Fluorescent techniques for discovery and characterization of phosphopantetheinyl transferase inhibitors.

Phosphopantetheinyl transferase (PPTase; E.C. 2.7.8.-) activates biosynthetic pathways that synthesize both primary and secondary metabolites in bacteria. Inhibitors of these enzymes have the potential to serve as antibiotic compounds that function through a unique mode of action and possess clinical utility. Here we report a direct and continuous assay for this enzyme class based upon monitoring polarization of a fluorescent phosphopantetheine analog as it is transferred from a low-molecular weight CoA substrate to higher-molecular weight protein acceptor. We demonstrate the utility of this method for the biochemical characterization of PPTase Sfp, a canonical representative from this class. We also establish the portability of this technique to other homologs by adapting the assay to function with the human PPTase, a target for which a microplate detection method does not currently exist. Comparison of these targets provides a basis to predict the therapeutic index of inhibitor candidates and offers a valuable characterization of enzyme activity.