Directed Evolution Mimics Allosteric Activation by Stepwise Tuning of the Conformational Ensemble.
Directed Evolution Mimics Allosteric Activation by Stepwise Tuning of the Conformational Ensemble.
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DOI：
10.1021/jacs.8b03490
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发表时间：
2018-06-13
影响因子：
15
通讯作者：
Arnold FH
中科院分区：
文献类型：
--
作者：
Buller AR;van Roye P;Cahn JKB;Scheele RA;Herger M;Arnold FH
变构酶含有丰富的催化多样性，但在生物催化中仍明显未得到充分利用。色氨酸合成酶是一种模式变构系统，是合成非经典氨基酸（ncAA）的重要酶。以前，我们从激烈火球菌PfTrpB中进化出β-亚基，用于在不存在其天然伴侣蛋白PfTrpA的情况下的ncAA合酶活性。然而，突变激活TrpB以提供独立催化剂的精确机制仍然是个谜。在这里，我们表明，定向进化引起的催化循环的限速步骤的逐渐变化。同时，中间体的稳态分布转移到有利于共价结合的色氨酸加合物，这是与这些物种的热力学稳定性增加。这些进化的，独立的TrpB的生化特性收敛于那些在天然系统中通过变构激活诱导的。野生型酶的高分辨率晶体结构，谱系中的中间体，以及最终的变体，包括五种不同的化学状态，表明激活突变对其直接环境只有轻微的结构影响。相反，突变稳定了子结构域的大规模运动，以有利于否则瞬时填充的封闭构象状态。这种稳定性的增加使得能够在催化活性TrpB中共价结合的Trp的第一个结构描述，证实了催化的关键特征。这些数据联合收割机表明，复杂的变构模型并不是通过定向进化重现其复杂效应的先决条件，这为工程化不同变构酶的独立版本开辟了道路。
Allosteric enzymes contain a wealth of catalytic diversity that remains distinctly underutilized for biocatalysis. Tryptophan synthase is a model allosteric system and a valuable enzyme for the synthesis of non-canonical amino acids (ncAA). Previously, we evolved the β-subunit from Pyrococcus furiosus, PfTrpB, for ncAA synthase activity in the absence of its native partner protein PfTrpA. However, the precise mechanism by which mutation activated TrpB to afford a stand-alone catalyst remained enigmatic. Here, we show that directed evolution caused a gradual change in the rate-limiting step of the catalytic cycle. Concomitantly, the steady-state distribution of intermediates shifts to favor covalently bound Trp adducts, which is associated with increased thermodynamic stability of these species. The biochemical properties of these evolved, stand-alone TrpBs converge on those induced in the native system by allosteric activation. High resolution crystal structures of the wild-type enzyme, an intermediate in the lineage, and the final variant, encompassing five distinct chemical states, show that activating mutations have only minor structural effects on their immediate environment. Instead, mutation stabilizes the large-scale motion of a sub-domain to favor an otherwise transiently populated closed conformational state. This increase in stability enabled the first structural description of Trp covalently bound in a catalytically active TrpB, confirming key features of catalysis. These data combine to show that sophisticated models of allostery are not a prerequisite to recapitulating its complex effects via directed evolution, opening the way to engineering stand-alone versions of diverse allosteric enzymes.
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影响因子：
21.8
作者：
Dodani SC;Kiss G;Cahn JK;Su Y;Pande VS;Arnold FH
通讯作者：
Arnold FH
影响因子：
--
作者：
Gao X;Jiang W;Jiménez-Osés G;Choi MS;Houk KN;Tang Y;Walsh CT
通讯作者：
Walsh CT
影响因子：
2.9
作者：
DREWE, WF;DUNN, MF
通讯作者：
DUNN, MF
影响因子：
15
作者：
Caulkins, Bethany G.;Bastin, Baback;Yang, Chen;Neubauer, Thomas J.;Young, Robert P.;Hilario, Eduardo;Huang, Yu-ming M.;Chang, Chia-en A.;Fan, Li;Dunn, Michael F.;Marsella, Michael J.;Mueller, Leonard J.
通讯作者：
Mueller, Leonard J.
影响因子：
2.9
作者：
Fan, YX;McPhie, P;Miles, EW
通讯作者：
Miles, EW