FRETmonitoring of a nonribosomal peptide synthetase

Jonas Alfermann, Xun Sun, Florian Mayerthaler, Thomas E. Morrell, Eva Dehling, Gerrit Volkmann, Tamiki Komatsuzaki, Haw Yang, Henning D. Mootz

Research output: Contribution to journalArticle

12 Scopus citations

Abstract

Nonribosomal peptide synthetases (NRPSs) are multidomain enzyme templates for the synthesis of bioactive peptides. Large-scale conformational changes during peptide assembly are obvious from crystal structures, yet their dynamics and coupling to catalysis are poorly understood. We have designed an NRPS FRET sensor to monitor, in solution and in real time, the adoption of the productive transfer conformation between phenylalanine-binding adenylation (A) and peptidyl-carrier-protein domains of gramicidin synthetase I from Aneurinibacillus migulanus. The presence of ligands, substrates or intermediates induced a distinct fluorescence resonance energy transfer (FRET) readout, which was pinpointed to the population of specific conformations or, in two cases, mixtures of conformations. A pyrophosphate switch and lysine charge sensors control the domain alternation of the A domain. The phenylalanine-thioester and phenylalanine-AMP products constitute a mechanism of product inhibition and release that is involved in ordered assembly-line peptide biosynthesis. Our results represent insights from solution measurements into the conformational dynamics of the catalytic cycle of NRPSs.

Original languageEnglish (US)
Pages (from-to)1009-1015
Number of pages7
JournalNature Chemical Biology
Volume13
Issue number9
DOIs
StatePublished - Sep 1 2017

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Cell Biology

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    Alfermann, J., Sun, X., Mayerthaler, F., Morrell, T. E., Dehling, E., Volkmann, G., Komatsuzaki, T., Yang, H., & Mootz, H. D. (2017). FRETmonitoring of a nonribosomal peptide synthetase. Nature Chemical Biology, 13(9), 1009-1015. https://doi.org/10.1038/nchembio.2435