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

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.

A Hydroxylated Tetracationic Viologen based on Dimethylaminoethanol as a Negolyte for Aqueous Flow Batteries

“…The procedure was modified from the literature [45] . In a typical experiment, 2‐dimethylaminoethanol (4.3 mL, 3.8 g, 43 mmol, 1 equiv.)…”

“…The procedure was modified from the literature. [45] In a typical experiment, 2-dimethylaminoethanol (4.3 mL, 3.8 g, 43 mmol, 1 equiv.) was dissolved in acetone (45 mL) to yield a < 1 M solution.…”

Tyrosinase-Mediated Oxidative Coupling of Tyrosine Tags on Peptides and Proteins

“…As another alternative, the widely used sortase enzyme links cargo groups bearing polyglycine ( n = 1–5) motifs to protein C-termini after the threonine residue in an LPXTG recognition sequence. , butelase, , subtiligase farnesyl transferase, , and SpyTag/SpyCatcher, , work similarly by recognizing and ligating specific peptide sequences. Tubulin tyrosine ligase , and AnkX , (a bacterial phosphocholine transferase) can also append synthetically modified small molecule substrates to proteins based on peptide recognition sequences. Each of these methods has advantages and disadvantages to consider when choosing a conjugation strategy, including the size of the recognition motif; the residual sequence left behind after modification; the required pH and buffer conditions; the synthetic accessibility of the coupling partners; and the kinetics, availability, stability, and selectivity of the enzyme catalyst.…”

The unity of opposites: Strategic interplay between bacterial effectors to regulate cellular homeostasis

“…Ser76 Rab1 modification by AnkX also negatively impacts the interactions of the modified Rab1 with its GDI, which are restored upon Lem3-catalyzed dephosphocholination of Rab1 ( 111 ). Surprisingly, Lem3 cannot hydrolyze AnkX-catalyzed phosphocholination of Rab35 on residue Thr76 ( 115 ) pointing to the existence of yet-to-be identified effector selective for the Rab35 modification. The importance of the AnkX/Lem3 pair in the hijacking of Rab1 is not as well understood as the role of the SidM/SidD pair.…”