CRYSTAL-STRUCTURE OF A EUKARYOTIC (PEA SEEDLING) COPPER-CONTAINING AMINE OXIDASE AT 2.2-ANGSTROM RESOLUTION

Background: Copper-containing amine oxidases catalyze the oxidative de amination of primary amines to aldehydes, in a reaction that requires free radicals. These enzymes are important in many biological processe s, including cell differentiation and growth, wound healing, detoxific ation and signalling. The catalytic reaction requires a redox cofactor , topa quinone (TPQ), which is derived by post-translational modificat ion of an invariant tyrosine residue. Both the biogenesis of the TPQ c ofactor and the reaction catalyzed by the enzyme require the presence of a copper atom at the active site. The crystal structure of a prokar yotic copper amine oxidase from E. coli (ECAO) has recently been repor ted. Results: The first structure of a eukaryotic (pea seedling) amine oxidase (PSAO) has been solved and refined at 2.2 Angstrom resolution . The crystallographic phases were derived from a single phosphotungst ic acid derivative. The positions of the tungsten atoms in the W-12 cl usters were obtained by molecular replacement using E. coli amine oxid ase as a search model. The methodology avoided bias from the search mo del, and provides an essentially independent view of a eukaryotic amin e oxidase. The PSAO molecule is a homodimer; each subunit has three do mains, The active site of each subunil: lies near an edge of the beta- sandwich of the largest domain, but is not accessible from the solvent . The essential active-site copper atom is coordinated by three histid ine side chains and two water molecules in an approximately square-pyr amidal arrangement, All the atoms of the TPQ cofactor are unambiguousl y defined, the shortest distance to the copper atom being similar to 6 Angstrom. Conclusions: There is considerable structural homology betw een PSAO and ECAO, A combination of evidence from both structures indi cates that the TPQ side chain is sufficiently flexible to permit the a romatic group to rotate about the C beta-C gamma bond, and to move bet ween bonding and non-bonding positions with respect to the Cu atom. Co nformational flexibility is also required at the surface of the molecu le to allow the substrates access to the active site, which is inacces sible to solvent, as expected for an enzyme that uses radical chemistr y.