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Kinetics Profiling of Gramicidin S Synthetase A, a Member of Nonribosomal Peptide Synthetases

Author(s): Sun, Xun; Li, Hao; Alfermann, Jonas; Mootz, Henning D; Yang, Haw

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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 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 the on-pathway counterpart remains generally elusive. Here, we introduce multi-platform 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 the 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.
Publication Date: 23-Dec-2014
Citation: Sun, Xun, Li, Hao, Alfermann, Jonas, Mootz, Henning D, Yang, Haw. "Kinetics Profiling of Gramicidin S Synthetase A, a Member of Nonribosomal Peptide Synthetases" Biochemistry, (50), 53, 7983 - 7989, doi:10.1021/bi501156m
DOI: doi:10.1021/bi501156m
ISSN: 0006-2960
EISSN: 1520-4995
Pages: 7983 - 7989
Type of Material: Journal Article
Journal/Proceeding Title: Biochemistry
Version: This is the author’s final manuscript. All rights reserved to author(s).

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