values for enzymatic activities of all the variants studied and support the conclusion that both Gly226 and Asp227 play crucial roles in the flavinylation of MAO B.

MAO B variants containing noncovalently bound [14C]FAD are not visualized on SDS-PAGE fluorograms because [14C]FAD that is not linked to MAO B will dissociate from the enzyme during protein denaturation in SDS-PAGE. Consequently, dot-blot assays were developed to measure the total amount of [14C]FAD that was bound to MAO B through noncovalent and covalent interactions. Since the procedure used for dot-blot assays does not denature proteins, the intensities of the dots on the autoradiogram reflect the total amount of [14C]FAD bound to the enzyme (both covalently and noncovalently). As seen in Fig. 4b, the intensities of the dots corresponding to the wild-type and variant enzymes (G226A, D227E, D227N, and D227A) were approximately equal, whereas untransfected COS-7 cells showed no dot, indicating the absence of [14C]FAD. Densitometric analysis of the autoradiograms showed that the intensities of the dots corresponding to the variants D227E, D227N, D227A, and G226A were 92, 89, 88, and 89%, respectively, of that of wild-type MAO B. These results indicate that mutations at positions 226 and 227 do not significantly alter the ability of the apoenzyme to bind [14C]FAD noncovalently. Collectively, the results of the fluorographic and dot-blot analyses indicate that the variant enzymes at positions 226 and 227 retain their ability to recruit FAD into the apoenzyme, but they exhibit reduced covalent linkage of FAD to the enzyme compared with the wild-type enzyme. Presumably, achievement of noncovalent binding of FAD does not assure that the flavin will be delivered in the correct orientation for the next step in forming the holoenzyme. It is noteworthy that the enzymatic activities of all variants studied thus far correlate well with covalently bound [14C]FAD, but not with the total amount of bound [14C]FAD. This indicates that only MAO B variants with covalently bound FAD are catalytically active. These results are in agreement with studies by others (29-31), who found that substitution of the FAD covalent binding residue (Cys397) in MAO B with alanine, serine, or histidine abolished catalytic activity when the mutants were expressed in mammalian systems. In contrast, Ito and co-workers (31, 32) reported that the variant proteins expressed in yeast retained some catalytic activity when the FAD covalent binding residues (i.e. Cys406 in MAO A and Cys397 in MAO B) were substituted with alanine.

In our previous studies (1, 2, 18), we identified two amino acid residues (Glu34 and Tyr44) that are essential for covalent FAD binding. Whether these variant enzymes can bind FAD through noncovalent interactions, however, was not established. In this study, we assessed the FAD-binding properties of these enzymes by applying dot-blot analysis. Quantitation of the fluorograms of SDS-PAGE and dot-blot autoradiograms showed that the intensity of the dots correlated well with the intensity of the bands on the fluorogram (Fig. 5). Furthermore, the total FAD binding (represented by the intensity of the dot) and covalent binding of FAD (represented by the band intensity of these variant enzymes) also correlated well with their enzymatic activities. Unlike the variants of Gly226 and Asp227 mentioned above, E34A and Y44A not only lost their ability to bind FAD covalently, but also lost their ability to recruit FAD noncovalently. In contrast, two conservative variants, Y44F and E34D (to a much lesser extent), still retained their partial ability to bind FAD covalently, and the extent of their covalent FAD binding correlated with their total FAD binding. These results indicate that Glu34 and Tyr44 in the N terminus of the molecule facilitate the first initial binding of FAD, thus positioning it in the correct orientation for the subsequent steps that lead to covalent binding of FAD. Alteration of either one of these sites of interaction affects MAO B enzymatic activity.

The expression of wild-type and variant MAO B enzymes was also analyzed by Western blot analysis (Fig. 3). The wild-type enzyme and G226A had bands of approximately equal intensity at a molecular mass of 59 kDa. Surprisingly, none of the variants at position 227 was recognized by the MAO B-specific monoclonal antibody, MAO B-1C2. Reduced expression of these variants was ruled out since the enzymatic activity assays, ELISA quantitation, and fluorography all revealed the presence of these proteins. MAO B-1C2 was produced previously, and it has been widely used for MAO B purification, immunostaining, quantitation, and Western blotting (12, 8, 10, 18). The inability of MAO B-1C2 to recognize the enzymes with substitutions at position 227 suggests that Asp227 may constitute part of the antigenic determinant. Interestingly, this recognition site appears to be uniquely specific since mutagenesis of the adjacent residue (G226A) had no effect on recognition.

Modification of the apoenzyme by flavinylation is obligatory for the generation of catalytic activity in many flavoproteins. Some examples include succinate dehydrogenase (33), dimethylglycine dehydrogenase (34), 6-hydroxy-D-nicotine oxidase (35), trimethylamine dehydrogenase (36), p-cresol methylhydroxylase (37), and MAOs A and B (18). Some studies on covalent flavinylation of proteins indicate that covalent coupling of FAD to its respective proteins appears to be an autocatalytic reaction (18, 35-38). However, the precise steps involved in this process remain unknown. In addition, whether flavinylation of apoflavoproteins is a co-translational or post-translational process remains to be definitively established (18, 37, 39).

Frieden (40) found that unfolded dihydrofolate reductase refolds and binds to its ligands dihydrofolate and NADP(H) in a multistep process. These ligands bind to the polypeptide at certain conformational states along the folding pathway (40). Brandsch et al. (39) proposed a similar mechanism for the covalent coupling of FAD to 6-hydroxy-D-nicotine oxidase. They suggested that covalent binding of FAD occurs only at specific conformational states in the polypeptide. Succinate dehydrogenase, with truncated C termini (minus 70 or 90 amino acid residues), cannot bind FAD covalently, although the FAD covalent linkage site is close to the N-terminal end of this enzyme (33). This suggests that FAD linkage to this enzyme occurs only when aposuccinate dehydrogenase has folded into certain conformations that can bind FAD. Studies on flavinylation of p-cresol methylhydroxylase (37) also suggest a sequential process for flavinylation of this tetrameric enzyme (alpha - and beta -subunits). In this pathway, FAD binds first to the apoflavoprotein subunit noncovalently, followed by interaction between the flavoprotein and the cytochrome subunits. The participation of the cytochrome subunit is required for eventual covalent FAD attachment to the flavoprotein subunit.

Sequential steps in flavinylation have also been proposed for flavoproteins that do not covalently link FAD. Massey and Curti (41) demonstrated that the reconstitution of catalytically active D-amino-acid oxidase is a two-stage process. Mixing FAD with the apo-D-amino-acid oxidase leads to the reconstitution of the holoenzyme, including a rapid binding of FAD by the apoenzyme followed by a slower change of the holoenzyme conformation. Massey and Curti (41) concluded that the slow conformational change is required for enzymatic activity. A similar observation was reported for glucose oxidase from Aspergillus niger (42). It was found that the reconstitution of glucose oxidase holoenzyme involves the initial binding of FAD by the apoenzyme and additional steps associated with changes in protein conformation. Swoboda (42) concluded that FAD incorporation can induce a conformational alteration in the apoenzyme that stabilizes the three-dimensional structure of the catalytically active enzyme, presumably through multiple interactions between FAD and the polypeptide.

Gondry et al. (43) studied the role of protein folding and FMN insertion in two flavocytochrome b2 mutants. They proposed that the polypeptide chain may fold without interaction with FMN to a stage where the scaffolding of a beta /alpha -barrel becomes competent to bind the cofactor. Furthermore, the interaction between FMN and the polypeptide may then induce final adjustments. In another example, Genzor et al. (44) determined the structures of apoflavodoxin and holoflavodoxin (with FMN). They hypothesized that the phosphate-binding site is predetermined by the protein backbone of the apoenzyme so that the phosphate-binding motif anchors FMN to the protein and leads to intercalation of the ribityl chain and isoalloxazine ring into flavodoxin. These examples support the notion that flavin binding depends upon the participation of conformational stages in the folding pathway of catalytically active flavoproteins.

We have measured selected parameters (enzymatic activity, covalent flavinylation, and noncovalent FAD binding) of several MAO B variants that possess single amino acid substitutions at key positions in two FAD-binding sites close to the N-terminal end and to the fingerprint site (residues 222-227). Based on the results of these studies, we propose that FAD couples to apo-MAO B in a stepwise process during the translation and folding of the polypeptide. The first step involves recruitment of FAD. The gamma -carboxyl group of Glu34 (at the dinucleotide-binding site) and the aromatic ring of Tyr44 (at the adjacent FAD-binding site) in the N terminus provide the initial topological dock for FAD binding (1, 2, 18). This interpretation can explain why mutations at either one of these functional residues result in a loss of both covalent and noncovalent linkages of FAD. We propose that FAD is then delivered to the fingerprint site (residues 222-227), which provides another topological dock to further secure the FAD molecule. The incoming FAD, which is held at multiple sites through interaction with several amino acid residues, can then be delivered to Cys397 in a position that places the 8alpha -methyl group of FAD in exact and close proximity to the thiol group of Cys397 to facilitate covalent flavinylation. Variants at Gly226 and Asp227 in the fingerprint site do not interfere with noncovalent binding of FAD because they are not involved in FAD recruitment, but they have reduced capability to couple FAD covalently, possibly because the conformation of these altered polypeptides does not support proper positioning of Cys397 for flavin linkage. Although the mechanism of FAD coupling is not completely understood, our studies provide insight into the complex set of steps that lead to activation of MAO B by flavinylation.

MAO inhibitors have long been used for the treatment of various psychiatric and neurological disorders, including depression (45) and Parkinson's disease (46). Our studies on the mechanism of flavinylation may lead to the development of new therapeutic drugs (e.g. analogs of FAD) for treatment of these disorders.

    ACKNOWLEDGEMENT

We thank Dr. Duane Lewis for critical reading of this manuscript and helpful suggestions.

    FOOTNOTES

* This work was supported in part by the Foundation for Research, NIH Grant NS24932, and a Granville Wrather fellowship (to C. W. A.) from the IC2 Institute (Austin, Texas).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger These authors contributed equally to this work.

§ Present address: Dept. of Tumor Biology, P. O. Box 79, University of Texas, M. D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030.

Supported by NIH Training Grant AAO7471.

parallel To whom correspondence should be addressed: Div. of Medicinal Chemistry, College of Pharmacy, University of Texas, Austin, TX 78712-1074. Tel.: 512-471-5715; Fax: 512-471-2181.

1 The abbreviations used are: MAOs, monoamine oxidases; ELISA, enzyme-linked immunosorbent assay; PAGE, polyacrylamide gel electrophoresis.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

  1. Kwan, S.-W., Lewis, D. A., Zhou, B. P., and Abell, C. W. (1995) Arch. Biochem. Biophys. 316, 385-391[CrossRef][Medline] [Order article via Infotrieve]
  2. Zhou, B. P., Lewis, D. A., Kwan, S.-W., Kirksey, T. J., and Abell, C. W. (1995) Biochemistry 34, 9526-9531[Medline] [Order article via Infotrieve]
  3. Eggink, G., Engel, H., Vriend, G., Terpstra, P., and Witholt, B. (1990) J. Mol. Biol. 212, 135-142[Medline] [Order article via Infotrieve]
  4. Van Driessche, G., Kol, M., Chen, Z.-W., Mathews, F. S., Meyer, T. E., Bartsch, R. G., Cusanovich, M. A., and Van Beeumen, J. J. (1996) Protein Sci. 5, 1753-1764[Abstract/Free Full Text]
  5. Von Korff, R. W. (1979) in Monoamine Oxidase: Structure, Function, and Altered Functions (Singer, T. P., Van Korff, R. W., and Murphy, D. L., eds), pp. 1-7, Academic Press, New York
  6. Greenawalt, J. W., and Schnaitman, C. (1970) J. Cell Biol. 46, 173-179[Free Full Text]
  7. Dostert, P. L., Beneditti, M. S., and Tipton, K. F. (1989) Med. Res. Rev. 9, 45-89[Medline] [Order article via Infotrieve]
  8. Westlund, K. N., Denney, R. M., Kochersperger, L. M., Rose, R. M., and Abell, C. W. (1985) Science 230, 181-183[Medline] [Order article via Infotrieve]
  9. Westlund, K. N., Kenney, R. M., Rose, R. M., and Abell, C. W. (1988) Neuroscience 25, 439-456[CrossRef][Medline] [Order article via Infotrieve]
  10. Denney, R. M., Fritz, R. R., Patel, N. T., and Abell, C. W. (1982) Science 215, 1400-1403[Medline] [Order article via Infotrieve]
  11. Kokersperger, L. M., Waguespack, A., Patterson, J. C., Hsieh, C. C. W., Weyler, W., Salach, J. I., and Denney, R. M. (1985) J. Neurosci. 11, 2874-2881
  12. Bach, A. W. J., Lan, N. C., Johnson, D. L., Abell, C. W., Bembenek, M. E., Kwan, S.-W., Seeburg, P. H., and Shih, J. C. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 4934-4938[Abstract]
  13. Weyler, W., Hsu, Y.-P. P., and Breakefield, X. O. (1990) Pharmacol. Ther. 47, 391-417[CrossRef][Medline] [Order article via Infotrieve]
  14. Tsugeno, Y., and Ito, A. (1997) J. Biol. Chem. 272, 14033-14036[Abstract/Free Full Text]
  15. Weyler, W. (1989) Biochem. J. 260, 725-732[Medline] [Order article via Infotrieve]
  16. Kearney, E. B., Salach, J. I., Walker, W. H., Seng, R. L., Kenney, W., Zeszotek, K., and Singer, T. P. (1971) Eur. J. Biochem. 24, 321-327[Medline] [Order article via Infotrieve]
  17. Shiloff, B. A., Behrens, P. Q., Kwan, S.-W., Lee, J. H., and Abell, C. W. (1996) Eur. J. Biochem. 242, 41-50[Abstract]
  18. Zhou, B. P., Lewis, D. A., Kwan, S.-W., and Abell, C. W. (1995) J. Biol. Chem. 270, 23653-23660[Abstract/Free Full Text]
  19. Wierenga, R. K., Terpstra, P., and Hol, W. G. J. (1986) J. Mol. Biol. 187, 101-107[Medline] [Order article via Infotrieve]
  20. Correll, C. C., Ludwig, M. L., Bruns, C. M., and Karplus, P. A. (1993) Protein Sci. 2, 2112-2133[Abstract/Free Full Text]
  21. Schultz, G. E. (1992) Curr. Opin. Struct. Biol. 2, 61-67[CrossRef]
  22. Manstein, D. J., and Pai, E. F. (1986) J. Biol. Chem. 261, 16169-16173[Abstract/Free Full Text]
  23. Deng, W. P., and Nickoloff, J. A. (1992) Anal. Biochem. 200, 81-88[Medline] [Order article via Infotrieve]
  24. Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 5463-5467[Abstract]
  25. Zimmerman, U., and Vienken, J. (1982) J. Membr. Biol. 67, 165-182[Medline] [Order article via Infotrieve]
  26. Yeomanson, K. B., and Billett, E. E. (1992) Biochim. Biophys. Acta 1116, 261-268[Medline] [Order article via Infotrieve]
  27. Wurtman, R. J., and Axelrod, J. (1963) Biochem. Pharmacol. 12, 1439-1440[CrossRef]
  28. Bonner, W. M., and Laskey, R. A. (1974) Eur. J. Biochem. 46, 83-88[Medline] [Order article via Infotrieve]
  29. Gottowik, J., Cesura, A. M., Malherbe, P., Lang, G., and Da Prada, M. (1993) FEBS Lett. 317, 152-156[CrossRef][Medline] [Order article via Infotrieve]
  30. Wu, H.-F., Chen, K., and Shih, J. C. (1993) Mol. Pharmacol. 43, 888-893[Abstract]
  31. Ogata, F., Tsugeno, Y., Hirasiki, I., Mitoma, J., and Ito, A. (1994) in Flavin and Flavoproteins 1993 (Yagi, K., ed), pp. 795-798, Walter de Gruyter & Co., Berlin
  32. Hiro, I., Tsugeno, Y., Hirashiko, I., Ogata, F., and Ito, A. (1996) J. Biochem. (Tokyo) 120, 759-765[Abstract]
  33. Robinson, K. M., and Lemire, B. D. (1996) J. Biol. Chem. 271, 4055-4060[Abstract/Free Full Text]
  34. Otto, A., Stoltz, M., Sailer, H.-P., and Brandsch, R. (1996) J. Biol. Chem. 271, 9823-9829[Abstract/Free Full Text]
  35. Stoltz, M., Rassow, J., Buckmann, A. F., and Brandsch, R. (1996) J. Biol. Chem. 271, 25208-25212[Abstract/Free Full Text]
  36. Packman, L. C., Mewies, M., and Scrutton, N. S. (1995) J. Biol. Chem. 270, 13186-13191[Abstract/Free Full Text]
  37. Kim, J., Fuller, J. H., Kuusk, V., Cunane, L., Chen, Z., Mathews, F. S., and McIntire, W. S. (1995) J. Biol. Chem. 270, 31202-31209[Abstract/Free Full Text]
  38. Brandsch, R., and Bichler, V. (1991) J. Biol. Chem. 266, 19056-19062[Abstract/Free Full Text]
  39. Brandsch, R., Bichler, V., Schmidt, M., and Buchner, J. (1992) J. Biol. Chem. 267, 20844-20849[Abstract/Free Full Text]
  40. Frieden, C. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 4413-4416[Abstract]
  41. Massey, V., and Curti, B. (1966) J. Biol. Chem. 241, 3417-3423[Abstract/Free Full Text]
  42. Swoboda, B. E. P. (1969) Biochim. Biophys. Acta 175, 365-379[Medline] [Order article via Infotrieve]
  43. Gondry, M., Diep Le, K. H., Manson, F. D. C., Chapman, S. K., Mathews, F. S., Reid, G. A., and Lederer, F. (1995) Protein Sci. 4, 925-935[Abstract/Free Full Text]
  44. Genzor, C. G., Perales-Alcon, A., Sancho, J., and Romero, A. (1996) Nat. Struct. Biol. 3, 329-332[Medline] [Order article via Infotrieve]
  45. Da Prada, M. D., Kettler, R., Keller, H. H., Burkard, W. P., and Haefely, W. E. (1989) J. Neural. Transm. 28, 5-20
  46. Tetrud, J. W., and Langston, J. W. (1989) Science 245, 519-522[Medline] [Order article via Infotrieve]
  47. Rasband, Wayne (1997) NIH Image, Version 1.62, NIH, Bethesda, MD


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