Structure-Function Relationships in Flavoenzyme-dependent Amine Oxidations (2002)

polyamine oxidase monoamine oxidase Amine oxidations are important in a number of basic biological processes ranging from lysyl oxidation in the cross-linking of collagen to the degradative metabolism of polyamines and neurotransmitters. The oxidations of biogenic amines to the corresponding imines are catalyzed by either the quinoprotein class of enzymes (usually primary amines) (1Hartmann C. McIntire W.S. Methods Enzymol. 1997; 280: 98-150Crossref PubMed Scopus (15) Google Scholar) or by the flavin-containing amine oxidases (primary, secondary, or tertiary amines) (2Massey V. Biochem. Soc. Trans. 2000; 28: 283-296Crossref PubMed Google Scholar). In both cases, molecular oxygen is the usual electron acceptor with hydrogen peroxide formed as reaction product. Flavin amine oxidases (2Massey V. Biochem. Soc. Trans. 2000; 28: 283-296Crossref PubMed Google Scholar) catalyze the oxidation of amines via an oxidative cleavage of the α-CH bond of the substrate to form an imine product with the concomitant reduction of the flavin cofactor (Fig. 1A). The imine product is then hydrolyzed (non-enzymatically in the cases investigated) to the corresponding aldehyde and ammonia (or amine for secondary or tertiary amine substrates). The reduced flavin coenzyme reacts with oxygen to form hydrogen peroxide and the oxidized form of the flavin to complete the catalytic cycle. The focus of this minireview is the structure and function of plant polyamine oxidase (PAO)1 and human monoamine oxidase (MAO), two thoroughly investigated members of the flavin-dependent class of amine oxidases. Both enzymes have been structurally characterized by x-ray crystallography (3Binda C. Coda A. Angelini R. Federico R. Ascenzi P. Mattevi A. Structure Fold Des. 1999; 7: 265-276Abstract Full Text Full Text PDF Scopus (163) Google Scholar, 4Binda C. Newton-Vinson P. Hubalek F. Edmondson D.E. Mattevi A. Nat. Struct. Biol. 2002; 9: 22-26Crossref PubMed Scopus (533) Google Scholar). Plant PAO is involved in the catabolism of polyamines (Fig.1 B) by catalyzing the oxidation of the secondary amino groups of spermine or spermidine and their acetyl derivatives (5Seiler N. Prog. Brain Res. 1995; 106: 333-344Crossref PubMed Scopus (124) Google Scholar). It is a soluble enzyme with a non-covalently bound FAD cofactor (6Sebela M. Radova A. Angelini R. Tavladoraki P. Frebort I.I. Pec P. Plant Sci. 2001; 160: 197-207Crossref PubMed Scopus (117) Google Scholar, 7Tavladoraki P. Schinina M.E. Cecconi F., Di Agostino S. Manera F. Rea G. Mariottini P. Federico R. FEBS Lett. 1998; 426: 62-66Crossref PubMed Scopus (91) Google Scholar). Very recently, the gene for human PAO has been identified (8Wang Y.L. Devereux W. Woster P.M. Stewart T.M. Hacker A. Casero R.A. Cancer Res. 2001; 61: 5370-5373PubMed Google Scholar), and the protein was investigated in preliminary work. Polyamines are essential for cell growth and differentiation (9Tabor C.W. Tabor H. Annu. Rev. Biochem. 1984; 53: 749-790Crossref PubMed Scopus (3233) Google Scholar), and their metabolism is the subject of extensive research to develop potential targets for antiproliferative drugs (10Casero R.A., Jr. Woster P.M. J. Med. Chem. 2001; 44: 1-26Crossref PubMed Scopus (218) Google Scholar). MAOs oxidize the primary amino groups of arylalkyl amines (Fig.1 C) and are widely distributed in higher eukaryotes. In mammals, MAO is present as two isoforms (MAO A and MAO B), which are separate gene products, that exhibit over 70% sequence identity and distinct but overlapping substrate specificities in the catabolism of neurotransmitters, such as dopamine and serotonin (11Weyler W. Hsu Y.P. Breakfield X. Pharmacol. Ther. 1990; 47: 391-417Crossref PubMed Scopus (291) Google Scholar,12Shih J.C. Chen K. Ridd M.J. Annu. Rev. Neurosci. 1999; 22: 197-217Crossref PubMed Scopus (1030) Google Scholar). Both MAO A and MAO B are implicated in a large number of neurological disorders and are a target for drugs against Parkinson's disease and depression (13Cesura A.M. Pletscher A. Prog. Drug Res. 1992; 38: 171-297PubMed Google Scholar). Mammalian MAOs are bound to the outer mitochondrial membrane and have a FAD molecule covalently bound to the protein via an 8α-thioether linkage to a cysteinyl residue (14Kearney E.B. Salach J.I. Walker W.H. Seng R.L. Kenney W. Zeszotek E. Singer T.P. Eur. J. Biochem. 1971; 24: 321-327Crossref PubMed Scopus (193) Google Scholar). They are expressed in both a tissue-dependent and an age-dependent manner and have been the subject of extensive clinical and pharmacological studies with more than 15,000 papers currently listed in the Medline index. A good deal of mechanistic information is available for these two isozymes (see below). The recent determination of the crystal structures of plant PAO and human MAO B have revealed valuable insights into the structure-function of the flavin-dependent amine oxidases. PAO (472 residues) and MAO B (520 residues) share ∼20% amino acid sequence identity that is distributed throughout their respective polypeptide chains with the FAD-binding regions exhibiting the highest level of homology (3Binda C. Coda A. Angelini R. Federico R. Ascenzi P. Mattevi A. Structure Fold Des. 1999; 7: 265-276Abstract Full Text Full Text PDF Scopus (163) Google Scholar). Mammalian MAOs contain a 50-residue C-terminal segment that is not found in the PAO sequence. Truncation experiments as well as sequence analysis have demonstrated that this C-terminal segment of MAO A or MAO B is involved in anchoring them to the outer mitochondrial membrane (15Mitoma J. Ito A. J. Biochem. (Tokyo). 1992; 111: 20-24Crossref PubMed Scopus (93) Google Scholar). The crystallization of human MAO B was facilitated by the development of a high level expression system of the human gene in Pichia pastoris (16Newton-Vinson P. Hubalek F. Edmodson D.E. Protein Expression Purif. 2000; 20: 334-345Crossref PubMed Scopus (105) Google Scholar). Recombinant MAO B is tightly bound to the membrane fraction of the expression host. The maize PAO used in the structural experiments was isolated from natural sources. The elucidations of the respective three-dimensional structures of both plant PAO and human MAO B have permitted insights into several common features that provide a structural framework for a more definitive understanding of their respective mechanisms of amine oxidation. The three-dimensional structure of maize PAO was solved at 1.9-Å resolution (3Binda C. Coda A. Angelini R. Federico R. Ascenzi P. Mattevi A. Structure Fold Des. 1999; 7: 265-276Abstract Full Text Full Text PDF Scopus (163) Google Scholar). The overall fold has a two-domain topology (Fig.2 A), essentially identical to that observed in the bacterial flavoenzyme p-hydroxybenzoate hydroxylase (the so-called “PHBH” fold) (17Fraaije M.W. Mattevi A. Trends Biochem. Sci. 2000; 25: 126-132Abstract Full Text Full Text PDF PubMed Scopus (419) Google Scholar). The interface of the two domains defines a U-shaped catalytic tunnel (Fig. 2 A), which is about 30 Å in length, being optimally configured to bind the linear polyamine substrates (Fig 1 B). The openings at each end of the tunnel are on the same side of the protein surface. One opening is lined by several acidic amino acid residues, which may have a role in steering the polycationic protonated amine substrate into the substrate-binding site. The interior of the tunnel is embedded by a number of aromatic residues, and its innermost section is located in front of the flavin to form the catalytic site for amine oxidation. Human MAO B is about 59 kDa with the FAD cofactor bound through the flavin C8α-position to a cysteine side chain (Cys-397). The crystal structure, which was solved to a resolution of 3.0 Å (4Binda C. Newton-Vinson P. Hubalek F. Edmondson D.E. Mattevi A. Nat. Struct. Biol. 2002; 9: 22-26Crossref PubMed Scopus (533) Google Scholar), reveals that the enzyme crystallizes as a dimer in two different crystal forms, which suggests that it may also occur as a dimer in its membrane environment. The overall fold of each monomer (Fig. 2 B) resembles that of PAO (Fig. 2 A). The main structural difference is related to the 50-residue C-terminal tail. This region forms an extended segment that traverses the protein surface and then folds into an α-helix. This helix protrudes from the basal face of the structure to anchor the protein to the mitochondrial outer membrane. Another prominent feature of the structure is the presence of two adjacent cavities in the interior of the protein (Fig.2 B). The largest cavity (a flat hydrophobic entity of 420 Å3 in volume) is directly in front of the covalent flavin ring and forms the substrate-binding site. For the substrate to enter this cavity, it must first bind to an “entrance cavity” (also a hydrophobic entity of 290 Å3 volume). This entrance cavity is situated near the point where the protein surface intersects with the surface of the outer mitochondrial membrane. The anionic membrane surface may facilitate the electrostatic channeling of the positively charged amine to this site for substrate admission in a manner similar to the negatively charged entrance site to the substrate tunnel in PAO. Both the entrance and substrate cavities are lined by aromatic and aliphatic residues that create a highly apolar environment for substrate binding. Whether such distinct cavities exist in the human MAO A structure is not known. It is perhaps worthwhile to point out that the amino acid residues separating the two cavities in MAO B are different in MAO A. Site-directed mutagenesis work (18Geha R.M. Rebrini I. Chen K. Shih J.C. J. Biol. Chem. 2001; 276: 9877-9882Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar) suggests Tyr-326 in MAO B (situated at the juncture of the two cavities) to be important. The current view for MAO is that the amine substrate is deprotonated to the free base on entering the catalytic substrate binding site. Thus, by extension, the deprotonated polyamine substrate also may be the form that traverses the tunnel in PAO. The mechanism for substrate deprotonation in this class of enzymes is not known. A striking feature emerging from the structural comparison is that, despite their overall structural folding similarity, PAO and MAO B differ substantially in the overall topology of their respective substrate-binding sites. In particular, the PAO U-shaped tunnel and the MAO B cavities follow entirely different pathways within the three-dimensional structures and are lined by residues that are not homologous in sequence. The only conserved features can be found in the sites for binding of the flavin and for recognition of the substrate amine group that undergoes oxidation. In both PAO and MAO B, the normally planar flavin ring is highly bent about the N5–N10 axis (about a 30° angle between the pyrimidine and the dimethylbenzene rings) with the active sites situated on there side of the flavin ring. Hydrogen-bonding interactions of the isoalloxazine ring of the cofactor with the protein are similar in the two enzymes. In particular, Lys-300 of PAO is bridged to the N5 atom of the flavin through a water molecule. Binding studies with inhibitors (19Binda C. Angelini R. Federico R. Ascenzi P. Mattevi A. Biochemistry. 2001; 40: 2766-2776Crossref PubMed Scopus (61) Google Scholar) have shown that this residue participates in catalysis by compensating for the change in the flavin protonation state (i.e. the addition of a hydrogen atom to the N5-position, which occurs on cofactor reduction). This interaction is strictly conserved also in MAO B where Lys-296 occupies a position identical to that of Lys-300 in PAO (Fig. 3, A andB). Recent 2.5-Å x-ray diffraction data on MAO B have shown a water molecule acting as a bridge between Lys-296 and the flavin N5 atom. This structural feature is not unique to these amine oxidases and has also been found in other flavoenzyme oxidases (but is not a general structural feature of flavoenzymes). In l-amino acid oxidase (20Pawelek P.D. Cheah J. Coulombe R. Macheroux P. Ghisla S. Vrielink A. EMBO J. 2000; 19: 4204-4215Crossref PubMed Scopus (227) Google Scholar), Lys-326 is structurally positioned with respect to the flavin as is Lys-300 in PAO. In the structure of monomeric sarcosine oxidase (21Trickey P. Wagner M.A. Jorns M.S. Mathews F.S. Structure Fold Des. 1999; 7: 331-345Abstract Full Text Full Text PDF Scopus (152) Google Scholar) (an enzyme that oxidizes N-methylglycine to glycine), Lys-265 is also hydrogen-bonded to the flavin N5-position through a water molecule. Taken together, these observations indicate the “Lys-H2O-flavin N5” element as a structural motif shared among flavin-dependent amine and amino acid oxidases. The PAO and MAO B substrate-binding sites, where the flavin-dependent amine oxidation takes place, display several conserved features. In both proteins, two aromatic amino acid side chains form an “aromatic sandwich” by facing each other in perpendicular orientations to the flavin on its re side. In PAO, residues Phe-403 and Tyr-439 are positioned parallel to each other and perpendicular to the flavin plane (Fig. 3 A). Likewise, in MAO B, there is an aromatic pair formed by Tyr-398 and Tyr-435, whose aromatic rings are slightly turned toward the flavin (Fig.3 B). The distance between these aromatic side chains in each pair is about 8 Å (Table I). Furthermore, in both PAO and MAO B the aromatic pairs are sheltered by additional aromatic residues (Fig. 3, A and B). Trp-60 in PAO and Tyr-60 in MAO B are positioned at the top of the flavin ring, being coplanar to the respective pyrimidine rings of the flavin coenzymes whereas Tyr-298 of PAO and Phe-343 of MAO B are both located on the same side of the flavin, in proximity of the lysine that is hydrogen-bonded to the FAD.Table IComparative distances in the catalytic sites of plant PAO and human MAO BPAOMAO ÅAromatic pair1-aThe aromatic sandwich is formed by Phe-403 and Tyr-439 in PAO and Tyr-398 and Tyr-435 in MAO B. The distance is between the centers of the side chain rings forming the aromatic pairs or between the nitrogen atom of the modeled substrate and the centers of the aromatic rings of the amino acids designated.8.27.8N(substrate)-aromatic1-aThe aromatic sandwich is formed by Phe-403 and Tyr-439 in PAO and Tyr-398 and Tyr-435 in MAO B. The distance is between the centers of the side chain rings forming the aromatic pairs or between the nitrogen atom of the modeled substrate and the centers of the aromatic rings of the amino acids designated.4.5 (Phe-403)4.4 (Tyr-398)4.6 (Tyr-439)3.9 (Tyr-435)N(substrate)-C4a(flavin)1-bThe distances refer to those of the nitrogen atoms of the amino groups undergoing oxidation in the modeled substrate of spermine in PAO and of benzylamine in MAO B (3,4) with the flavin C4a position.3.83.71-a The aromatic sandwich is formed by Phe-403 and Tyr-439 in PAO and Tyr-398 and Tyr-435 in MAO B. The distance is between the centers of the side chain rings forming the aromatic pairs or between the nitrogen atom of the modeled substrate and the centers of the aromatic rings of the amino acids designated.1-b The distances refer to those of the nitrogen atoms of the amino groups undergoing oxidation in the modeled substrate of spermine in PAO and of benzylamine in MAO B (3Binda C. Coda A. Angelini R. Federico R. Ascenzi P. Mattevi A. Structure Fold Des. 1999; 7: 265-276Abstract Full Text Full Text PDF Scopus (163) Google Scholar,4Binda C. Newton-Vinson P. Hubalek F. Edmondson D.E. Mattevi A. Nat. Struct. Biol. 2002; 9: 22-26Crossref PubMed Scopus (533) Google Scholar) with the flavin C4a position. Open table in a new tab On the basis of the structures of enzyme-inhibitor complexes (3Binda C. Coda A. Angelini R. Federico R. Ascenzi P. Mattevi A. Structure Fold Des. 1999; 7: 265-276Abstract Full Text Full Text PDF Scopus (163) Google Scholar, 19Binda C. Angelini R. Federico R. Ascenzi P. Mattevi A. Biochemistry. 2001; 40: 2766-2776Crossref PubMed Scopus (61) Google Scholar), models of the bound substrates in the active sites have been proposed with reference to spermine for PAO (Figs. 1 B and3 A) and to benzylamine for MAO B (Figs. 1 C and3 B). In these models, the amino groups are positioned between the side chains of the aromatic sandwiches of the two enzymes with distances between the amine nitrogen and each aromatic ring (TableI) within the range generally observed in amine-aromatic recognition sites (22Levitt M. Perutz M.F. J. Mol. Biol. 1988; 201: 751-754Crossref PubMed Scopus (658) Google Scholar). An additional element in binding the substrate amino group is the flavin ring, whose C4a atom is in van der Waals contact with the substrate nitrogen (Fig. 3 C). The combination of the flavin and aromatic sandwich generates an “aromatic cage” that is suggested to recognize the deprotonated amine group of the substrate. Binding of the substrate amino group through aromatic side chains is also observed in bacterial trimethylamine dehydrogenase (a 6-S-cysteinyl- FMN-dependent dehydrogenase that catalyzes the oxidative N-demethylation of trimethylamine to dimethylamine and formaldehyde) (23 and references therein). The three-dimensional structure of this protein reveals that the binding site for the substrate amino group is in the form of an “aromatic bowl.” Thus, it appears that recognition of amine substrates via the placement of the amine moiety in an aromatic cage may represent a common feature among flavoenzymes catalyzing the oxidation of amines, which may have mechanistic implications. A number of mechanisms have been proposed to describe the chemical events involved in flavin-dependent amine oxidations. One mechanism that has achieved considerable notoriety is the single electron transfer mechanism (also termed the aminium cation radical or single electron transfer mechanism) that was suggested by Silverman and colleagues (24Silverman R.B. Accts. Chem. Res. 1995; 28: 335-342Crossref Scopus (244) Google Scholar) based on their work on the interaction of MAO B with N-cyclopropyl-N-benzylamine substrate analogues as well as other analogues that serve as mechanism-based inhibitors of the enzyme. This mechanism is shown in Fig4 A. A key feature of this mechanism is that the flavin serves as a one-electron oxidant of the amine to form the aminium cation radical as the first initial reversible step in catalysis. The formation of such an intermediate would render the α-proton sufficiently acidic as to allow a basic amino acid residue at the active site to abstract the proton with subsequent radical recombination occurring to form the imine product and reduced flavin as products. Although chemically attractive and able to account for the formation of ring-opened products observed, little direct evidence from rapid reaction studies has been found to support this scheme. Stopped flow studies show no evidence for any flavin radical intermediates during catalysis (25Walker M.C. Edmondson D.E. Biochemistry. 1994; 33: 7088-7098Crossref PubMed Scopus (143) Google Scholar, 26Miller J.R. Edmondson D.E. Biochemistry. 1999; 38: 13670-13683Crossref PubMed Scopus (167) Google Scholar). No influence of magnetic field on the reaction rate is observed as would be expected if a radical pair (flavin radical and substrate radical) were formed as transient intermediates (27Miller J.R. Edmondson D.E. Grissom C.B. J. Am. Chem. Soc. 1995; 117: 7830-7831Crossref Scopus (42) Google Scholar). Thermodynamic consideration also suggests that the one-electron oxidation of a primary amine (E = +1.5 V) by the flavin moiety in MAO B (E = +0.04 V) is unlikely (28Newton-Vinson P. Edmondson D.E. Ghisla S. Kroneck P. Macheroux P. Sund H. Flavins and Flavoproteins. Agency for Scientific Research, Berlin2000: 431-434Google Scholar). Current structural data on MAO B or on PAO also show there to be no amino acid residue at the catalytic site that would be expected to function as an active site base for any proposed H+abstraction mechanisms of catalysis. Mechanism-based inactivation studies of MAO B by cyclopropylbenzylamine analogues show inhibition of the enzyme to occur by modification of Cys-365, which was suggested to be the active site base in MAO B (29Zhong B. Silverman R.B. J. Am. Chem. Soc. 1997; 119: 6690-6691Crossref Scopus (42) Google Scholar). However, the structural data on MAO B show this residue not to be at the active site on the enzyme but instead to be at the surface of the molecule some 20 Å in distance (4Binda C. Newton-Vinson P. Hubalek F. Edmondson D.E. Mattevi A. Nat. Struct. Biol. 2002; 9: 22-26Crossref PubMed Scopus (533) Google Scholar). Although the structural data provide no evidence for a proton-abstraction mechanism, recent quantitative structure-activity relationships (QSAR) data on MAO A with a series ofpara-substituted benzylamine analogues (26Miller J.R. Edmondson D.E. Biochemistry. 1999; 38: 13670-13683Crossref PubMed Scopus (167) Google Scholar) provides definitive evidence that α-CH bond cleavage indeed does occur by a proton abstraction mechanism. The reconciliation of these apparently disparate observations is shown by the mechanism suggested for MAO A catalysis as shown in Fig. 4 B. In this mechanism, the amine functionality adds to the 4a-position of the flavin in a nucleophilic manner, which activates the N5-position to function as a strong active site base because the pK a of reduced flavins approximates that of benzyl protons. 2S. Ghisla, personal communication. This proposed mechanism would imply that the reaction is concerted; a conclusion that remains to be experimentally verified. The mechanism shown in Fig. 4 B would appear to be more consistent with available mechanistic and structural data than the radical mechanism shown in Fig. 4 A. The lysine-H2O-flavin N5 motif discussed above for the amine oxidases poses an additional question with regard to the proposed mechanism is Fig 4 B. Why should the flavin C4a adduct abstract a proton from the substrate when it has a water molecule in close proximity? Currently there is no answer to this question, and further work is required to provide an explanation. Flavin-dependent amine oxidases catalyze the oxidative dehydrogenation of a –CH2–NH– group in a manner mechanistically similar to the quinoprotein amine oxidases in that a H+ abstraction mechanism is the initial C–H bond cleavage event. These classes of enzymes differ in the substrate specificities observed and in the detailed mechanisms of catalysis. The flavin moiety is central to both substrate dehydrogenation and to the reduction of O2 to H2O2. The folding of the PAO amino acid chain relative to that of MAO B is crucial in defining their relative substrate binding sites, and these differences account for their respective substrate diversities. Similarities in active site structures suggest a crucial role for an aromatic cage in the oxidation of amines, which has yet to be mechanistically defined. The observed Lys-H2O-flavin N5 hydrogen bonding motif appears to be a consistent motif among several flavoenzyme oxidases, and the mechanistic significance of this motif awaits further work. The structural information available should allow for more detailed investigations, which should provide further insights into the catalytic mechanisms of flavin-dependent amine oxidations.