Nonribosomal peptide synthetases (NRPSs): Understanding functional domain interplay and biosynthetic directionality using FRET spectroscopy

• Applicant: Professor Dr. Henning D. Mootz
• Subject Area: Biochemistry ; Biophysics
• Term: from 2019 to 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 434700456

Non-ribosomal peptide synthetases (NRPSs) are mega-enzymes that act as protein templates and catalysts for the biosynthesis of many important peptide natural products like daptomycin and cyclosporine. The single amino acid building blocks are activated by adenylation domains (A domains) under the consumption of ATP, covalently bound as thioesters on 4’-phosphopantetheine-equipped peptidyl-carrier proteins (PCP domains) and linked by condensation domains (C domains). Further domains like the epimerization domain (E domain) provide additional structural diversification. A thioesterase domain (TE domain) releases the final product. In a typical linear NRPS simple rules dictate the necessary domain arrangements for a certain peptide product. To incorporate a single amino acid, all necessary domains are integrated into one module and the order of modules in the multifunctional enzyme corresponds to the sequence of the amino acids in the product. However, only very little is known about the temporal and spatial nature of intramolecular domain-domain interactions and how these translate into the essential directionality of the step-wise peptide assembly. Crystal structures of multi-domain NRPS fragments provide important insight, but only represent static snap-shots that lack dynamic information. Very recently, we have reported the first FRET sensor of a simplified A-PCP di-domain to investigate conformational changes of the catalytically active enzyme in real time using time-course experiments. These studies have already led to important new insights and models. Here, we propose to significantly expand the concept to study the dynamics of NRPSs using FRET. We expect to gain novel mechanistic insights into how directional NRPS biosynthesis is driven.

• DFG Programme: Research Grants