Crystal Structures of Copper-depleted and Copper-bound Fungal Pro-tyrosinase INSIGHTS INTO ENDOGENOUS CYSTEINE-DEPENDENT COPPER INCORPORATION
Crystal Structures of Copper-depleted and Copper-bound Fungal Pro-tyrosinase INSIGHTS INTO ENDOGENOUS CYSTEINE-DEPENDENT COPPER INCORPORATION
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DOI：
10.1074/jbc.m113.477612
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发表时间：
2013-07-26
影响因子：
4.8
通讯作者：
Itoh, Shinobu
中科院分区：
文献类型：
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作者：
Fujieda, Nobutaka;Yabuta, Shintaro;Itoh, Shinobu
酪氨酸酶是一种双核铜单加氧酶/氧化酶，在黑色素的生物合成中起重要作用。酪氨酸酶的结构和功能已被广泛研究，但从前体形式到活性形式的翻译后成熟过程却很少被探索。在这项研究中，我们分别以1.39和2.05的分辨率提供了曲霉菌全长酪氨酸酶原的全和脱辅基形式的晶体结构，揭示了C-末端结构域上的Phe（513）作为底物类似物被容纳在底物结合位点中，以保护双铜活性位点免受底物接近（C-末端结构域的蛋白水解切割）。末端结构域或通过酸处理使C-末端结构域变形将原酪氨酸酶转化为活性酶（Fujieda，N.，Murata，M.，Yabuta，S.，Ikeda，T.，Shimokawa，C.，中村，Y.，Hata，Y.，和Itoh，S.等人（2012）ChemBioChem. 13，193-201和Fujieda，N.，Murata，M.，Yabuta，S.，Ikeda，T.，Shimokawa，C.，中村，Y.，Hata，Yl和Itoh，S.（2013）J.Biol.Inorg.Chem.18，19-26）。详细的晶体学分析和基于结构的突变研究表明，铜掺入活性位点是由以下三个半胱氨酸控制的：Cys（92），其通过不寻常的全形式硫醚键与His（94）共价结合，Cys（522）和Cys（525）位于C-末端结构域的CXXC基序。根据详细的结构信息，讨论了真菌酪氨酸酶成熟过程的分子机制，包括双核铜单元的容纳、翻译后His-Cys硫醚交联的形成以及蛋白水解C-末端断裂产生活性酪氨酸酶。
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 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.