Characterization of the first naturally thermostable terpene synthases and development of strategies to improve thermostability in this family of enzymes.
Characterization of the first naturally thermostable terpene synthases and development of strategies to improve thermostability in this family of enzymes.
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DOI：
10.1111/febs.14072
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发表时间：
2017-06
期刊：
影响因子：
--
通讯作者：
Leak DJ
中科院分区：
文献类型：
--
作者：
Styles MQ;Nesbitt EA;Marr S;Hutchby M;Leak DJ
萜类天然产物家族正成为异源微生物生产的目标，作为从植物中提取的更便宜、更可靠的替代品。导致萜烯结构多样化的关键酶是一类萜烯合成酶（TS），在适合工业应用之前，这些酶通常需要通过工程来改善其热稳定性、鲁棒性和催化活性等性能。提高热稳定性通常依赖于筛选大量的突变体，因为没有自然的热稳定性TSs描述，基于合理的设计决策。我们已经表征了具有热稳定性的天然TSs的第一个例子，它在高达78°C的温度下催化倍半萜τ‐muurolol的形成。我们还报道了65°C时k cat值为0.95 s−1的酶，这是细菌倍半萜合成酶记录的最高k cat值。反过来，这些耐热酶被用作模型，为另一个具有相同特异性但序列同源性较低的TS的合理工程提供信息。新设计的变种显示增加热稳定性和周转。考虑到一类TS结构域的高度同源性，该方法可以普遍适用于改善该类其他酶的性能。模型数据可在PMDB数据库中获得，登录号为PM0080780。本研究首次发现了天然耐热的I类倍半萜合成酶；催化倍半萜形成τ‐muurolol的酶，其活性可保持在78°C，并且具有迄今为止观察到的最高k cat值（65°C时为0.95 s−1）的细菌萜烯合成酶。
The terpenoid family of natural products is being targeted for heterologous microbial production as a cheaper and more reliable alternative to extraction from plants. The key enzyme responsible for diversification of terpene structure is the class‐I terpene synthase (TS), and these often require engineering to improve properties such as thermostability, robustness and catalytic activity before they are suitable for industrial use. Improving thermostability typically relies on screening a large number of mutants, as there are no naturally thermostable TSs described upon which to base rational design decisions. We have characterized the first examples of natural TSs exhibiting thermostability, which catalyse the formation of the sesquiterpene τ‐muurolol at temperatures up to 78 °C. We also report an enzyme with a k cat value of 0.95 s−1 at 65 °C, the highest k cat recorded for a bacterial sesquiterpene synthase. In turn, these thermostable enzymes were used as a model to inform the rational engineering of another TS, with the same specificity but low sequence identity to the model. The newly engineered variant displayed increased thermostability and turnover. Given the high structural homology of the class‐I TS domain, this approach could be generally applicable to improving the properties of other enzymes in this class. Model data are available in the PMDB database under the accession number PM0080780. This study has characterized the first naturally thermostable class‐I sesquiterpene synthases; enzymes that catalyse the formation of the sesquiterpene τ‐muurolol and can retain activity as high as 78 °C, and have the highest k cat value (0.95 s−1 at 65 °C) observed for a bacterial terpene synthase to date.
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影响因子：
14.9
作者：
Finn RD;Clements J;Arndt W;Miller BL;Wheeler TJ;Schreiber F;Bateman A;Eddy SR
通讯作者：
Eddy SR
DOI：
10.1093/protein/9.8.629
发表时间：
1996-08-01
期刊：
PROTEIN ENGINEERING
影响因子：
--
作者：
Danson, MJ;Hough, DW;Pearl, L
通讯作者：
Pearl, L
影响因子：
8
作者：
Diaz, Juan E.;Lin, Chun-Shi;Weiss, Gregory A.
通讯作者：
Weiss, Gregory A.
影响因子：
14.8
作者：
通讯作者：
--
影响因子：
4.7
作者：
Sarria, Stephen;Wong, Betty;Peralta-Yahya, Pamela
通讯作者：
Peralta-Yahya, Pamela