Toward a Halophenol Dehalogenase from Iodotyrosine Deiodinase via Computational Design
Toward a Halophenol Dehalogenase from Iodotyrosine Deiodinase via Computational Design
复制标题
通过计算设计从碘酪氨酸脱碘酶开发卤代酚脱卤酶
DOI：
10.1021/acscatal.8b03587
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发表时间：
2018
期刊：
影响因子：
12.9
通讯作者：
Rokita, Steven E.
中科院分区：
文献类型：
--
作者：
Sun, Zuodong;Rokita, Steven E.
还原脱卤提供了一种有吸引力的方法，从环境中去除卤化污染物，碘酪氨酸脱碘酶（IYD）可能有助于这一过程后，它可以接受广泛的底物。IYD的选择性部分地由一个由1026个氨基酸组成的活性位点环控制。在不存在底物的情况下，环是无序的，并且仅在卤代酪氨酸缔合时折叠成紧凑的螺旋-转角-螺旋。应用Rosetta的设计算法重新设计该回路，以响应2-碘苯酚而不是碘酪氨酸。一种使用有限数量的取代来增加螺旋区域的固有稳定性的策略未能产生具有所需性质的变体。一系列的点突变确定了强大的上位相互作用，阻碍了适应IYD。第二种策略克服了这一限制，该策略对Rosetta的侧链取代没有任何限制。九个代表性的设计，包含14和18之间的取代超过26个连续的网站进行了实验评估。表现最好的催化剂（UD 08）支持2-碘苯酚的周转增加4.5倍，并且相对于天然酶抑制碘酪氨酸的周转2000倍。在不存在底物的情况下，UD 08的活性位点环似乎比天然序列更不无序，这从它们对蛋白水解的相对敏感性中可以看出。在2-碘苯酚的存在下，UD 08的蛋白水解保护增加了9倍，几乎与野生型IYD对碘酪氨酸的等效反应相媲美。因此，Rosetta设计实现了创建在碘苯酚存在下获得结构的活性位点序列的目标。虽然有限数量的点突变足以增加2-碘苯酚脱卤的催化效率，但只有Rosetta成功地创建了响应于该底物的环结构。
Reductive dehalogenation offers an attractive approach for removing halogenated pollutants from the environment, and iodotyrosine deiodinase (IYD) may contribute to this process after it can be engineered to accept a broad range of substrates. The selectivity of IYD is controlled in part by an active site loop of ∼26 amino acids. In the absence of a substrate, the loop is disordered and only folds into a compact helix-turn-helix upon halotyrosine association. The design algorithm of Rosetta was applied to redesign this loop for response to 2-iodophenol rather than iodotyrosine. One strategy using a restricted number of substitutions for increasing the inherent stability of the helical regions failed to generate variants with the desired properties. A series of point mutations identified strong epistatic interactions that impeded adaptation of IYD. This limitation was overcome by a second strategy that placed no restrictions on side-chain substitution by Rosetta. Nine representative designs containing between 14 and 18 substitutions over 26 contiguous sites were evaluated experimentally. The top performing catalyst (UD08) supported a 4.5-fold increase in turnover of 2-iodophenol and suppressed turnover of iodotyrosine by 2000-fold relative to the native enzyme. The active site loop of UD08 appeared less disordered than the native sequence in the absence of substrate, as evident from their relative sensitivity to proteolysis. Protection from proteolysis increased 9-fold for UD08 in the presence of 2-iodophenol and nearly rivaled the equivalent response of wild-type IYD to iodotyrosine. Thus, the Rosetta designs achieved the goal of creating an active site sequence that gained structure in the presence of iodophenol. Although a limited number of point mutations was sufficient to increase the catalytic efficiency for 2-iodophenol dehalogenation, only Rosetta successfully created a loop structure responsive to this substrate.
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DOI：
10.1073/pnas.1706849114
发表时间：
2017-11-07
影响因子：
11.1
作者：
Akiva E;Copp JN;Tokuriki N;Babbitt PC
通讯作者：
Babbitt PC
基于毒性底物的卤代烷脱卤酶活性选择测定
影响因子：
5
作者：
Michael P. C. Fibinger;Timo Davids;D. Böttcher;U. Bornscheuer
通讯作者：
U. Bornscheuer
哺乳动物还原性去二世酶具有脱盐酸盐氯化和溴化底物具有广泛的能力。
影响因子：
15
作者：
McTamney, Patrick M.;Rokita, Steven E.
通讯作者：
Rokita, Steven E.
酶促卤化和脱征反应：普遍和机械上的多样化。
影响因子：
62.1
作者：
Agarwal V;Miles ZD;Winter JM;Eustáquio AS;El Gamal AA;Moore BS
通讯作者：
Moore BS
影响因子：
3.9
作者：
Sun Z;Su Q;Rokita SE
通讯作者：
Rokita SE