Crystal structure of a bacterial endospore coat component - A laccase with enhanced thermostability properties
Crystal structure of a bacterial endospore coat component - A laccase with enhanced thermostability properties
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DOI：
10.1074/jbc.m301251200
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发表时间：
2003-05-23
影响因子：
4.8
通讯作者：
Carrondo, MA
中科院分区：
文献类型：
--
作者：
Enguita, FJ;Martins, LO;Carrondo, MA
由革兰氏阳性土壤细菌枯草芽孢杆菌产生的内生孢子被由超过30种结构组分形成的蛋白质外壳所屏蔽，所述结构组分自组装成层状内涂层和较厚的条纹状电致密外涂层。65-kDa科塔蛋白是外被膜层的丰富组分。科塔是一种高度热稳定的漆酶，其组装到涂层中是孢子抵抗过氧化氢和紫外线所必需的。在这里，我们报告的结构科塔在1.7埃的分辨率，由X射线晶体学确定。这是内生孢子外壳组分的第一个结构，也是细菌漆酶的第一个结构。科塔的整体折叠包括三个铜氧还蛋白样结构域，包括一个单核和一个三核铜中心。这种排列与其他多铜氧化酶的排列相似，并且与大肠杆菌的铜耐受蛋白CueO的排列最相似。然而，科塔中的三个铜氧还蛋白结构域通过外部结构域间环进一步连接，这增加了结构的堆积水平。我们建议，这些域间循环有助于显着的酶的热稳定性，但我们的研究结果表明，其他因素可能发挥作用。与其他含有四个铜原子的单体多铜氧化酶的结构比较表明，科塔可能接受任何已知的漆酶的最大底物。此外，与其他漆酶不同，科塔似乎有一个灵活的盖状区域接近底物结合位点，可能介导底物的可及性。讨论了这些发现对科塔特性、其组装和细菌孢子外壳结构特性的影响。
Endospores produced by the Gram-positive soil bacterium Bacillus subtilis are shielded by a proteinaceous coat formed by over 30 structural components, which self-assemble into a lamellar inner coat and a thicker striated electrodense outer coat. The 65-kDa CotA protein is an abundant component of the outer coat layer. CotA is a highly thermostable laccase, assembly of which into the coat is required for spore resistance against hydrogen peroxide and UV light. Here, we report the structure of CotA at 1.7-Angstrom resolution, as determined by x-ray crystallography. This is the first structure of an endospore coat component, and also the first structure of a bacterial laccase. The overall fold of CotA comprises three cupredoxin-like domains and includes one mononuclear and one trinuclear copper center. This arrangement is similar to that of other multicopper oxidases and most similar to that of the copper tolerance protein CueO of Escherichia coli. However, the three cupredoxin domains in CotA are further linked by external interdomain loops, which increase the packing level of the structure. We propose that these interdomain loops contribute to the remarkable thermostability of the enzyme, but our results suggest that additional factors are likely to play a role. Comparisons with the structure of other monomeric multicopper oxidases containing four copper atoms suggest that CotA may accept the largest substrates of any known laccase. Moreover, and unlike other laccases, CotA appears to have a flexible lidlike region close to the substrate-binding site that may mediate substrate accessibility. The implications of these findings for the properties of CotA, its assembly and the properties of the bacterial spore coat structure are discussed.