Monitoring multiple active sites on thiotemplate enzymes in parallel: A molecular movie of yersiniabactin bioassembly

Shaun M. McLoughlin, Neil L. Kelleher*

*Corresponding author for this work

Research output: Contribution to journalArticle

8 Scopus citations

Abstract

For the uninterrupted observation of natural product bioassembly on nonribosomal peptide synthetases, Quadrupole Fourier Transform Mass Spectrometry (Q-FTMS) was utilized to directly interrogate peptides harboring covalently modified residues in yersiniabactin synthetase. After proteolysis in CNBr, the peptides corresponding to each carrier site were identified and visualized using a continuous kinetic assay. Overall, complex intermediate formation was rapid, with observation of the HPTT-β-keto-2,2-dimethyl-S-ACP intermediate within 4 s, while each active site reached saturation within ∼20 s. Reduction of the β-keto group at the ACP domain was found to have the slowest rate, accumulating only after 40 s. This represents the first study to correlate five active sites in tandem with kinetic and structural resolution of the complex intermediates in addition to regiospecific information preserved in the assay.

Original languageEnglish (US)
Pages (from-to)14984-14985
Number of pages2
JournalJournal of the American Chemical Society
Volume127
Issue number43
DOIs
StatePublished - Nov 2 2005

ASJC Scopus subject areas

  • Catalysis
  • Chemistry(all)
  • Biochemistry
  • Colloid and Surface Chemistry

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