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| Citation key | 161.2017.Schumacher |
|---|---|
| Author | Schumacher, D., and Lemke, O., and Helma, J., and Gerszonowicz, L., and Waller, V., and Stoschek, T., and Durkin, P. M., and Budisa, N. and Leonhardt, H., and Keller, B., and Hackenberger, C. |
| Pages | 3471-3478 |
| Year | 2017 |
| DOI | 10.1039/C7SC00574A |
| Journal | Chem. Sci. [Edge Article] |
| Volume | 8 |
| Abstract | Broad substrate tolerance of tubulin tyrosine ligase is the basic rational behind its wide applicability for chemoenzymatic protein functionalization. In this context, we report that the wild type enzyme enables ligation of various unnatural amino acids substantially bigger and structurally unrelated to the natural substrate tyrosine without the need for extensive protein engineering. The notion of this unusual substrate flexibility is due to the fact that the enzyme’s catalytic pocket forms an extended cavity during ligation as confirmed by docking experiments and all atom molecular dynamic simulations. This feature enabled one-step C-terminal biotinylation and fluorescent coumarin labeling of various functional proteins as demonstrated with Ubiquitin, an antigen binding nanobody and the apoptosis marker Annexin V. Broad substrate tolerance establishes tubulin tyrosine ligase as a powerful tool for in vitro enzyme-mediated protein modification with single functional amino acids in a specific structural context. |