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Issue 5, 2017
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Broad substrate tolerance of tubulin tyrosine ligase enables one-step site-specific enzymatic protein labeling

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Abstract

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 in vitro enzyme-mediated protein modification with single functional amino acids in a specific structural context.

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

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Supplementary files

Article information


Submitted
07 Feb 2017
Accepted
18 Mar 2017
First published
20 Mar 2017

This article is Open Access
All publication charges for this article have been paid for by the Royal Society of Chemistry

Chem. Sci., 2017,8, 3471-3478
Article type
Edge Article

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

D. Schumacher, O. Lemke, J. Helma, L. Gerszonowicz, V. Waller, T. Stoschek, P. M. Durkin, N. Budisa, H. Leonhardt, B. G. Keller and C. P. R. Hackenberger, Chem. Sci., 2017, 8, 3471
DOI: 10.1039/C7SC00574A

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