Copper amine oxidase from Hansenula polymorpha: the crystal structure determined at 2.4 A resolution reveals the active conformation.

BACKGROUND: Copper-containing amine oxidases (CAOs) are widespread in nature. These enzymes oxidize primary amine substrates to the aldehyde product, reducing molecular oxygen to hydrogen peroxide in the process. CAOs contain one type 2 copper atom and topaquinone (TPQ), a modified tyrosine sidechain utilized as a redox cofactor. The methylamine oxidase from the yeast Hansenula polymorpha (HPAO) is an isoform of CAO with a preference for small aliphatic amine or phenethylamine substrates. The enzyme is dimeric with a subunit molecular weight of 78 kDa. Structural studies are directed at understanding the basis for cofactor biogenesis and catalytic efficiency.
RESULTS: The X-ray crystal structure of HPAO has been solved at 2.4 A resolution by a combination of molecular replacement and single isomorphous replacement followed by refinement using sixfold symmetry averaging. The electron density at the catalytic site shows that the TPQ conformation corresponds to that of the active form of the enzyme. Two channels, one on either side of TPQ, are observed in the structure that provide access between the active site and the bulk solvent.
CONCLUSIONS: The structure shows TPQ in a position poised for catalysis. This is the first active CAO structure to reveal this conformation and may help further our understanding of the catalytic mechanism. On the substrate side of TPQ a water-containing channel leading to the protein surface can serve as an entrance or exit for substrate and product. On the opposite side of TPQ there is direct access from the bulk solvent of the dimer interface by which molecular oxygen may enter and hydrogen peroxide depart. In addition, a network of conserved water molecules has been identified which may function in the catalytic mechanism.