title: Kinetics profiling of gramicidin S synthetase A, a member of nonribosomal peptide synthetases.
abstract: Nonribosomal peptide synthetases (NRPS) incorporate assorted amino acid substrates into complex natural products. The substrate is activated via the formation of a reactive aminoacyl adenylate and is subsequently attached to the protein template via a thioester bond. The reactive nature of such intermediates, however, leads to side reactions that also break down the high-energy anhydride bond. The off-pathway kinetics or their relative weights compared to that of the on-pathway counterpart remains generally elusive. Here, we introduce multiplatform kinetics profiling to quantify the relative weights of on- and off-pathway reactions. Using the well-defined stoichiometry of thioester formation, we integrate a mass spectrometry (MS) kinetics assay, a high-performance liquid chromatography (HPLC) assay, and an ATP-pyrophosphate (PPi) exchange assay to map out a highly efficient on-pathway kinetics profile of the substrate activation and intermediate uploading (>98% relative weight) for wide-type gramicidin S synthetase A (GrsA) and a 87% rate profile for a cysteine-free GrsA mutant. Our kinetics profiling approach complements the existing enzyme-coupled byproduct-release assays, unraveling new mechanistic insights of substrate activation/channeling in NRPS enzymes.
authors.affiliation: Department of Chemistry, Princeton University , Princeton, New Jersey 08544, United States.
authors.forename: Xun
authors.initials: X
authors.lastname: Sun
authors.forename: Hao
authors.initials: H
authors.lastname: Li
authors.forename: Jonas
authors.initials: J
authors.lastname: Alfermann
authors.forename: Henning D
authors.initials: HD
authors.lastname: Mootz
authors.forename: Haw
authors.initials: H
authors.lastname: Yang
mesh_terms: D000591:Amino Acid Isomerases / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry*; D007700:Kinetics; D013058:Mass Spectrometry; D010453:Peptide Synthases / Q000737:chemistry*
chemical_list: D001426:Bacterial Proteins; D000591:Amino Acid Isomerases; C022343:phenylalanine racemase (ATP-hydrolyzing); D010453:Peptide Synthases