Exploring the Substrate Scope of the Bacterial Phosphocholine Transferase AnkX for Versatile Protein Functionalization

Chembiochem. 2019 Sep 16;20(18):2336-2340. doi: 10.1002/cbic.201900200. Epub 2019 Aug 21.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Proteins / metabolism*
  • Cytidine Diphosphate Choline / analogs & derivatives
  • Cytidine Diphosphate Choline / metabolism
  • Diacylglycerol Cholinephosphotransferase / metabolism*
  • Fluoresceins / chemistry
  • Fluorescent Dyes / chemistry
  • Protein Engineering
  • Substrate Specificity
  • rab1 GTP-Binding Proteins / chemistry*


  • Bacterial Proteins
  • Fluoresceins
  • Fluorescent Dyes
  • Cytidine Diphosphate Choline
  • Diacylglycerol Cholinephosphotransferase
  • rab1 GTP-Binding Proteins