Characterization and structural analysis of a leucine aminopeptidase using site-directed mutagenesis
Abstract Amp0279 (EC 3.4.11.24, GenBank: CP000817.1) is a Co2+-dependent leucine aminopeptidase from the Lysinibacillus sphaericus C3-41 genome. After analyses using molecular docking and spatial structure analysis, site-directed mutagenesis mutants were performed as Amp0279-R131E, Amp0279-R131H, Am...
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SpringerOpen
2024-12-01
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| Series: | AMB Express |
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| Online Access: | https://doi.org/10.1186/s13568-024-01793-2 |
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| author | Yuqi Men Yang Liu Dongjie Yin Guan Wang Rui Qin Hairong Xiong Yawei Wang |
| author_facet | Yuqi Men Yang Liu Dongjie Yin Guan Wang Rui Qin Hairong Xiong Yawei Wang |
| author_sort | Yuqi Men |
| collection | DOAJ |
| description | Abstract Amp0279 (EC 3.4.11.24, GenBank: CP000817.1) is a Co2+-dependent leucine aminopeptidase from the Lysinibacillus sphaericus C3-41 genome. After analyses using molecular docking and spatial structure analysis, site-directed mutagenesis mutants were performed as Amp0279-R131E, Amp0279-R131H, Amp0279-R131A and Amp0279-E349D. The optimum pH of Amp0279-R131E was shifted from the original 8.5 to 7.5, and the overall electrostatic potential was shifted towards acidic. Compared with the original enzyme, the mutant proteins all gained better structural stability, especially the apparent melting temperature (Tm) of Amp0279-R131H increased from 41.8 to 45.5 °C. Morever, when protein was bound to the substrate, the Tm of Amp0279-R131E was increased by 7.3 °C and Amp0279-R131H increased by 5.4 °C, compared to the original enzyme. This is consistent with the results that the mutants acquired higher binding energies to the substrates, and an increase the hydrogen bonding force. In addition, the molecular docking of mutant and substrate revealed that the truncation of R131 contributes to the increase in the binding capacity of the substrate molecules to the active centre. In contrast, the presence of π-Cation interactions generated by R131 with the substrate has an important effect on the ability of Amp0279 to hydrolyse the substrate. This study demostrated that R131 is a key site for activity and stability, which is important in the future exploration of the functional structure of Amp0279. |
| format | Article |
| id | doaj-art-53eb8e8ed88e4f29b58790a2332fb569 |
| institution | OA Journals |
| issn | 2191-0855 |
| language | English |
| publishDate | 2024-12-01 |
| publisher | SpringerOpen |
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| series | AMB Express |
| spelling | doaj-art-53eb8e8ed88e4f29b58790a2332fb5692025-08-20T02:32:26ZengSpringerOpenAMB Express2191-08552024-12-0114111210.1186/s13568-024-01793-2Characterization and structural analysis of a leucine aminopeptidase using site-directed mutagenesisYuqi Men0Yang Liu1Dongjie Yin2Guan Wang3Rui Qin4Hairong Xiong5Yawei Wang6College of Life Science, South-Central Minzu UniversityCollege of Life Science, Wuchang University of TechnologyCollege of Life Science, South-Central Minzu UniversityCollege of Life Science, South-Central Minzu UniversityCollege of Life Science, South-Central Minzu UniversityCollege of Life Science, South-Central Minzu UniversityCollege of Life Science, South-Central Minzu UniversityAbstract Amp0279 (EC 3.4.11.24, GenBank: CP000817.1) is a Co2+-dependent leucine aminopeptidase from the Lysinibacillus sphaericus C3-41 genome. After analyses using molecular docking and spatial structure analysis, site-directed mutagenesis mutants were performed as Amp0279-R131E, Amp0279-R131H, Amp0279-R131A and Amp0279-E349D. The optimum pH of Amp0279-R131E was shifted from the original 8.5 to 7.5, and the overall electrostatic potential was shifted towards acidic. Compared with the original enzyme, the mutant proteins all gained better structural stability, especially the apparent melting temperature (Tm) of Amp0279-R131H increased from 41.8 to 45.5 °C. Morever, when protein was bound to the substrate, the Tm of Amp0279-R131E was increased by 7.3 °C and Amp0279-R131H increased by 5.4 °C, compared to the original enzyme. This is consistent with the results that the mutants acquired higher binding energies to the substrates, and an increase the hydrogen bonding force. In addition, the molecular docking of mutant and substrate revealed that the truncation of R131 contributes to the increase in the binding capacity of the substrate molecules to the active centre. In contrast, the presence of π-Cation interactions generated by R131 with the substrate has an important effect on the ability of Amp0279 to hydrolyse the substrate. This study demostrated that R131 is a key site for activity and stability, which is important in the future exploration of the functional structure of Amp0279.https://doi.org/10.1186/s13568-024-01793-2AminopeptidaseSite-directed mutagenesispH shiftMolecular dockingHydrogen bondπ-Cation interaction |
| spellingShingle | Yuqi Men Yang Liu Dongjie Yin Guan Wang Rui Qin Hairong Xiong Yawei Wang Characterization and structural analysis of a leucine aminopeptidase using site-directed mutagenesis AMB Express Aminopeptidase Site-directed mutagenesis pH shift Molecular docking Hydrogen bond π-Cation interaction |
| title | Characterization and structural analysis of a leucine aminopeptidase using site-directed mutagenesis |
| title_full | Characterization and structural analysis of a leucine aminopeptidase using site-directed mutagenesis |
| title_fullStr | Characterization and structural analysis of a leucine aminopeptidase using site-directed mutagenesis |
| title_full_unstemmed | Characterization and structural analysis of a leucine aminopeptidase using site-directed mutagenesis |
| title_short | Characterization and structural analysis of a leucine aminopeptidase using site-directed mutagenesis |
| title_sort | characterization and structural analysis of a leucine aminopeptidase using site directed mutagenesis |
| topic | Aminopeptidase Site-directed mutagenesis pH shift Molecular docking Hydrogen bond π-Cation interaction |
| url | https://doi.org/10.1186/s13568-024-01793-2 |
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