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| Format: | Artículo Open Access |
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Wiley
2025
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| Accès en ligne: | https://onlinelibrary.wiley.com/doi/10.1002/pro.70021 |
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| _version_ | 1867018514625200128 |
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| author | Kathryn K. Crone Jason W. Labonte Mikael H. Elias Michael F. Freeman |
| author_facet | Kathryn K. Crone Jason W. Labonte Mikael H. Elias Michael F. Freeman Kathryn K. Crone Jason W. Labonte Mikael H. Elias Michael F. Freeman |
| collection | Wiley Open Access |
| contents | α‐N‐Methyltransferase regiospecificity is mediated by proximal, redundant enzyme–substrate interactions Kathryn K. Crone Jason W. Labonte Mikael H. Elias Michael F. Freeman Protein Science AbstractN‐Methylation of the peptide backbone confers pharmacologically beneficial characteristics to peptides that include greater membrane permeability and resistance to proteolytic degradation. The borosin family of ribosomally synthesized and post‐translationally modified peptides offer a post‐translational route to install amide backbone α‐N‐methylations. Previous work has elucidated the substrate scope and engineering potential of two examples of type I borosins, which feature autocatalytic precursors that encode N‐methyltransferases that methylate their own C‐termini in trans. We recently reported the first discrete N‐methyltransferase and precursor peptide from Shewanella oneidensis MR‐1, a minimally iterative, type IV borosin that allowed the first detailed kinetic analyses of borosin N‐methyltransferases. Herein, we characterize the substrate scope and resilient regiospecificity of this discrete N‐methyltransferase by comparison of relative rates and methylation patterns of over 40 precursor peptide variants along with structure analyses of nine enzyme–substrate complexes. Sequences critical to methylation are identified and demonstrated in assaying minimal peptide substrates and non‐native peptide sequences for assessment of secondary structure requirements and engineering potential. This work grants understanding towards the mechanism of substrate recognition and iterative activity by discrete borosin N‐methyltransferases. 10.1002/pro.70021 http://creativecommons.org/licenses/by-nc-nd/4.0/ |
| doi_str_mv | 10.1002/pro.70021 |
| format | Artículo Open Access |
| id | wiley_oa_10_1002_pro_70021 |
| institution | Wiley Open Access |
| license_str_mv | http://creativecommons.org/licenses/by-nc-nd/4.0/ |
| publishDate | 2025 |
| publisher | Wiley |
| record_format | wiley_oa |
| spellingShingle | α‐N‐Methyltransferase regiospecificity is mediated by proximal, redundant enzyme–substrate interactions Kathryn K. Crone Jason W. Labonte Mikael H. Elias Michael F. Freeman Protein Science α‐N‐Methyltransferase regiospecificity is mediated by proximal, redundant enzyme–substrate interactions Kathryn K. Crone Jason W. Labonte Mikael H. Elias Michael F. Freeman Protein Science AbstractN‐Methylation of the peptide backbone confers pharmacologically beneficial characteristics to peptides that include greater membrane permeability and resistance to proteolytic degradation. The borosin family of ribosomally synthesized and post‐translationally modified peptides offer a post‐translational route to install amide backbone α‐N‐methylations. Previous work has elucidated the substrate scope and engineering potential of two examples of type I borosins, which feature autocatalytic precursors that encode N‐methyltransferases that methylate their own C‐termini in trans. We recently reported the first discrete N‐methyltransferase and precursor peptide from Shewanella oneidensis MR‐1, a minimally iterative, type IV borosin that allowed the first detailed kinetic analyses of borosin N‐methyltransferases. Herein, we characterize the substrate scope and resilient regiospecificity of this discrete N‐methyltransferase by comparison of relative rates and methylation patterns of over 40 precursor peptide variants along with structure analyses of nine enzyme–substrate complexes. Sequences critical to methylation are identified and demonstrated in assaying minimal peptide substrates and non‐native peptide sequences for assessment of secondary structure requirements and engineering potential. This work grants understanding towards the mechanism of substrate recognition and iterative activity by discrete borosin N‐methyltransferases. 10.1002/pro.70021 http://creativecommons.org/licenses/by-nc-nd/4.0/ |
| title | α‐N‐Methyltransferase regiospecificity is mediated by proximal, redundant enzyme–substrate interactions |
| topic | Protein Science |
| url | https://onlinelibrary.wiley.com/doi/10.1002/pro.70021 |