Caught in the Act: Trapping an Acyl Carrier Protein Interacting with a Ketosynthase

Abstract

Polyketide synthases (PKSs) and fatty acid synthases (FASs) in microorganisms rely on selective and specific protein-protein interactions to build structurally complex molecules. These synthases utilize acyl carrier proteins (ACPs) to carry reactive intermediates and substrates to different stops on the enzyme assembly line. ACPs collaborate with their partner enzymes by tethering substrates and intermediates onto its highly conserved 4′-phosphopantetheine (4′-Ppant) arm. Previously, we showed that the installation of a site-specific thiocyanate vibrational spectroscopy probe onto the 4′-Ppant arm allows us to visualize and evaluate the solvation environment of the 4′-Ppant arm using infrared spectroscopy. Herein, we extend these studies to show that the thiocyanate probe can be used to probe ACP-protein interactions. We characterized a potential ACP-enzyme interaction using a range of biochemical and biophysical techniques including FTIR spectroscopy, SDS PAGE, exclusion chromatography, and tandem proteolysis mass spectrometry. The ability to induce a covalent link between an ACP and its catalytic partner could represent a powerful tool for exploring the possibility of synthesizing “unnatural” natural products through the bioengineering of PKSs and FASs.