Crystal structure of CotA laccase complexed with 2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonate) at a novel binding site.
Crystal structure of CotA laccase complexed with 2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonate) at a novel binding site.
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DOI：
10.1107/s2053230x1600426x
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发表时间：
2016-04
期刊：
影响因子：
--
通讯作者：
Wang G
中科院分区：
文献类型：
--
作者：
Liu Z;Xie T;Zhong Q;Wang G
科塔与2，2-连氮基双-（3-乙基苯并噻唑啉-6-磺酸盐）在空穴基序中形成复合物的晶体结构已得到解决，这种新的结合位点可能是科塔漆酶蛋白质工程的潜在结构靶点。枯草芽孢杆菌科塔漆酶是芽孢外被膜中丰富的组分，是一种典型的漆酶。本文研究了科塔与2，2-连氮双-（3-乙基苯并噻唑啉-6-磺酸盐）（ABTS）配合物的空穴结构。新的结合位点距离T1结合口袋约26 nm。 与已知的其他漆酶结构的比较表明，孔是科塔的一个特定功能。关键残基Arg 476和Ser 360直接与ABTS结合。定点突变研究表明，位于新结合位点底部的残基Arg 146、Arg 429和Arg 476是ABTS和双胍基哒嗪氧化所必需的。特别地，与野生型相比，Thr 480 Phe变体被鉴定为对ABTS的特异性几乎是对阿糖腺苷的3.5倍。这些结果表明，ABTS的新的结合位点可能是一个潜在的目标，为科塔漆酶的蛋白工程。
The crystal structure of CotA complexed with 2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonate) in a hole motif has been solved; this novel binding site could be a potential structure-based target for protein engineering of CotA laccase. The CotA laccase from Bacillus subtilis is an abundant component of the spore outer coat and has been characterized as a typical laccase. The crystal structure of CotA complexed with 2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS) in a hole motif has been solved. The novel binding site was about 26 Å away from the T1 binding pocket. Comparison with known structures of other laccases revealed that the hole is a specific feature of CotA. The key residues Arg476 and Ser360 were directly bound to ABTS. Site-directed mutagenesis studies revealed that the residues Arg146, Arg429 and Arg476, which are located at the bottom of the novel binding site, are essential for the oxidation of ABTS and syringaldazine. Specially, a Thr480Phe variant was identified to be almost 3.5 times more specific for ABTS than for syringaldazine compared with the wild type. These results suggest this novel binding site for ABTS could be a potential target for protein engineering of CotA laccases.