Crystal Structures of Copper-depleted and Copper-bound Fungal Pro-tyrosinase

• Published in: The Journal of biological chemistry 2013-07, Vol.288 (30), p.22128-22140
• Main Authors: Fujieda, Nobutaka, Yabuta, Shintaro, Ikeda, Takuya, Oyama, Takuji, Muraki, Norifumi, Kurisu, Genji, Itoh, Shinobu
• Format: Article
• Language: English
• Subjects: Copper; Copper Chaperone; Copper Transport; Post-translational Modification; Protein Cross-linking; Protein-derived Cofactor; Tyrosinase; Zymogen
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M113.477612

Tyrosinase, a dinuclear copper monooxygenase/oxidase, plays a crucial role in the melanin pigment biosynthesis. The structure and functions of tyrosinase have so far been studied extensively, but the post-translational maturation process from the pro-form to the active form has been less explored. In this study, we provide the crystal structures of Aspergillus oryzae full-length pro-tyrosinase in the holo- and the apo-forms at 1.39 and 2.05 Å resolution, respectively, revealing that Phe513 on the C-terminal domain is accommodated in the substrate-binding site as a substrate analog to protect the dicopper active site from substrate access (proteolytic cleavage of the C-terminal domain or deformation of the C-terminal domain by acid treatment transforms the pro-tyrosinase to the active enzyme (Fujieda, N., Murata, M., Yabuta, S., Ikeda, T., Shimokawa, C., Nakamura, Y., Hata, Y., and Itoh, S. (2012) ChemBioChem. 13, 193–201 and Fujieda, N., Murata, M., Yabuta, S., Ikeda, T., Shimokawa, C., Nakamura, Y., Hata, Yl, and Itoh, S. (2013) J. Biol. Inorg. Chem. 18, 19–26). Detailed crystallographic analysis and structure-based mutational studies have shown that the copper incorporation into the active site is governed by three cysteines as follows: Cys92, which is covalently bound to His94 via an unusual thioether linkage in the holo-form, and Cys522 and Cys525 of the CXXC motif located on the C-terminal domain. Molecular mechanisms of the maturation processes of fungal tyrosinase involving the accommodation of the dinuclear copper unit, the post-translational His-Cys thioether cross-linkage formation, and the proteolytic C-terminal cleavage to produce the active tyrosinase have been discussed on the basis of the detailed structural information. Background: Fungal tyrosinase maturation involves multiple processes of the dinuclear copper assembly and proteolytic activation. Results: Structural examinations and mutational studies of the pro-tyrosinases revealed that three endogenous cysteines contribute to the copper incorporation. Conclusion: The three highly flexible cysteines are essential for assembly of the active site across the protein shell. Significance: Elucidation of such a copper incorporation process provides useful insights into metal homeostasis.