A Panel of TrpB Biocatalysts Derived from Tryptophan Synthase through the Transfer of Mutations that Mimic Allosteric Activation.
A Panel of TrpB Biocatalysts Derived from Tryptophan Synthase through the Transfer of Mutations that Mimic Allosteric Activation.
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DOI：
10.1002/anie.201606242
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发表时间：
2016-09-12
影响因子：
16.6
通讯作者：
Arnold, Frances H.
中科院分区：
文献类型：
--
作者：
Murciano-Calles, Javier;Romney, David K.;Brinkmann-Chen, Sabine;Buller, Andrew R.;Arnold, Frances H.
天然存在的酶同源物通常与非天然底物表现出高度不同的活性。然而，利用为新的或改变功能而设计的酶来开发这种多样性是很困难的，因为必须为每个同源物复制工程。我们证明了来自火球菌的色氨酸合成酶β-亚基（TrpB）的一小组突变，模仿α-亚基结合所提供的激活，在TrpB同源物中具有类似的激活作用，序列同源性只有57%。动力学和光谱分析表明，突变通过相同的机制起作用，模仿α-亚基结合。从这些单独的酶中，我们发现了一种新的催化剂，它在合成5-取代色氨酸（一类重要的生物化合物）方面显示出非常广泛的活性。这项研究证明了变构活化如何在整个蛋白质家族中重演，以有效地探索自然序列多样性，以实现理想的生物催化转化。色氨酸合成酶复合物对一些吲哚类似物有活性。β-亚基（TrpB）完成了合成有用的反应，但需要α-亚基充分活跃。我们已经将来自焦球菌的重新激活的TrpB变体的突变转移到同源的TrpB中，以生成一组独立的TrpB催化剂，其中一组对于制造5-取代色氨酸（一种重要的生物基序）特别有用。
Naturally occurring enzyme homologs often display highly divergent activity with non-natural substrates. Exploiting this diversity with enzymes engineered for new or altered function, however, is laborious because the engineering must be replicated for each homolog. We demonstrate that a small set of mutations of the tryptophan synthase β-subunit (TrpB) from Pyrococcus furiosus, which mimic the activation afforded by binding of the α-subunit, has a similar activating effect in TrpB homologs with as little as 57% sequence identity. Kinetic and spectroscopic analyses indicate that the mutations function through the same mechanism, mimicry of α-subunit binding. From this collection of stand-alone enzymes, we identified a new catalyst that displays a remarkably broad activity profile in the synthesis of 5-substituted tryptophans, a biologically important class of compounds. This investigation demonstrates how allosteric activation can be recapitulated throughout a protein family to efficiently explore natural sequence diversity for desirable biocatalytic transformations. The tryptophan synthase enzyme complex is active toward a number of indole analogs. The β-subunit (TrpB) performs the synthetically useful reaction, but requires the α-subunit to be fully active. We have transferred mutations from a re-activated TrpB variant from Pyrococcus furiosus into homologous TrpBs to generate a panel of stand-alone TrpB catalysts, one of which is especially useful for making 5-substituted tryptophans, an important biological motif.
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影响因子：
3.2
作者：
Heel, Thomas;McIntosh, John A.;Dodani, Sheel C.;Meyerowitz, Joseph T.;Arnold, Frances H.
通讯作者：
Arnold, Frances H.
影响因子：
2.1
作者：
Corr, Michael J.;Smith, Duncan R. M.;Goss, Rebecca J. M.
通讯作者：
Goss, Rebecca J. M.
影响因子：
4.7
作者：
Han, Han;Davis, Mark E.
通讯作者：
Davis, Mark E.
影响因子：
15
作者：
Roy, Abhijeet Deb;Grueschow, Sabine;Goss, Rebecca J. M.
通讯作者：
Goss, Rebecca J. M.
影响因子：
4.9
作者：
Goss, Rebecca J. M.;Newill, Philip L. A.
通讯作者：
Newill, Philip L. A.