Structure and function of the engineered multicopper oxidase CueO from Escherichia coli -: Deletion of the methionine-rich helical region covering the substrate-binding site
Structure and function of the engineered multicopper oxidase CueO from Escherichia coli -: Deletion of the methionine-rich helical region covering the substrate-binding site
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DOI：
10.1016/j.jmb.2007.07.041
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发表时间：
2007-10-12
影响因子：
5.6
通讯作者：
Sakurai, Takeshi
中科院分区：
文献类型：
--
作者：
Kataoka, Kunishige;Komori, Hirofumi;Sakurai, Takeshi
CueO 是一种多铜氧化酶 (MCO)，参与大肠杆菌中铜的稳态，并且是迄今为止发现的唯一的亚铜氧化酶。与其他 MCO 不同，CueO 的底物结合位点深埋在富含蛋氨酸的螺旋区域下，包括干扰有机底物进入的 α 螺旋 5、6 和 7。我们删除了 Pro357-His406 区域，并将其替换为 Gly-Gly 连接子。存在和不存在过量 Cu(II) 的情况下截短突变体的晶体结构表明，与 CueO 相比，CueO 分子的支架和金属结合位点被保留。此外，由于四个Cu中心，蛋白质分子的高热稳定性及其光谱和磁性在截短后也得以保留。就功能而言，由于不稳定的 Cu 位点对 Cu(I) 离子的亲和力降低，突变体的亚铜氧化酶活性降低至重组 CueO 的约 10%，尽管漆酶底物如 2,2'-azino-bis-(3-乙基苯并噻唑啉-6-磺酸)、对苯二胺和 2,6-二甲氧基苯酚的活性由于这些有机物的获取变化而增加。底物到 I 型 Cu 位点。 CueO 目前的工程表明，富含甲硫氨酸的 α 螺旋起到了阻碍大体积有机底物进入的屏障的作用，这为 CueO 提供了作为亚铜氧化酶的特异性。 (C) 2007 Elsevier Ltd. 保留所有权利。
CueO is a multicopper oxidase (MCO) that is involved in the homeostasis of Cu in Escherichia coli and is the sole cuprous oxidase to have ever been found. Differing from other MCOs, the substrate-binding site of CueO is deeply buried under a methionine-rich helical region including alpha-helices 5, 6, and 7 that interfere with the access of organic substrates. We deleted the region Pro357-His406 and replaced it with a Gly-Gly linker. The crystal structures of a truncated mutant in the presence and in the absence of excess Cu(II) indicated that the scaffold of the CueO molecule and metal-binding sites were reserved in comparison with those of CueO. In addition, the high thermostability of the protein molecule and its spectroscopic and magnetic properties due to four Cu centers were also conserved after truncation. As for functions, the cuprous oxidase activity of the mutant was reduced to ca 10% that of recombinant CueO owing to the decrease in the affinity of the labile Cu site for Cu(I) ions, although activities for laccase substrates such as 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid), p-phenylenediamine, and 2,6-dimethoxyphenol increased due to changes in the access of these organic substrates to the type I Cu site. The present engineering of CueO indicates that the methionine-rich a-helices function as a barrier to the access of bulky organic substrates, which provides CueO with specificity as a cuprous oxidase. (C) 2007 Elsevier Ltd. All rights reserved.