Contents:

I. Introduction
II. General Structure
III. Active Site
IV. FAD-Binding Domain
V. Conclusion
VI. References

I. Introduction

Monoamine oxidase (MAO) is an FAD-dependent enzyme located in the outer mitochondrial membrane of cells in both the central nervous system and peripheral tissues [10, 12]. It is involved in the oxidative deamination of neurotransmitters and other primary and secondary amines, and is found in two isoforms, A and B. MAO A preferentially oxidizes serotonin, adrenaline, and dopamine, and defects have been linked to depression and abnormally aggressive behavior [1, 5, 10].

MAO B, on the other hand, selectively oxidizes ß-phenylethylamine and benzylamine, as well as dopamine, tyramine, and tryptamine [1, 5, 10]. It contains 520 amino acid residues, sharing 70% of its sequence with MAO A, and has a molecular weight of approximately 58, 000 kDa [10]. MAO B expression in the brain increases during aging and may be linked to disorders such as Alzheimer’s, while a particular allele of the MAO B gene has been linked to Parkinson’s disease [10, 12]. While Parkinson’s is often treated with L-DOPA, the precursor amino acid to dopamine, the addition of an MAO B inhibitor such as deprenyl dramatically increases its neuroprotective effects [10].

The mechanistic pathway of catalysis by MAO B is similar to that of other flavoenyzme oxidases, in that it can proceed by either a binary or a ternary complex mechanism dependent on the substrate that is present. Benzylamine and its meta and para substituted analogues are oxidized by a ternary pathway, while phenylethylamine oxidation occurs via a binary (or ping-pong) pathway. The difference in pathway is due to variance in the rate of release of the protonated imine product relative to the rate of oxidation of the reduced flavin-imine complex by diatomic oxygen. In benzylamine oxidation, the rate of oxidation of the imine complex is many times faster than the rate of imine dissociation, while the opposite is true when phenylethylamine is the substrate.

Mechanisms of MAO B Catalysis [7]

II. General Structure

Monoamine oxidase B is an integral outer membrane protein in the mitochondria. MAO B is a dimeric enzyme < > consisting of 520 amino acids < > [1-3, 10]. Each monomer consists of a large solvent-exposed globular structure (residues 1-488 < >) and a short helical C-terminal domain (residues 489-520 < >) that is anchored to the lipid bilayer of the mitochondrial outer membrane [1, 2]. The solvent-exposed domain of the protein is representative of similar structures in other flavin-dependent enzymes consisting of an FAD-binding domain and a substrate-binding domain [6]. The substrate-binding domain consists of a large cavity (700 cubic Å) which serves as the active site for the protein. This cavity opens on one side of the protein and extends deep into the core until it reaches the inner face of the flavin cofactor < >[13].

An extended loop (residues 461-488 < >) originates from the FAD-binding domain and connects the main body of the protein to the C-terminal a-helix. The C-terminus is a transmembrane a-helix that functions to anchor the protein to the phospholipids bilayer of the cell membrane. As previously mentioned, the helical domain of the C-terminus is orientated perpendicularly to the axis of the monomer [6]. Association with the membrane is established through the electrostatic interactions of the side chain of Arg494 < > with the polar head groups of the phospholipids. This loop is stabilized by a number of interactions with the amino acid side chains of both substrate- and cofactor-binding domains. < > The helical axis of the C-terminal domain is then positioned perpendicular to the plane of axis of the globular domain of the monomer. Two structures have been proposed for the membrane-embedded portion of the C-terminal domain (residues 500-520; Figure 1); however, it has been impossible to determine which is correct, as their embeddedness in the membrane makes crystallography impossible.

Two possible structures for the C-terminal domain of MAO B [2]

Pro109-Ile110 and Trp157 < > are solvent-exposed hydrophobic sites in the substrate-binding domain that are in close proximity to the C-terminal helix. Studies that truncated the C-terminal tail (residues 461-520) show that this truncation does not completely prevent binding of MAO B to the mitochondrial membrane, suggesting that there are other interactions that aid in the protein's association with the membrane. The aforementioned hydrophobic residues may help to create a more rigid orientation of the protein to the membrane surface and aid the C-terminus in anchoring the protein [8].

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III. Active Site

The active site of MAO B is located within the substrate-binding domain of each monomer and consists of two solvent-inaccessible cavities < > [1, 2, 3]. Substrates must pass through the entrance cavity, which opens onto the membrane-bound side of the protein, before they enter the substrate-binding cavity on the re side of the flavin cofactor < > [2, 3]. Both the entrance cavity and the substrate-binding cavity are lined with hydrophobic aromatic and aliphatic residues [3].

The entrance cavity is shielded from the solvent by loop 99-112. < > Ile110 and Pro109, < > located at the end of the loop, are surface-exposed and may interact with the mitochondrial membrane to serve as a gating mechanism for the two cavities of the active site. The entrance cavity < > has a volume of 290 cubic Å and is lined by the residues Phe103, Pro104, Trp119, Leu164, Leu167, Phe168, Leu171, Ile199, Ile316, and Tyr326 < >[3]. These residues are part of a sequence that confers substrate and inhibitor specificity [8].

Residues Tyr326, Leu171, Phe168, and Ile199 < > serve as a gating mechanism for substrate entrance to the substrate-binding site [2, 3]. In particular, the side chain of Ile199 < > adopts a closed or open conformation in the presence of bulky ligands that allows their diffusion into the active site [2]. The total distance that substrates must travel from the surface of the entrance cavity to the flavin ring, where catalysis occurs, is approximately 20 Å [3].

The substrate cavity is in the shape of an ellipsoidal disk. One side is lined by residues Leu171, Cys172, and Tyr398; the other, by Phe168, Ile198, Ile199, Gln206, and Tyr435 < >[2, 3]. The floor of the cavity is formed by the side chain of Tyr188, while the ceiling is made up of the aromatic residues Tyr60, Tyr326, and Phe343 < >[2]. The flat shape of the cavity restricts the orientation of substrate or inhibitor during binding, such that the carbon atom being oxidized is bound in a highly conserved position near the flavin N5-C4 locus. The amine of benzylamine, for example, is held in an aromatic caged environment between the phenolic side chains of Tyr398 and Tyr435 [3].

Crystallization studies of MAO-B in complex with N-propargylaminoindan and other inhibitors have provided evidence for a network of water molecules in the active site (Figure 2). Two of these molecules are H-bonded to each other at the bottom of the substrate cavity between Tyr435 and Tyr398. The other two water molecules are found in the lateral side of the cavity, H-bonding to the oxygen atom of Gln206 and the O4 atom of the flavin ring [2]. Further research is necessary to determine whether these water molecules are also in place during substrate binding, or if their presence is a factor in the inhibitory properties of molecules such as rasigiline and S-PAI [2].

Stereo view of MAO B binding site with inhibitors [2]

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IV. FAD-Binding Domain

The flavin adenine dinucleotide (FAD cofactor) is covalently bound by a thioester link to the protein between the 8a-position and Cys397, < > which is located in the C-terminal region of MAO B [4]. There are also extensive non-covalent interactions of the FAD side chain with MAO B. A hydrophobic environment surrounds the coenzyme within the protein, and hydrogen-bonding interactions dominate the bonding to both amino acid side chains oand peptide bonds. The only electrostatic interaction is between the anionic pyrophosphate of FAD and the positively charged guanidine group of Arg42. < > The pyrophosphate bond also experiences extensive H-bonding to water molecules and to the peptide bonds of Thr426 and Ser15. < > The carboxyate of Glu34 is H-bonded to the ribose ring of the FAD adenosine moiety. < > This is important for FAD binding before covalent incorporation as well as for maintaining the structural integrity of the covalent FAD in MAO B. The ribose ring of the FAD adenosine also forms H-bonds with a guanidine group of Arg36 < > and to a water molecule. The specific adenine ring interactions with the protein include only H-bonds to the peptide bond of Val235 < > and an additional H-bond to a water molecule. The ribityl side chain of the flavin moiety has very few interactions with the protein. The only specific interactions observed are H-bonds between the 3'-OH and the carbonyl oxygen of Gly434 < > as well as between the 4'-OH and a phosphate oxygen of the pyrophosphate linkage [6, 7].

FAD binding site [2]

Interactions of the isoalloxazine ring of the covalent FAD with the MAO B exhibit important function with the flavin reactivity that do not involve specific interactions, but steric constraints, instead. The flavin ring is located in an area of the enzyme that is not accessible to bulk solvent. The substrate binding site identifies the face of the flavin ring that interacts with the amine substrate in catalysis. Here, many H-bonding systems are observed. < > The first is between the 2-carbonyl oxygen of the pyrimidine ring and the N-H of Met436 peptide bond and a water molecule. Another involves the pyrimidine ring again, and is between the N(3)-H and the carbonyl of Tyr60. At the same time, the Tyr60 and the 4-carbonyl form an H-bond. Many other hydrogen bonds also interact with the isoalloxazine ring to add to the stability of the structure. This is a common motif throughout flavoprotein oxidases [6].

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V. Conclusion

Monoamine oxidase B is an FAD-dependent enzyme that is active in the oxidative deamination of neurotransmitters, including dopamine and noradrenaline, and of other arylakylamines, such as benzylamine, which might otherwise function as false neurotransmitters [1, 5, 12]. It is found in high concentrations in the brain and central nervous system, as well as in peripheral tissues [10, 12]. Crystallography has definitively demonstrated that MAO B occurs naturally as a dimer and does not retain its activity in the monomeric form [1-3]. However, work conducted using MAO B isolated from bovine liver suggests that the enzyme may in fact be present in larger oligomeric complexes, with a hexameric configuration producing the highest rate of activity [12].

The gene encoding MAO B is located on the X chromosome at Xp11.23-11.4; particular alleles have been linked to Parkinson's disease, while increased overall expression has been found in aging brains, especially in Alzheimer's patients [10, 12]. Parkinson's is often treated using L-DOPA and MAO B inhibitors such as deprenyl [10]. As overactivity of MAO B may also be occuring in Alzheimer's disease, future pharmaceutical research is necessary into the use of MAO B inhibitors as therapy for this devastating disorder.

VII. References

1. Binda, C., Hubálek, F., Li, M., Edmondson, D. E., & Mattevi, A. (2004). Crystal structure of human monoamine oxidase B, a drug target enzyme monotopically inserted into the mitochondrial outer membrane. Federation of European Biochemical Societies Letters, 564, 225-228.

2. Binda, C., Hubálek, F., Li, M., Herzig, Y., Sterling, J., Edmondson, D. E., & Mattevi, A. (2004). Crystal structures of monoamine oxidase B in complex with four inhibitors of the N-propargylaminoindan class. Journal of Medicinal Chemistry, 47, 1767-1774.

3. Binda, C., Newton-Vinson, P., Hubálek, F., Edmondson, D. E., & Mattevi, A. (2002). Structure of human monoamine oxidase B, a drug target for the treatment of neurological disorders. Nature Structural Biology, 9, 22-26.

4. Cesura, A. M., Gottowik, J., Lahm, H.-W., Lang, G., Imhof, R., Malherbe, P., Röthlisberger, U., & Da Prada, M. (1996). Investigation on the structure of the active site of monoamine oxidase-B by affinity labeling with the selective inhibitor lazabemide and by site-directed mutagenesis. European Journal of Biochemistry, 236, 996-1002.

5. Chen, K., Wu, H.-F., & Shih, J. C. (1996). Influence of C terminus on monoamine oxidase A and B catalytic activity. Journal of Neurochemistry, 66, 797-803.

6. Edmondson, D.E., Binda, C., Mattevi, A. (2004). The FAD binding sites of human monoamine oxidases A and B. Neurotoxicology, 25, 63-72.

7. Edmondson, D.E. (1995). Structure activity of the substrate binding site in monoamine oxidase B. Biochimie, 77, 643-650.

8. Gottowik, J., Malherbe, P., Lang, G., Da Prada, M., & Cesura, A. M. (1995). Structure/function relationships of mitochondrial monoamine oxidase A and B chimeric forms. European Journal of Biochemistry, 230, 934-942.

9. Miller, J.R., Guan, N., Hubalek, F., Edmondson, D.E. (2000). The FAD binding sites of human liver monoamine oxidases A and B: investigation of the role of flavin ribityl side chain hydroxyl groups in the covalent flavinylation reaction and catalytic activities. Biochimica et Biophysica Acta, 1476, 27-32.

10. Nagatsu, T. (2004). Progress in monoamine oxidase (MAO) research in relation to genetic engineering. NeuroToxicology, 25, 11-20.

11. Palmer, S. L., Mabic, S., & Castagnoli, N. (1997). Probing the active sites of monoamine oxidase A and B with 1,4-disubstituted tetrahydropyridine substrates and inactivators. Journal of Medicinal Chemistry, 40, 1982-1989.

12. Shihloff, B. A., Behrens, P. Q., Kwan, S.-W., Lee, J. H., & Abell, C. W. (1996). Monoamine oxidase B isolated from bovine liver exists as large oligomeric complexes in vitro. European Journal of Biochemistry, 242, 41-50.

13. Veselovsky, A. V., Ivanov, A. S., Medvedev, A. E. (2004). Computer modeling and visulaization of active site of monoamine oxidases. Neurotoxicology, 25, 37-46.