Broad substrate tolerance of tubulin tyrosine ligase enables one-step site-specific enzymatic protein labeling

The broad substrate tolerance of tubulin tyrosine ligase is the basic rationale 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 that are substantially bigger than and st...

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Bibliographic Details
Published in:Chemical science (Cambridge) 2017-05, Vol.8 (5), p.3471-3478
Main Authors: Schumacher, Dominik, Lemke, Oliver, Helma, Jonas, Gerszonowicz, Lena, Waller, Verena, Stoschek, Tina, Durkin, Patrick M, Budisa, Nediljko, Leonhardt, Heinrich, Keller, Bettina G, Hackenberger, Christian P R
Format: Article
Language:English
Subjects:
Amino acids
Enzymes
Labels
Marking
Proteins
Substrates
Tolerances
Tyrosine
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Summary:The broad substrate tolerance of tubulin tyrosine ligase is the basic rationale 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 that are substantially bigger than and structurally unrelated to the natural substrate, tyrosine, without the need for extensive protein engineering. 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 dynamics 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. Its broad substrate tolerance establishes tubulin tyrosine ligase as a powerful tool for enzyme-mediated protein modification with single functional amino acids in a specific structural context.
ISSN:2041-6520
2041-6539
DOI:10.1039/c7sc00574a