Installing hydrolytic activity into a completely de novo protein framework
Installing hydrolytic activity into a completely de novo protein framework
复制标题
DOI：
10.1038/nchem.2555
复制
发表时间：
2016-09-01
期刊：
影响因子：
21.8
通讯作者：
Woolfson, Derek N.
中科院分区：
文献类型：
--
作者：
Burton, Antony J.;Thomson, Andrew R.;Woolfson, Derek N.
类酶催化剂的设计测试了我们对蛋白质中序列与结构/功能关系的理解。在这里，我们将水解活性可预见地安装到一个完全从头和热稳定的α-螺旋桶中，该桶包括围绕可进入通道排列的七个螺旋。我们发现，内腔的桶接受21个突变的功能极性残基。由此产生的变体在每个螺旋上具有半胱氨酸-组氨酸-谷氨酸三联体，其水解对硝基苯基乙酸酯的催化效率与基于天然蛋白质支架的最有效的重新设计的水解酶相匹配。这是第一次报告的功能催化三联体工程改造成从头蛋白质框架。我们的系统的灵活性还允许容易地掺入非天然侧链，以提高活性和探测催化机制。这种可预测的和稳健的真正从头生物催化剂的构建为化学和生物化学合成中的应用提供了希望。
The design of enzyme-like catalysts tests our understanding of sequence-to-structure/function relationships in proteins. Here we install hydrolytic activity predictably into a completely de novo and thermostable a-helical barrel, which comprises seven helices arranged around an accessible channel. We show that the lumen of the barrel accepts 21 mutations to functional polar residues. The resulting variant, which has cysteine-histidine-glutamic acid triads on each helix, hydrolyses p-nitrophenyl acetate with catalytic efficiencies that match the most-efficient redesigned hydrolases based on natural protein scaffolds. This is the first report of a functional catalytic triad engineered into a de novo protein framework. The flexibility of our system also allows the facile incorporation of unnatural side chains to improve activity and probe the catalytic mechanism. Such a predictable and robust construction of truly de novo biocatalysts holds promise for applications in chemical and biochemical synthesis.