Ensemble-based enzyme design can recapitulate the effects of laboratory directed evolution in silico.
Ensemble-based enzyme design can recapitulate the effects of laboratory directed evolution in silico.
复制标题
DOI：
10.1038/s41467-020-18619-x
复制
发表时间：
2020-09-23
影响因子：
16.6
通讯作者：
Chica RA
中科院分区：
文献类型：
--
作者：
Broom A;Rakotoharisoa RV;Thompson MC;Zarifi N;Nguyen E;Mukhametzhanov N;Liu L;Fraser JS;Chica RA
人工酶的创造是计算蛋白质设计的一个关键目标。尽管从头酶已被成功设计，但它们的催化效率较低，需要定向进化来提高活性。在这里，我们使用室温 X 射线晶体学来研究设计的 Kemp 消除酶 HG3 (kcat/KM 146 M−1s−1) 进化过程中构象整体的变化。我们观察到催化残基越来越刚性，活性位点变得更好预组织，并且其入口变宽。基于这些观察，我们设计了 HG4，一种高效的生物催化剂（kcat/KM 103,000 M−1s−1），包含进化过程中发现的关键第一和第二壳突变。 HG4 结构表明其活性位点是预先组织和刚性化的，可实现高效催化。我们的结果表明，定向进化如何通过改变构象系综以有利于催化生产亚状态来规避酶设计固有的挑战，并建议改进结合晶体学数据系综建模的设计方法。 Kemp 消除酶是催化苯并异恶唑协同去质子化和开环的人工酶。在这里，作者使用室温 X 射线晶体学来研究 Kemp 消除酶 HG3 构象整体沿着定向进化轨迹的变化，并开发了一种实验引导的、基于整体的计算酶设计程序。
The creation of artificial enzymes is a key objective of computational protein design. Although de novo enzymes have been successfully designed, these exhibit low catalytic efficiencies, requiring directed evolution to improve activity. Here, we use room-temperature X-ray crystallography to study changes in the conformational ensemble during evolution of the designed Kemp eliminase HG3 (kcat/KM 146 M−1s−1). We observe that catalytic residues are increasingly rigidified, the active site becomes better pre-organized, and its entrance is widened. Based on these observations, we engineer HG4, an efficient biocatalyst (kcat/KM 103,000 M−1s−1) containing key first and second-shell mutations found during evolution. HG4 structures reveal that its active site is pre-organized and rigidified for efficient catalysis. Our results show how directed evolution circumvents challenges inherent to enzyme design by shifting conformational ensembles to favor catalytically-productive sub-states, and suggest improvements to the design methodology that incorporate ensemble modeling of crystallographic data. Kemp eliminases are artificial enzymes that catalyze the concerted deprotonation and ring-opening of benzisoxazoles. Here, the authors use room-temperature X-ray crystallography to investigate changes to the conformational ensemble of the Kemp eliminase HG3 along a directed evolutionary trajectory, and develop an experimentally guided, ensemble-based computational enzyme design procedure.
登录
查看更多内容
DOI：
10.1107/s0907444905036693
发表时间：
2006-01-01
影响因子：
2.2
作者：
Evans, P
通讯作者：
Evans, P
DOI：
10.1073/pnas.1013910107
发表时间：
2010-11-23
影响因子：
11.1
作者：
Chica, Roberto A.;Moore, Matthew M.;Mayo, Stephen L.
通讯作者：
Mayo, Stephen L.
影响因子：
8
作者：
Althoff, Eric A.;Wang, Ling;Jiang, Lin;Giger, Lars;Lassila, Jonathan K.;Wang, Zhizhi;Smith, Matthew;Hari, Sanjay;Kast, Peter;Herschlag, Daniel;Hilvert, Donald;Baker, David
通讯作者：
Baker, David
DOI：
10.1107/s0907444909052925
发表时间：
2010-02
期刊：
Acta crystallographica. Section D, Biological crystallography
影响因子：
--
作者：
Adams PD;Afonine PV;Bunkóczi G;Chen VB;Davis IW;Echols N;Headd JJ;Hung LW;Kapral GJ;Grosse-Kunstleve RW;McCoy AJ;Moriarty NW;Oeffner R;Read RJ;Richardson DC;Richardson JS;Terwilliger TC;Zwart PH
通讯作者：
Zwart PH
影响因子：
14.8
作者：
Giger L;Caner S;Obexer R;Kast P;Baker D;Ban N;Hilvert D
通讯作者：
Hilvert D