Nature Chemical Biology
Nature Chemical Biology
5 years ago

FRET monitoring of a nonribosomal peptide synthetase

FRET monitoring of a nonribosomal peptide synthetase
Florian Mayerthaler, Henning D Mootz, Xun Sun, Gerrit Volkmann, Tamiki Komatsuzaki, Thomas E Morrell, Haw Yang, Jonas Alfermann, Eva Dehling
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.

Publisher URL: http://dx.doi.org/10.1038/nchembio.2435

DOI: 10.1038/nchembio.2435

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