Slow dynamics orchestrate communication between binding sites in the condensation domain of a non-ribosomal peptide synthetase.

Non-ribosomal peptide synthetases (NRPSs) are complex molecular machineries that synthesize non-proteinaceous peptides in microorganisms. These peptides (NRPs) usually present a wide range of biological activities and are highly regarded as potential anti-cancer and anti-infective agents. Because of their chemical complexity, derivatives of NRPs with tailored pharmacological properties are difficult to synthesize chemically, which has triggered efforts to understand the functional mechanisms of NRPS systems and develop protein engineering strategies aimed at enabling enzymatic synthesis of non-natural NRPs. A fundamental reaction step of NRPS systems is the formation of peptide bonds between amino-acid-like building blocks. This reaction is catalysed by so-called condensation domains. The structures of several condensation domains and their complexes have been solved by crystallography and electron microscopy, but these structures have failed to provide the key to the design of artificial condensation domains. Here, we use NMR spectroscopy to reveal a complex network of dynamics in the condensation domain of the NRPS responsible for the synthesis of Tomaymycin and reveal how these motions mediate communication between the two substrate binding sites, providing a means to synchronize interactions for efficient catalysis. Our results underline the impact of dynamics, next to structure, on the function of enzymatic units and reinforce the need to consider conformational flexibility in the design of proteins with altered functions.