Semi-rational design of an aromatic dioxygenase by substrate tunnel redirection
Summary: Lignin valorization is crucial for achieving economic and sustainable biorefinery processes. However, the enzyme substrate preferences involved in lignin degradation remain poorly understood, and low activity toward specific substrates presents a significant challenge to the efficient utili...
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| Language: | English |
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Elsevier
2025-01-01
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| Series: | iScience |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2589004224027974 |
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| author | Jiawei Wang Xingyu Ouyang Shiyu Meng Jiayi Li Liangxu Liu Chaofeng Li Hengrun Li Haotian Zheng Chao Liao Yi-Lei Zhao Jun Ni |
| author_facet | Jiawei Wang Xingyu Ouyang Shiyu Meng Jiayi Li Liangxu Liu Chaofeng Li Hengrun Li Haotian Zheng Chao Liao Yi-Lei Zhao Jun Ni |
| author_sort | Jiawei Wang |
| collection | DOAJ |
| description | Summary: Lignin valorization is crucial for achieving economic and sustainable biorefinery processes. However, the enzyme substrate preferences involved in lignin degradation remain poorly understood, and low activity toward specific substrates presents a significant challenge to the efficient utilization of lignin. In this study, we investigated the substrate promiscuity of ThAdo, a key enzyme involved in lignin valorization. Pre-reaction state analysis revealed that a hydrogen bond network is critical in determining substrate selectivity. By performing targeted saturation mutagenesis on residues surrounding the substrate tunnels, we identified the Y205W and Y205Q mutants, which demonstrated 0.73-fold and 0.72-fold enhancements in activity, respectively. Structural analysis indicated that the redirection of the original substrate tunnel may be responsible for the improved activity. Our study provides essential insights into the substrate preference mechanisms of lignin degrading enzymes and suggests that this tunnel-redirection strategy can be extended to other promiscuous enzymes. |
| format | Article |
| id | doaj-art-c974ac4308ec48efaf13f58fd135cc40 |
| institution | DOAJ |
| issn | 2589-0042 |
| language | English |
| publishDate | 2025-01-01 |
| publisher | Elsevier |
| record_format | Article |
| series | iScience |
| spelling | doaj-art-c974ac4308ec48efaf13f58fd135cc402025-08-20T02:39:38ZengElsevieriScience2589-00422025-01-0128111157010.1016/j.isci.2024.111570Semi-rational design of an aromatic dioxygenase by substrate tunnel redirectionJiawei Wang0Xingyu Ouyang1Shiyu Meng2Jiayi Li3Liangxu Liu4Chaofeng Li5Hengrun Li6Haotian Zheng7Chao Liao8Yi-Lei Zhao9Jun Ni10State Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic and Developmental Sciences, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China; Zhangjiang Institute for Advanced Study, Shanghai Jiao Tong University, Shanghai 200240, ChinaState Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic and Developmental Sciences, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, ChinaInnovation Center for Synthetic Biotechnology, Lumy Biotechnology, Changzhou 213200, Jiangsu, ChinaState Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic and Developmental Sciences, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, ChinaState Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic and Developmental Sciences, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China; Zhangjiang Institute for Advanced Study, Shanghai Jiao Tong University, Shanghai 200240, ChinaState Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic and Developmental Sciences, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China; Zhangjiang Institute for Advanced Study, Shanghai Jiao Tong University, Shanghai 200240, ChinaState Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic and Developmental Sciences, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China; Zhangjiang Institute for Advanced Study, Shanghai Jiao Tong University, Shanghai 200240, ChinaState Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic and Developmental Sciences, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China; Zhangjiang Institute for Advanced Study, Shanghai Jiao Tong University, Shanghai 200240, ChinaState Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic and Developmental Sciences, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China; Zhangjiang Institute for Advanced Study, Shanghai Jiao Tong University, Shanghai 200240, ChinaState Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic and Developmental Sciences, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, ChinaState Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic and Developmental Sciences, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China; Zhangjiang Institute for Advanced Study, Shanghai Jiao Tong University, Shanghai 200240, China; Innovation Center for Synthetic Biotechnology, Lumy Biotechnology, Changzhou 213200, Jiangsu, China; Corresponding authorSummary: Lignin valorization is crucial for achieving economic and sustainable biorefinery processes. However, the enzyme substrate preferences involved in lignin degradation remain poorly understood, and low activity toward specific substrates presents a significant challenge to the efficient utilization of lignin. In this study, we investigated the substrate promiscuity of ThAdo, a key enzyme involved in lignin valorization. Pre-reaction state analysis revealed that a hydrogen bond network is critical in determining substrate selectivity. By performing targeted saturation mutagenesis on residues surrounding the substrate tunnels, we identified the Y205W and Y205Q mutants, which demonstrated 0.73-fold and 0.72-fold enhancements in activity, respectively. Structural analysis indicated that the redirection of the original substrate tunnel may be responsible for the improved activity. Our study provides essential insights into the substrate preference mechanisms of lignin degrading enzymes and suggests that this tunnel-redirection strategy can be extended to other promiscuous enzymes.http://www.sciencedirect.com/science/article/pii/S2589004224027974biochemistryenzyme engineeringstructural biology |
| spellingShingle | Jiawei Wang Xingyu Ouyang Shiyu Meng Jiayi Li Liangxu Liu Chaofeng Li Hengrun Li Haotian Zheng Chao Liao Yi-Lei Zhao Jun Ni Semi-rational design of an aromatic dioxygenase by substrate tunnel redirection iScience biochemistry enzyme engineering structural biology |
| title | Semi-rational design of an aromatic dioxygenase by substrate tunnel redirection |
| title_full | Semi-rational design of an aromatic dioxygenase by substrate tunnel redirection |
| title_fullStr | Semi-rational design of an aromatic dioxygenase by substrate tunnel redirection |
| title_full_unstemmed | Semi-rational design of an aromatic dioxygenase by substrate tunnel redirection |
| title_short | Semi-rational design of an aromatic dioxygenase by substrate tunnel redirection |
| title_sort | semi rational design of an aromatic dioxygenase by substrate tunnel redirection |
| topic | biochemistry enzyme engineering structural biology |
| url | http://www.sciencedirect.com/science/article/pii/S2589004224027974 |
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