A homogeneous resonance energy transfer assay for phosphopantetheinyl transferase.
Phosphopantetheinyl transferase plays an essential role in activating fatty acid, polyketide, and nonribosomal peptide biosynthetic pathways, catalyzing covalent attachment of a 4'-phosphopantetheinyl group to a conserved residue within carrier protein domains. This enzyme has been validated as an essential gene to primary metabolism and presents a target for the identification of antibiotics with a new mode of action. Here we report the development of a homogeneous resonance energy transfer assay using fluorescent coenzyme A derivatives and a surrogate peptide substrate that can serve to identify inhibitors of this enzyme class. This assay lays a blueprint for translation of these techniques to other transferase enzymes that accept fluorescent substrate analogues.
Foley
Timothy L
TL
Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92093, USA.
Burkart
Michael D
MD
D000818:Animals; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000032:analysis / Q000494:pharmacology; D004795:Enzyme Stability / Q000187:drug effects; D029968:Escherichia coli Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000378:metabolism; D008958:Models, Molecular; D010204:Pantetheine / Q000031:analogs & derivatives / Q000378:metabolism; D010455:Peptides / Q000378:metabolism; D011487:Protein Conformation; D012710:Serum Albumin, Bovine / Q000494:pharmacology; D014166:Transferases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D017855:Transferases (Other Substituted Phosphate Groups) / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*
D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D010455:Peptides; C082662:phosphopantetheinyl transferase; C073125:acpS protein, E coli; D012710:Serum Albumin, Bovine; D010204:Pantetheine; D014166:Transferases; D017855:Transferases (Other Substituted Phosphate Groups); C021364:holo-(acyl-carrier-protein) synthase; C003129:4'-phosphopantetheine; D004121:Dimethyl Sulfoxide