title: Crystal structures of copper-depleted and copper-bound fungal pro-tyrosinase: insights into endogenous cysteine-dependent copper incorporation.
abstract: 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 Phe(513) 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: Cys(92), which is covalently bound to His(94) via an unusual thioether linkage in the holo-form, and Cys(522) and Cys(525) 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.
authors.affiliation: Department of Material and Life Science, Division of Advanced Science and Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamada-oka, Suita, Osaka 565-0871, Japan.
authors.forename: Nobutaka
authors.initials: N
authors.lastname: Fujieda
authors.forename: Shintaro
authors.initials: S
authors.lastname: Yabuta
authors.forename: Takuya
authors.initials: T
authors.lastname: Ikeda
authors.forename: Takuji
authors.initials: T
authors.lastname: Oyama
authors.forename: Norifumi
authors.initials: N
authors.lastname: Muraki
authors.forename: Genji
authors.initials: G
authors.lastname: Kurisu
authors.forename: Shinobu
authors.initials: S
authors.lastname: Itoh
mesh_terms: D000595:Amino Acid Sequence; D001051:Apoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001236:Aspergillus oryzae / Q000201:enzymology / Q000235:genetics; D020134:Catalytic Domain; D003300:Copper / Q000737:chemistry* / Q000378:metabolism; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004792:Enzyme Precursors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020035:Holoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D014442:Monophenol Monooxygenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid
chemical_list: D001051:Apoenzymes; D004792:Enzyme Precursors; D005656:Fungal Proteins; D020035:Holoenzymes; D003300:Copper; D014442:Monophenol Monooxygenase; D003545:Cysteine
keywords: Copper; Copper Chaperone; Copper Transport; Post-translational Modification; Protein Cross-linking; Protein-derived Cofactor; Tyrosinase; Zymogen