Engineering of Ancestors as a Tool to Elucidate Structure, Mechanism, and Specificity of Extant Terpene Cyclase.
Engineering of Ancestors as a Tool to Elucidate Structure, Mechanism, and Specificity of Extant Terpene Cyclase.
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DOI：
10.1021/jacs.0c10214
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发表时间：
2021-03-17
影响因子：
15
通讯作者：
Syrén PO
中科院分区：
文献类型：
--
作者：
Schriever K;Saenz-Mendez P;Rudraraju RS;Hendrikse NM;Hudson EP;Biundo A;Schnell R;Syrén PO
结构信息对于理解催化机制和指导生物催化剂（如萜烯环化酶）的酶工程工作至关重要。然而，低序列相似性会阻碍同源性建模，并且固有的蛋白质不稳定性对结构研究提出了挑战。我们假设，工程热稳定的祖先酶的X射线晶体学可以访问可靠的同源模型现存的生物催化剂。我们已经应用这个概念与分子建模和酶的测定，以了解螺紫烯合酶，一类萜环化酶的结构活性关系，旨在工程师的特异性。工程表面补丁重建的祖先提供了一个模板结构的生成一个高置信度的同源模型的现存酶。在结构考虑的基础上，我们设计和结晶的祖先变体与单一的残基交换，表现出定制的底物特异性和保存的热稳定性。我们展示了如何在祖先的支架中确定的两个单一的氨基酸改变可以转移到现存的酶，赋予一个特异性开关，影响现存的酶的特异性形成的二萜螺紫烯超过形成的倍半萜hedycaryol和法尼醇高达25倍。这项研究强调了祖先序列重建与酶工程相结合作为化学生物学中的通用工具的价值。
Structural information is crucial for understanding catalytic mechanisms and to guide enzyme engineering efforts of biocatalysts, such as terpene cyclases. However, low sequence similarity can impede homology modeling, and inherent protein instability presents challenges for structural studies. We hypothesized that X-ray crystallography of engineered thermostable ancestral enzymes can enable access to reliable homology models of extant biocatalysts. We have applied this concept in concert with molecular modeling and enzymatic assays to understand the structure activity relationship of spiroviolene synthase, a class I terpene cyclase, aiming to engineer its specificity. Engineering a surface patch in the reconstructed ancestor afforded a template structure for generation of a high-confidence homology model of the extant enzyme. On the basis of structural considerations, we designed and crystallized ancestral variants with single residue exchanges that exhibited tailored substrate specificity and preserved thermostability. We show how the two single amino acid alterations identified in the ancestral scaffold can be transferred to the extant enzyme, conferring a specificity switch that impacts the extant enzyme’s specificity for formation of the diterpene spiroviolene over formation of sesquiterpenes hedycaryol and farnesol by up to 25-fold. This study emphasizes the value of ancestral sequence reconstruction combined with enzyme engineering as a versatile tool in chemical biology.
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影响因子：
15
作者：
Devamani T;Rauwerdink AM;Lunzer M;Jones BJ;Mooney JL;Tan MA;Zhang ZJ;Xu JH;Dean AM;Kazlauskas RJ
通讯作者：
Kazlauskas RJ
DOI：
10.1107/s2053230x15024619
发表时间：
2016-02
期刊：
Acta crystallographica. Section F, Structural biology communications
影响因子：
--
作者：
Deller MC;Kong L;Rupp B
通讯作者：
Rupp B
影响因子：
37.8
作者：
Gumulya, Yosephin;Baek, Jong-Min;Gillam, Elizabeth M. J.
通讯作者：
Gillam, Elizabeth M. J.
影响因子：
4.8
作者：
Bart, Aaron G.;Harris, Kurt L.;Scott, Emily E.
通讯作者：
Scott, Emily E.
DOI：
10.1073/pnas.0601605103
发表时间：
2006-06-27
影响因子：
11.1
作者：
Greenhagen, Bryan T.;O'Maille, Paul E.;Chappell, Joe
通讯作者：
Chappell, Joe