Abstract
At high resolution, we determined the crystal structures of copper-bound and metal-free tyrosinase in a complex with ORF378 designated as a "caddie" protein because it assists with transportation of two CuII ions into the tyrosinase catalytic center. These structures suggest that the caddie protein covers the hydrophobic molecular surface of tyrosinase and interferes with the binding of a substrate tyrosine to the catalytic site of tyrosinase. The caddie protein, which consists of one six-strandedbeta-sheet and one alpha-helix, has no similarity with all proteins deposited into the Protein Data Bank. Although tyrosinase and catechol oxidase are classified into the type 3 copper protein family, the latter enzyme lacks monooxygenase activity. The difference in catalytic activity is based on the structural observations that a large vacant space is present just above the active center of tyrosinase and that one of the six His ligands for the two copper ions is highly flexible. These structural characteristics of tyrosinase suggest that, in the reaction that catalyzes the ortho-hydroxylation of monophenol, one of the two Cu(II) ions is coordinated by the peroxide-originated oxygen bound to the substrate. Our crystallographic study shows evidence that the tyrosinase active center formed by dinuclear coppers is flexible during catalysis.
Highlights
When the S. castaneoglobisporus tyrosinase is overexpressed using an Escherichia coli host-vector system [20], the coexpression of ORF378 is indispensable to obtain tyrosinase that has catalytic activity; the transport of Cu(II) ions to the catalytic center of tyrosinase, which is mediated by ORF378, may be necessary to convert tyrosinase to its active form
Crystallography of the Met Form of Cu(II)-bound Tyrosinase Complexed with ORF378—To investigate the copper-binding site in the model and the structural change caused by Cu(II) incorporation, we soaked the crystal in the reservoir solution containing 1 mM CuSO4 for the given times
Overview of the Structure—We determined the first crystal structure of tyrosinase, which is complexed with the caddie protein ORF378, at Native 1 Mercury
Summary
Crystallography of the Met Form of Cu(II)-bound Tyrosinase Complexed with ORF378—To investigate the copper-binding site in the model and the structural change caused by Cu(II) incorporation, we soaked the crystal in the reservoir solution containing 1 mM CuSO4 for the given times. Its phenolic hydroxyl forms a hydrogen bond with solvent atoms (water molecule or peroxide ion), which form a bridge with CuA and CuB in the active center of tyrosinase (Fig. 7, B–E).
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