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

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 β-strand facilitates a sharp turn and extends the β-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 β-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. 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 β-strand facilitates a sharp turn and extends the β-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 β-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. Monoamine oxidases A and B (MAOs 1The abbreviations used are: MAOs, monoamine oxidases; ELISA, enzyme-linked immunosorbent assay; PAGE, polyacrylamide gel electrophoresis. 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 (5Von Korff R.W. Singer T.P. Van Korff R.W. Murphy D.L. Monoamine Oxidase: Structure, Function, and Altered Functions. Academic Press, New York1979: 1-7Google Scholar). These isozymes are integral proteins of the outer mitochondrial membrane (6Greenawalt J.W. Schnaitman C. J. Cell Biol. 1970; 46: 173-179Crossref PubMed Scopus (115) Google Scholar) and can be distinguished by differences in substrate and inhibitor preferences and in cell and tissue distribution (7Dostert P.L. Beneditti M.S. Tipton K.F. Med. Res. Rev. 1989; 9: 45-89Crossref PubMed Scopus (142) Google Scholar, 8Westlund K.N. Denney R.M. Kochersperger L.M. Rose R.M. Abell C.W. Science. 1985; 230: 181-183Crossref PubMed Scopus (419) Google Scholar, 9Westlund K.N. Kenney R.M. Rose R.M. Abell C.W. Neuroscience. 1988; 25: 439-456Crossref PubMed Scopus (305) Google Scholar, 10Denney R.M. Fritz R.R. Patel N.T. Abell C.W. Science. 1982; 215: 1400-1403Crossref PubMed Scopus (121) Google Scholar, 11Kokersperger L.M. Waguespack A. Patterson J.C. Hsieh C.C.W. Weyler W. Salach J.I. Denney R.M. J. Neurosci. 1985; 11: 2874-2881Crossref Google Scholar). 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 (12Bach A.W.J. Lan N.C. Johnson D.L. Abell C.W. Bembenek M.E. Kwan S.-W. Seeburg P.H. Shih J.C. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 4934-4938Crossref PubMed Scopus (701) Google Scholar,13Weyler W. Hsu Y.-P.P. Breakefield X.O. Pharmacol. Ther. 1990; 47: 391-417Crossref PubMed Scopus (292) Google Scholar). Recent studies (14Tsugeno Y. Ito A. J. Biol. Chem. 1997; 272: 14033-14036Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar) 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 (15Weyler W. Biochem. J. 1989; 260: 725-732Crossref PubMed Scopus (34) Google Scholar), which is covalently linked at Cys397 (12Bach A.W.J. Lan N.C. Johnson D.L. Abell C.W. Bembenek M.E. Kwan S.-W. Seeburg P.H. Shih J.C. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 4934-4938Crossref PubMed Scopus (701) Google Scholar, 16Kearney E.B. Salach J.I. Walker W.H. Seng R.L. Kenney W. Zeszotek K. Singer T.P. Eur. J. Biochem. 1971; 24: 321-327Crossref PubMed Scopus (193) Google Scholar). Detergent-extracted bovine liver MAO B appears to operate as an oligomeric complex (17Shiloff B.A. Behrens P.Q. Kwan S.-W. Lee J.H. Abell C.W. Eur. J. Biochem. 1996; 242: 41-50Crossref PubMed Scopus (9) Google Scholar). 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 (1Kwan S.-W. Lewis D.A. Zhou Abell C.W. Arch. Biochem. Biophys. PubMed Scopus Google Scholar, Lewis D.A. Kwan S.-W. Abell C.W. PubMed Scopus Google Scholar) and is covalently linked to Lewis D.A. Kwan S.-W. Abell C.W. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the MAOs are integral proteins in the outer mitochondrial it to a crystal to the precise residues that the site and that interact with FAD. we identified two regions in MAO B that are for noncovalent FAD binding in previous studies (1Kwan S.-W. Lewis D.A. Zhou Abell C.W. Arch. Biochem. Biophys. PubMed Scopus Google Scholar, Lewis D.A. Kwan S.-W. Abell C.W. PubMed Scopus Google Scholar). is a which is in the of MAO B (residues This site is in with and is to of a J. Mol. Biol. PubMed Scopus Google Scholar), in which the with the group of the of FAD. mutagenesis which Glu34 with or in a of FAD coupling a of MAO B activity (1Kwan S.-W. Lewis D.A. Zhou Abell C.W. Arch. Biochem. Biophys. PubMed Scopus Google Lewis D.A. Kwan S.-W. Abell C.W. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). A FAD-binding found adjacent to the in MAO B (residues in MAO B). This by comparing sequences in several Protein Sci. PubMed Scopus Google Scholar). The consensus sequence contains a residue which is to in FAD binding Van with the found that the of the residue is for FAD binding and catalytic activity of MAO B Lewis D.A. Kwan S.-W. Abell C.W. PubMed Scopus Google Scholar). In to the two adjacent FAD-binding regions on the terminus and the covalent binding site near the we found that MAO B contains a FAD-binding which by sequence in B. J. Mol. Biol. 1990; PubMed Scopus Google Scholar). This site found in the oxidoreductase of by B. J. Mol. Biol. 1990; PubMed Scopus Google Scholar) and in several M. Van Protein Sci. 1996; PubMed Scopus Google Scholar). In is of a β-strand by an glycine residue that a sharp turn to the aspartate residue to the ribityl chain of FAD. The β-carbonyl group of highly conserved aspartate a with the 3′-hydroxyl group of the ribityl chain of FAD Biol. Scopus Google Scholar). This is highly conserved in MAOs A and B across species (human, bovine, and but the of in MAOs A and B To the hypothesis that these two residues are involved in FAD binding in MAO B, we Gly226 to and Asp227 to or by mutagenesis and expressed the in COS-7 cells. The of these substitutions were by activity of covalent and noncovalent Our results indicate that the sharp turn at Gly226 and the group of Asp227 are required for covalent flavinylation and catalytic activity of MAO B, but not for noncovalent FAD also the of MAO B at Glu34 and Tyr44 to FAD This in with previous studies (1Kwan S.-W. Lewis D.A. Zhou Abell C.W. Arch. Biochem. Biophys. PubMed Scopus Google Scholar, Lewis D.A. Kwan S.-W. Abell C.W. PubMed Scopus Google Scholar, Lewis D.A. Kwan S.-W. Abell C.W. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), that the MAO B at Glu34 and which MAO B catalytic activity and the to FAD also FAD noncovalently in it appears that the of Gly226 and Asp227 in MAO B flavinylation the initial noncovalent binding of FAD to the consensus sequences of MAO B near the amino but to covalent to Cys397. In we that at by the MAO that Asp227 of the by a of and J. Biol. Chem. Full Text PDF PubMed Google Scholar) as Lewis D.A. Kwan S.-W. Abell C.W. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). by the of and Biochem. PubMed Scopus Google Scholar) a as (1Kwan S.-W. Lewis D.A. Zhou Abell C.W. Arch. Biochem. Biophys. PubMed Scopus Google Scholar). MAO B the site of the used to MAO B The and the amino acid are shown in Gly at with in the and Asp at with or the of were to a site the sequence for amino site a to the site in the The were for the of the site by the The of the in by F. S. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). and were to COS-7 used for MAO B were in with bovine and at COS-7 were by these in bovine for Lewis D.A. Kwan S.-W. Abell C.W. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). by U. J. J. Biol. 1982; PubMed Scopus Google Scholar) of or MAO B COS-7 as (1Kwan S.-W. Lewis D.A. Zhou Abell C.W. Arch. Biochem. Biophys. PubMed Scopus Google Scholar). In covalent flavinylation or noncovalent FAD of or MAO B of and of MAO B were COS-7 in were in of bovine bovine and at with were at and in a and of MAO B by to a of and for at Protein of or MAO B were determined a were to MAO B by with a to MAO B a of the of and Biophys. PubMed Scopus Google Scholar) as (1Kwan S.-W. Lewis D.A. Zhou Abell C.W. Arch. Biochem. Biophys. PubMed Scopus Google Scholar). of or MAO B were determined in for MAO B activity by a of the of and J. Biochem. Pharmacol. PubMed Scopus Google Scholar) as (1Kwan S.-W. Lewis D.A. Zhou Abell C.W. Arch. Biochem. Biophys. PubMed Scopus Google Scholar). The of and MAO were determined in in MAO B activity expressed as activity of of and as activity of of MAO B). COS-7 with or MAO B were in of and MAO B the with for at The at for and were to MAO B were with of a B at by with of for The of and MAO B or were by at for and with and the or MAO B proteins in the were with and the or MAO B proteins were the with of a for at the by with of and were to of or MAO B proteins the The were for proteins were to and by with the MAO MAO R.M. Fritz R.R. Patel N.T. Abell C.W. Science. 1982; 215: 1400-1403Crossref PubMed Scopus (121) Google Scholar), as (1Kwan S.-W. Lewis D.A. Zhou Abell C.W. Arch. Biochem. Biophys. PubMed Scopus Google Scholar). and MAO B enzymes were to on a The gel acid and for and for to and Eur. J. Biochem. 46: PubMed Scopus Google Scholar) with as Lewis D.A. Kwan S.-W. Abell C.W. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The gel to at for and MAO B enzymes were in a to the the membrane with of and the or MAO B, an the membrane and the membrane a the the membrane The were with of and The membrane in an at and to at for The of and the of were and the Scholar). and MAO B enzymes were expressed in mammalian COS-7 these not MAO B (1Kwan S.-W. Lewis D.A. Zhou Abell C.W. Arch. Biochem. Biophys. PubMed Scopus Google Scholar). of and MAO B enzymes are shown in The of MAO B expressed for of MAO of to that of MAO B of of Asp227 with or to a or in of Asp227 with in a of activity A in which the glycine with at also a of activity the of the with their of and of and MAO B B MAO MAO of are shown in of or MAO B expressed in COS-7 and the were at with for The were and and MAO B proteins were the cell with the in MAO B and activity were were in in the of are shown in in a of or MAO B expressed in COS-7 and the were at with for The were and and MAO B proteins were the cell with the in MAO B and activity were were in in the To the of MAO B by and MAO B enzymes were with B to and MAO as R.M. Fritz R.R. Patel N.T. Abell C.W. Science. 1982; 215: 1400-1403Crossref PubMed Scopus (121) Google Scholar). shown in MAO B and have of to the prepared COS-7 MAO B at not a of and MAO B enzymes with substitutions at Asp227 and Gly226 by of and MAO B The and MAO B enzymes were to and a of the that covalent of and and of that of the These results indicate that MAO B and the can covalently to to and to covalently and The of MAO B covalently linked FAD and noncovalently FAD. The of the MAO B on the of only FAD covalently to the a and a noncovalently FAD be the to of proteins to and we a to the of that is covalently and noncovalently the or MAO B in the and enzymes in the of the that the of to and were and of that of MAO B, that the of to the apoenzyme and noncovalent is the in the and enzymes with substitutions at positions and In previous studies (1Kwan S.-W. Lewis D.A. Zhou Abell C.W. Arch. Biochem. Biophys. PubMed Scopus Google Scholar, Lewis D.A. Kwan S.-W. Abell C.W. PubMed Scopus Google Scholar, Lewis D.A. Kwan S.-W. Abell C.W. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), we found that Glu34 in the site and Tyr44 in the adjacent FAD-binding are for initial FAD of Glu34 with aspartate or in an of activity and covalent FAD coupling a (1Kwan S.-W. Lewis D.A. Zhou Abell C.W. Arch. Biochem. Biophys. PubMed Scopus Google Scholar, Lewis D.A. Kwan S.-W. Abell C.W. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of Tyr44 with on activity or covalent FAD of Tyr44 with in a of activity and covalent FAD binding a Lewis D.A. Kwan S.-W. Abell C.W. PubMed Scopus Google Scholar). these FAD noncovalently not In we used to assess the of FAD binding for these The of and the of the were and with the The results of that the FAD binding for these with their covalent binding and of FAD of covalent binding of FAD, and activity as with MAO B. FAD covalent binding of FAD, and activity with MAO B. and to their to FAD not only covalently but also noncovalently of the for MAO B were The of these MAO with their and that Tyr44 are residues that in the of MAO B the covalent flavinylation process. have the of a site in MAO B that is highly conserved oxidoreductases that FAD as a B. J. Mol. Biol. 1990; PubMed Scopus Google Scholar, M. Van Protein Sci. 1996; PubMed Scopus Google Scholar, Biol. Scopus Google Scholar). site have and the of FAD-binding is B. J. Mol. Biol. 1990; PubMed Scopus Google Scholar, M. Van Protein Sci. 1996; PubMed Scopus Google Scholar). This FAD-binding of a β-strand that with an glycine residue that a sharp the aspartate residue in to FAD. The β-carbonyl group of highly conserved aspartate a with the 3′-hydroxyl group of the ribityl of FAD. To the of the residues (Gly226 and in MAO B, the glycine with and aspartate with or the activity a group is in the the of the chain is not as as a group is at A of activity for which also the of aspartate by to a the β-carbonyl group of and the 3′-hydroxyl group of the ribityl of FAD. the the chain a with a group in a but not of activity A reduction of activity in the is by the chain of that residue a sharp turn at The of reduction the as in that the sharp turn by Gly226 and the the β-carbonyl group of Asp227 and the 3′-hydroxyl group of the ribityl chain are for the activity of MAO B. The of and MAO B enzymes determined by an B is by at major to a the of a MAO B and Our results that the of MAO B enzymes determined by are to the of MAO B that activity of the MAO B enzymes were to be to or in the on covalent flavinylation of these enzymes and that the MAO B covalently to the as MAO B. a of covalently of the but and covalently and of MAO B, These results are in with the for of the and the that Gly226 and Asp227 in the flavinylation of MAO B. MAO B noncovalently are not on that is not linked to MAO B the in were to the of that to MAO B noncovalent and covalent the used for not the of the on the the of to the covalently and in the of the to the and enzymes and were COS-7 the of of the that the of the to the and were and of that of MAO B. These results indicate that at positions and not the of the apoenzyme to the results of the and indicate that the enzymes at positions and their to FAD the but covalent of FAD to the with the of noncovalent binding of FAD not that the be in the for the step in the is that the of with covalently but not with the of This that only MAO B with covalently FAD are These results are in with studies by J. M. PubMed Scopus Google Scholar, K. Shih J.C. Mol. Pharmacol. Google Scholar, F. Y. J. Ito A. K. and Scholar), found that of the FAD covalent binding residue in MAO B with or catalytic activity the mutants were expressed in mammalian In Ito and F. Y. J. Ito A. K. and Scholar, Y. F. Ito A. J. Biochem. 1996; PubMed Scopus Google Scholar) that the proteins expressed in catalytic activity the FAD covalent binding residues in MAO A and Cys397 in MAO B) were with In previous studies (1Kwan S.-W. Lewis D.A. Zhou Abell C.W. Arch. Biochem. Biophys. PubMed Scopus Google Scholar, Lewis D.A. Kwan S.-W. Abell C.W. PubMed Scopus Google Scholar, Lewis D.A. Kwan S.-W. Abell C.W. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), we identified two amino acid residues and that are for covalent FAD these enzymes can FAD noncovalent not In we the FAD-binding of these enzymes by of the of and that the of the with the of the on the Furthermore, the FAD binding by the of the and covalent binding of FAD by the of these also with their the of Gly226 and Asp227 and not only their to FAD but also their to FAD In two and a their to FAD and the of their covalent FAD binding with their FAD These results indicate that Glu34 and Tyr44 in the terminus of the the initial binding of FAD, it in the for the steps that to covalent binding of FAD. of one of these of MAO B The of and MAO B enzymes also by The and of at a of of the at by the MAO MAO of these the activity and the of these MAO and it used for MAO B and (12Bach A.W.J. Lan N.C. Johnson D.L. Abell C.W. Bembenek M.E. Kwan S.-W. Seeburg P.H. Shih J.C. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 4934-4938Crossref PubMed Scopus (701) Google Scholar, 8Westlund K.N. Denney R.M. Kochersperger L.M. Rose R.M. Abell C.W. Science. 1985; 230: 181-183Crossref PubMed Scopus (419) Google Scholar, 10Denney R.M. Fritz R.R. Patel N.T. Abell C.W. Science. 1982; 215: 1400-1403Crossref PubMed Scopus (121) Google Scholar, Lewis D.A. Kwan S.-W. Abell C.W. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The of MAO to the enzymes with substitutions at that Asp227 of the site appears to be mutagenesis of the adjacent residue on of the apoenzyme by flavinylation is obligatory for the of catalytic activity in J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar), A. M. R. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar), oxidase M. J. R. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar), M. J. Biol. Chem. 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Biol. 1996; PubMed Scopus Google Scholar) determined the structures of and that the site is by the of the apoenzyme that the to the and to of the ribityl chain and These the that binding the of in the of have covalent and noncovalent FAD of several MAO B that single amino acid substitutions at positions in two FAD-binding to the end and to the site (residues Based on the results of these we propose that FAD to B in a the and of the The step of FAD. The group of Glu34 the and the of the adjacent FAD-binding in the terminus the initial for FAD binding (1Kwan S.-W. Lewis D.A. Zhou Abell C.W. Arch. Biochem. Biophys. PubMed Scopus Google Scholar, Lewis D.A. Kwan S.-W. Abell C.W. PubMed Scopus Google Scholar, Lewis D.A. Kwan S.-W. Abell C.W. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). This can at one of these residues in a of covalent and noncovalent of FAD. propose that FAD is to the site (residues 222–227), which to the FAD The FAD, which is at with several amino acid can be to Cys397 in a that the group of FAD in and to the group of Cys397 to covalent at Gly226 and Asp227 in the site not with noncovalent binding of FAD are not involved in FAD but have to FAD the of these not of Cys397 for Although the of FAD coupling is not studies the complex of steps that to of MAO B by MAO have used for the of and R. J. 1989; Scholar) and J.W. J.W. Science. 1989; PubMed Scopus Google Scholar). Our studies on the of flavinylation to the of of for of these Lewis for of and