Abstract
Non-ribosomal peptide synthetases (NRPSs) are large, modular enzymes that produce bioactive peptides of tremendous structural and chemical diversity, due to the incorporation, alongside the canonical 20 amino acids, of non-proteinogenic amino acids, fatty acids, sugars and heterocyclic rings. For linear NRPSs, the size and composition of the peptide product is dictated by the number, order and specificity of the individual modules, each made of several domains. Given the size and complexity of NRPSs, most in vitro studies have focused on individual domains, di-domains or single modules extracted from the full-length proteins. However, intermodular interactions could play a critical role and regulate the activity of the domains and modules in unpredictable ways. Here we investigate in vitro substrate activation by three A domains of the tyrocidine synthetase TycC enzyme, systematically comparing their activity when alone (with the respective PCP domain), in pairs (di-modular constructs) or all together (tri-modular construct). Furthermore, we study the impact of mutations in the A or PCP domains in these various constructs. Our results suggest that substrate adenylation and effects of mutations largely depend on the context in which the domains/modules are. Therefore, generalizing properties observed for domains or modules in isolation should be done with caution.
Highlights
Non-ribosomal peptide synthetases (NRPSs) are modular mega-enzymes that produce peptides independently of the ribosome
This diversity is achieved in numerous ways, some of which include: (i) A domain specificity is not limited to proteinogenic amino acids; (ii) initial C domains can incorporate fatty acids; (iii) auxiliary domains can modify the building blocks along the way and (iv) the TE domain can release the product as a cyclic peptide
From studies carried out on individual modules extracted from multi-modular NRPSs, the A domain was reported to adopt three different conformations: (i) an open conformation, where no ligand is bound; (ii) an adenylation conformation, where the small C-terminal Asub domain is reconfigured by ~48° to complex with ATP and the substrate to perform the adenylation reaction; and, (iii) a thiolation state, in which the C-terminal Asub domain is rotated by ~140° around the large N-terminal Acore to tether the activated substrate to the PPE arm of the peptidyl carrier protein (PCP) domain[13,14,15,16]
Summary
Non-ribosomal peptide synthetases (NRPSs) are modular mega-enzymes that produce peptides independently of the ribosome. From studies carried out on individual modules extracted from multi-modular NRPSs, the A domain was reported to adopt three different conformations: (i) an open conformation, where no ligand is bound; (ii) an adenylation conformation, where the small C-terminal Asub domain is reconfigured by ~48° to complex with ATP and the substrate to perform the adenylation reaction; and, (iii) a thiolation state, in which the C-terminal Asub domain is rotated by ~140° around the large N-terminal Acore to tether the activated substrate to the PPE arm of the PCP domain[13,14,15,16] This A domain alternation mechanism is expected to contribute most to the NRP elongation process. It is licit to ask whether there is an intrinsic communication between modules that ensures proper directionality and progression of NRP synthesis and peptide identity
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