Multiple Substrate and Domain Binding in Non-Ribosomal Peptide Synthetases

Abstract

Non-ribosomal peptide synthetases (NRPSs) are enzymatic systems that synthesize important natural products in bacteria and fungi, often with pharmaceutical applications (antibiotics, antitumor agents or immunosuppressants). NRPSs use a series of domains, organized in contiguous modules, to covalently load substrates and condense those loaded on adjacent modules in an assembly line fashion. The substrates may be further modified by tailoring domains. Such multiple catalytic steps require a series of transient, sequential domain/domain and domain/substrate interactions, which are currently poorly understood. We have used NMR to probe and characterize binding sites for several NRPS domains. We show that chemical substrates do interact with NRPS domains and may be involved in modulating domain interactions. We also present the solution structures of hitherto uncharacterized domains and discuss the role of protein dynamics. We introduce novel NMR approaches necessary for such challenging systems (53 kDa domains, dynamic proteins). Understanding NRPS domain communication is a prerequisite to understand the biosynthesis of many natural products and offers prospect for reprogramming NRPS assembly lines.View Large Image | View Hi-Res Image | Download PowerPoint Slide