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PDBsum entry 2c75
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Oxidoreductase
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PDB id
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2c75
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References listed in PDB file
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Key reference
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Title
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Functional role of the "aromatic cage" in human monoamine oxidase b: structures and catalytic properties of tyr435 mutant proteins.
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Authors
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M.Li,
C.Binda,
A.Mattevi,
D.E.Edmondson.
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Ref.
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Biochemistry, 2006,
45,
4775-4784.
[DOI no: ]
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PubMed id
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Abstract
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Current structural results of several flavin-dependent amine oxidizing enzymes
including human monoamine oxidases A and B (MAO A and MAO B) show aromatic amino
acid residues oriented approximately perpendicular to the flavin ring,
suggesting a functional role in catalysis. In the case of human MAO B, two
tyrosyl residues (Y398 and Y435) are found in the substrate binding site on the
re face of the covalent flavin ring [Binda et al. (2002) J. Biol. Chem. 277,
23973-23976]. To probe the functional significance of this structure, Tyr435 in
MAO B was mutated with the amino acids Phe, His, Leu, or Trp, the mutant
proteins expressed in Pichia pastoris, and purified to homogeneity. Each mutant
protein contains covalent FAD and exhibits a high level of catalytic
functionality. No major alterations in active site structures are detected on
comparison of their respective crystal structures with that of WT enzyme. The
relative k(cat)/K(m) values for each mutant enzyme show Y435 > Y435F = Y435L
= Y435H > Y435W. A similar behavior is also observed with the membrane-bound
forms of MAO A and MAO B (MAO A Y444 mutant enzymes are found to be unstable on
membrane extraction). p-Nitrobenzylamine is found to be a poor substrate while
p-nitrophenethylamine is found to be a good substrate for all WT and mutant
forms of MAO B. Analysis of these kinetic and structural data suggests the
function of the "aromatic cage" in MAO to include a steric role in substrate
binding and access to the flavin coenzyme and to increase the nucleophilicity of
the substrate amine moiety. These results are consistent with a proposed polar
nucleophilic mechanism for catalytic amine oxidation.
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