Related Experiment Video
Updated: Jul 12, 2026

A Facile Protocol to Generate Site-Specifically Acetylated Proteins in Escherichia Coli
Published on: December 9, 2017
Loading peptidyl-coenzyme A onto peptidyl carrier proteins: a novel approach in characterizing macrocyclization by
Stephan A Sieber1, Christopher T Walsh, Mohamed A Marahiel
1Fachbereich Chemie/Biochemie, Philipps-Univerität Marburg, Hans-Meerwein-Strasse, 35032 Marburg, Germany.
Abstract:
Here we report a new experimental approach to characterize recombinant nonribosomal peptide cyclases that do not show activity with conventional N-acetylcysteamine (SNAC) substrates. To explore the great potential of these domains for the catalysis of cell-free cyclization reactions, the new strategy takes advantage of the direct interaction between the natural substrate where the peptide chain is attached to the phosphopantetheine arm of the peptidyl carrier protein (PCP) and the peptide cyclase. A prerequisite for this reaction is the promiscuity of the Bacillus subtilis phosphopantetheinyl transferase Sfp for loading chemically synthesized peptidyl-coenzyme A substrates instead of the smaller natural substrate coenzyme A (CoASH) onto apoPCP. With this novel method we were able to characterize the regioselectivity of branched-chain cyclization catalyzed by the fengycin cyclase, which displays no activity with peptidyl-SNAC substrates.
Related Concept Videos
Mechanical Protein Functions
ATP Synthase: Mechanism
Tail-anchoring of Proteins in the ER Membrane
GPI Anchoring of Proteins in the ER Membrane
GPI-anchor structure
A sequence of 11 enzymatic reactions results in the synthesis of the complete GPI anchor consisting of a hydrophobic and a hydrophilic portion. The hydrophobic portion comprises phosphatidylinositol, while the hydrophilic part comprises polar groups like phosphoethanolamine,...
Post-translational Translocation of Proteins to the RER
Targeting proteins to the ER
Hsp40 and Hsp70 chaperone molecules bind the translated proteins in the cytosol to prevent their folding. The chaperone binding helps to keep the signal...
Amino Acid Biosynthetic Pathways

