Kinetics profiling of gramicidin S synthetase A, a member of nonribosomal peptide synthetases

Xun Sun, Hao Li, Jonas Alfermann, Henning D. Mootz, Haw Yang

Research output: Contribution to journalArticle

7 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)7983-7989
Number of pages7
JournalBiochemistry
Volume53
Issue number50
DOIs
StatePublished - Dec 23 2014

All Science Journal Classification (ASJC) codes

  • Biochemistry

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