Crystal Structure of Agaricus bisporus Mushroom Tyrosinase: Identity of the Tetramer Subunits and Interaction with Tropolone
Crystal Structure of Agaricus bisporus Mushroom Tyrosinase: Identity of the Tetramer Subunits and Interaction with Tropolone
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DOI：
10.1021/bi200395t
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发表时间：
2011-06-21
期刊：
影响因子：
2.9
通讯作者：
Dijkstra, Bauke W.
中科院分区：
文献类型：
--
作者：
Ismaya, Wangsa T.;Rozeboom, Henriette J.;Dijkstra, Bauke W.
酪氨酸酶催化酚类化合物转化为它们的醌衍生物，醌衍生物是形成黑色素的前体，黑色素是生物体中普遍存在的色素。由于酪氨酸酶在食品工业中对布朗宁反应的重要性，人们对蘑菇双孢蘑菇中的酪氨酸酶进行了深入的研究。在以前的研究中，酪氨酸酶复合物被证明是一个H2L2四聚体，但没有得到任何线索的身份的亚基，其模式的协会，和三维结构的复杂。在这里，我们解开这个四聚体在分子水平上。其2.3埃分辨率的晶体结构是完整真菌酪氨酸酶复合物的第一个结构。该复合物由两个类似于392个残基的H亚基和两个类似于150个残基的L亚基组成。H亚基来源于ppo3基因，具有与其他酪氨酸酶相似的折叠，但它类似于大100个残基。L亚基似乎是orf239342的产物，并具有凝集素样折叠。H亚基在脱氧状态下含有双核铜结合位点，其中三个组氨酸残基协调每个铜离子。这些组氨酸的侧链通过氢键或在His85的情况下通过与Cys83的侧链的硫醚桥固定其取向。特异性酪氨酸酶抑制剂托酚酮与酶形成前米氏复合物。它在双核铜位点附近结合，而不直接与铜离子配位。ORF239342亚基的功能尚不清楚。在其他凝集素中鉴定的碳水化合物结合位点在ORF 239342中不保守，并且亚基距离活性位点超过25埃，使得不太可能在活性中起作用。这些结构解释了钙离子如何稳定酶的四聚体状态。
Tyrosinase catalyzes the conversion of phenolic compounds into their quinone derivatives, which are precursors for the formation of melanin, a ubiquitous pigment in living organisms. Because of its importance for browning reactions in the food industry, the tyrosinase from the mushroom Agaricus bisporus has been investigated in depth. In previous studies the tyrosinase enzyme complex was shown to be a H2L2 tetramer, but no clues were obtained of the identities of the subunits, their mode of association, and the 3D structure of he complex. Here we unravel this tetramer at the molecular level. Its 2.3 angstrom resolution crystal structure is the first structure of the full fungal tyrosinase complex. The complex comprises two H subunits of similar to 392 residues and two L subunits of similar to 150 residues. The H subunit originates from the ppo3 gene and has a fold similar to other tyrosinases, but it is similar to 100 residues larger. The L subunit appeared to be the product of orf239342 and has a lectin-like fold. The H subunit contains a binuclear copper-binding site in the deoxy-state, in which three histidine residues coordinate each copper ion. The side chains of these histidines have their orientation fixed by hydrogen bonds or, in the case of His85, by a thioether bridge with the side chain of Cys83. The specific tyrosinase inhibitor tropolone forms a pre-Michaelis complex with the enzyme. It binds near the binuclear copper site without directly coordinating the copper ions. The function of the ORF239342 subunits is not known. Carbohydrate binding sites identified in other lectins are not conserved in ORF239342, and the subunits are over 25 angstrom away from the active site, making a role in activity unlikely. The structures explain how calcium ions stabilize the tetrameric state of the enzyme.