Engineering the Active Site Lid Dynamics to Improve the Catalytic Efficiency of Yeast Cytosine Deaminase
Conformational dynamics is important for enzyme catalysis. However, engineering dynamics to achieve a higher catalytic efficiency is still challenging. In this work, we develop a new strategy to improve the activity of yeast cytosine deaminase (yCD) by engineering its conformational dynamics. Specif...
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MDPI AG
2023-04-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/24/7/6592 |
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| author | Hanzhong Deng Mingming Qin Zhijun Liu Ying Yang Yefei Wang Lishan Yao |
| author_facet | Hanzhong Deng Mingming Qin Zhijun Liu Ying Yang Yefei Wang Lishan Yao |
| author_sort | Hanzhong Deng |
| collection | DOAJ |
| description | Conformational dynamics is important for enzyme catalysis. However, engineering dynamics to achieve a higher catalytic efficiency is still challenging. In this work, we develop a new strategy to improve the activity of yeast cytosine deaminase (yCD) by engineering its conformational dynamics. Specifically, we increase the dynamics of the yCD C-terminal helix, an active site lid that controls the product release. The C-terminal is extended by a dynamical single α-helix (SAH), which improves the product release rate by up to ~8-fold, and the overall catalytic rate <i>k</i><sub>cat</sub> by up to ~2-fold. It is also shown that the <i>k</i><sub>cat</sub> increase is due to the favorable activation entropy change. The NMR H/D exchange data indicate that the conformational dynamics of the transition state analog complex increases as the helix is extended, elucidating the origin of the enhanced catalytic entropy. This study highlights a novel dynamics engineering strategy that can accelerate the overall catalysis through the entropy-driven mechanism. |
| first_indexed | 2024-03-11T05:35:10Z |
| format | Article |
| id | doaj.art-af1340a35f984c93a6f5a9689ee78e8c |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-11T05:35:10Z |
| publishDate | 2023-04-01 |
| publisher | MDPI AG |
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| series | International Journal of Molecular Sciences |
| spelling | doaj.art-af1340a35f984c93a6f5a9689ee78e8c2023-11-17T16:53:19ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672023-04-01247659210.3390/ijms24076592Engineering the Active Site Lid Dynamics to Improve the Catalytic Efficiency of Yeast Cytosine DeaminaseHanzhong Deng0Mingming Qin1Zhijun Liu2Ying Yang3Yefei Wang4Lishan Yao5Qingdao New Energy Shandong Laboratory, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao 266101, ChinaQingdao New Energy Shandong Laboratory, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao 266101, ChinaNational Facility for Protein Science, Shanghai Advanced Research Institute, Chinese Academy of Sciences, Shanghai 201210, ChinaQingdao New Energy Shandong Laboratory, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao 266101, ChinaQingdao New Energy Shandong Laboratory, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao 266101, ChinaQingdao New Energy Shandong Laboratory, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao 266101, ChinaConformational dynamics is important for enzyme catalysis. However, engineering dynamics to achieve a higher catalytic efficiency is still challenging. In this work, we develop a new strategy to improve the activity of yeast cytosine deaminase (yCD) by engineering its conformational dynamics. Specifically, we increase the dynamics of the yCD C-terminal helix, an active site lid that controls the product release. The C-terminal is extended by a dynamical single α-helix (SAH), which improves the product release rate by up to ~8-fold, and the overall catalytic rate <i>k</i><sub>cat</sub> by up to ~2-fold. It is also shown that the <i>k</i><sub>cat</sub> increase is due to the favorable activation entropy change. The NMR H/D exchange data indicate that the conformational dynamics of the transition state analog complex increases as the helix is extended, elucidating the origin of the enhanced catalytic entropy. This study highlights a novel dynamics engineering strategy that can accelerate the overall catalysis through the entropy-driven mechanism.https://www.mdpi.com/1422-0067/24/7/6592dynamics engineeringcytosine deaminaseprodrugprotein structure |
| spellingShingle | Hanzhong Deng Mingming Qin Zhijun Liu Ying Yang Yefei Wang Lishan Yao Engineering the Active Site Lid Dynamics to Improve the Catalytic Efficiency of Yeast Cytosine Deaminase International Journal of Molecular Sciences dynamics engineering cytosine deaminase prodrug protein structure |
| title | Engineering the Active Site Lid Dynamics to Improve the Catalytic Efficiency of Yeast Cytosine Deaminase |
| title_full | Engineering the Active Site Lid Dynamics to Improve the Catalytic Efficiency of Yeast Cytosine Deaminase |
| title_fullStr | Engineering the Active Site Lid Dynamics to Improve the Catalytic Efficiency of Yeast Cytosine Deaminase |
| title_full_unstemmed | Engineering the Active Site Lid Dynamics to Improve the Catalytic Efficiency of Yeast Cytosine Deaminase |
| title_short | Engineering the Active Site Lid Dynamics to Improve the Catalytic Efficiency of Yeast Cytosine Deaminase |
| title_sort | engineering the active site lid dynamics to improve the catalytic efficiency of yeast cytosine deaminase |
| topic | dynamics engineering cytosine deaminase prodrug protein structure |
| url | https://www.mdpi.com/1422-0067/24/7/6592 |
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