Dynamics of loops surrounding the active site architecture in GH5_2 subfamily TfCel5A for cellulose degradation
Abstract Background Lignocellulose is the most abundant natural biomass resource for the production of biofuels and other chemicals. The efficient degradation of cellulose by cellulases is a critical step for the lignocellulose bioconversion. Understanding the structure-catalysis relationship is vit...
| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2023-10-01
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| Series: | Biotechnology for Biofuels and Bioproducts |
| Subjects: | |
| Online Access: | https://doi.org/10.1186/s13068-023-02411-2 |
| _version_ | 1797453522062540800 |
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| author | Xiuyun Wu Sha Zhao Zhennan Tian Chao Han Xukai Jiang Lushan Wang |
| author_facet | Xiuyun Wu Sha Zhao Zhennan Tian Chao Han Xukai Jiang Lushan Wang |
| author_sort | Xiuyun Wu |
| collection | DOAJ |
| description | Abstract Background Lignocellulose is the most abundant natural biomass resource for the production of biofuels and other chemicals. The efficient degradation of cellulose by cellulases is a critical step for the lignocellulose bioconversion. Understanding the structure-catalysis relationship is vital for rational design of more stable and highly active enzymes. Glycoside hydrolase (GH) family 5 is the largest and most functionally diverse group of cellulases, with a conserved TIM barrel structure. The important roles of the various loop regions of GH5 enzymes in catalysis, however, remain poorly understood. Results In the present study, we investigated the relationship between the loops surrounding active site architecture and its catalytic efficiency, taking TfCel5A, an enzyme from GH5_2 subfamily of Thermobifida fusca, as an example. Large-scale computational simulations and site-directed mutagenesis experiments revealed that three loops (loop 8, 3, and 7) around active cleft played diverse roles in substrate binding, intermediate formation, and product release, respectively. The highly flexible and charged residue triad of loop 8 was responsible for capturing the ligand into the active cleft. Severe fluctuation of loop 3 led to the distortion of sugar conformation at the − 1 subsite. The wobble of loop 7 might facilitate product release, and the enzyme activity of the mutant Y361W in loop 7 was increased by approximately 40%. Conclusion This study unraveled the vital roles of loops in active site architecture and provided new insights into the catalytic mechanism of the GH5_2 cellulases. |
| first_indexed | 2024-03-09T15:24:04Z |
| format | Article |
| id | doaj.art-180af8bd627a44b59ed757f404cb5b36 |
| institution | Directory Open Access Journal |
| issn | 2731-3654 |
| language | English |
| last_indexed | 2024-03-09T15:24:04Z |
| publishDate | 2023-10-01 |
| publisher | BMC |
| record_format | Article |
| series | Biotechnology for Biofuels and Bioproducts |
| spelling | doaj.art-180af8bd627a44b59ed757f404cb5b362023-11-26T12:36:09ZengBMCBiotechnology for Biofuels and Bioproducts2731-36542023-10-0116111210.1186/s13068-023-02411-2Dynamics of loops surrounding the active site architecture in GH5_2 subfamily TfCel5A for cellulose degradationXiuyun Wu0Sha Zhao1Zhennan Tian2Chao Han3Xukai Jiang4Lushan Wang5State Key Laboratory of Microbial Technology, Institute of Microbial Technology, Shandong UniversityState Key Laboratory of Microbial Technology, Institute of Microbial Technology, Shandong UniversityState Key Laboratory of Microbial Technology, Institute of Microbial Technology, Shandong UniversityShandong Key Laboratory of Agricultural Microbiology, Shandong Agricultural UniversityNational Glycoengineering Research Center, Shandong UniversityState Key Laboratory of Microbial Technology, Institute of Microbial Technology, Shandong UniversityAbstract Background Lignocellulose is the most abundant natural biomass resource for the production of biofuels and other chemicals. The efficient degradation of cellulose by cellulases is a critical step for the lignocellulose bioconversion. Understanding the structure-catalysis relationship is vital for rational design of more stable and highly active enzymes. Glycoside hydrolase (GH) family 5 is the largest and most functionally diverse group of cellulases, with a conserved TIM barrel structure. The important roles of the various loop regions of GH5 enzymes in catalysis, however, remain poorly understood. Results In the present study, we investigated the relationship between the loops surrounding active site architecture and its catalytic efficiency, taking TfCel5A, an enzyme from GH5_2 subfamily of Thermobifida fusca, as an example. Large-scale computational simulations and site-directed mutagenesis experiments revealed that three loops (loop 8, 3, and 7) around active cleft played diverse roles in substrate binding, intermediate formation, and product release, respectively. The highly flexible and charged residue triad of loop 8 was responsible for capturing the ligand into the active cleft. Severe fluctuation of loop 3 led to the distortion of sugar conformation at the − 1 subsite. The wobble of loop 7 might facilitate product release, and the enzyme activity of the mutant Y361W in loop 7 was increased by approximately 40%. Conclusion This study unraveled the vital roles of loops in active site architecture and provided new insights into the catalytic mechanism of the GH5_2 cellulases.https://doi.org/10.1186/s13068-023-02411-2CellulaseGH5_2 subfamilyActive site architectureLoop dynamicsEnzyme catalysis |
| spellingShingle | Xiuyun Wu Sha Zhao Zhennan Tian Chao Han Xukai Jiang Lushan Wang Dynamics of loops surrounding the active site architecture in GH5_2 subfamily TfCel5A for cellulose degradation Biotechnology for Biofuels and Bioproducts Cellulase GH5_2 subfamily Active site architecture Loop dynamics Enzyme catalysis |
| title | Dynamics of loops surrounding the active site architecture in GH5_2 subfamily TfCel5A for cellulose degradation |
| title_full | Dynamics of loops surrounding the active site architecture in GH5_2 subfamily TfCel5A for cellulose degradation |
| title_fullStr | Dynamics of loops surrounding the active site architecture in GH5_2 subfamily TfCel5A for cellulose degradation |
| title_full_unstemmed | Dynamics of loops surrounding the active site architecture in GH5_2 subfamily TfCel5A for cellulose degradation |
| title_short | Dynamics of loops surrounding the active site architecture in GH5_2 subfamily TfCel5A for cellulose degradation |
| title_sort | dynamics of loops surrounding the active site architecture in gh5 2 subfamily tfcel5a for cellulose degradation |
| topic | Cellulase GH5_2 subfamily Active site architecture Loop dynamics Enzyme catalysis |
| url | https://doi.org/10.1186/s13068-023-02411-2 |
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