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Exploring the Substrate Scope of the Bacterial Phosphocholine Transferase AnkX for Versatile Protein Functionalization |
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| Authors: | Dr Philipp Ochtrop Stefan Ernst Prof Dr Aymelt Itzen Prof Dr Christian Hedberg |
| Affiliation: | 1. Chemical Biology Center (KBC), Department of Chemistry, Umeå University, Linnaeus väg 10, 90187 Umeå, Sweden
These authors contributed equally to this work.;2. Department of Biochemistry and Signal Transduction, University Medical Centre Hamburg–Eppendorf (UKE), Martinistrasse 52, 20246 Hamburg, Germany These authors contributed equally to this work.;3. Department of Biochemistry and Signal Transduction, University Medical Centre Hamburg–Eppendorf (UKE), Martinistrasse 52, 20246 Hamburg, Germany;4. Chemical Biology Center (KBC), Department of Chemistry, Umeå University, Linnaeus väg 10, 90187 Umeå, Sweden |
| Abstract: | Site-specific protein functionalization has become an indispensable tool in modern life sciences. Here, tag-based enzymatic protein functionalization techniques are among the most versatilely applicable approaches. However, many chemo-enzymatic functionalization strategies suffer from low substrate scopes of the enzymes utilized for functional labeling probes. We report on the wide substrate scope of the bacterial enzyme AnkX towards derivatized CDP-choline analogues and demonstrate that AnkX-catalyzed phosphocholination can be used for site-specific one- and two-step protein labeling with a broad array of different functionalities, displaying fast second-order transfer rates of 5×102 to 1.8×104 m −1 s−1. Furthermore, we also present a strategy for the site-specific dual labeling of proteins of interest, based on the exploitation of AnkX and the delabeling function of the enzyme Lem3. Our results contribute to the wide field of protein functionalization, offering an attractive chemo-enzymatic tag-based modification strategy for in vitro labeling. |
| Keywords: | CDP-choline dual labeling phosphocholination protein modifications transferases |