Influence of condensation domains on activity and specificity of adenylation domains

Abstract

Many clinically used natural products are produced by non-ribosomal peptide synthetases (NRPSs), which due to their modular nature should be accessible to modification and engineering approaches. While the adenylation domain (A) plays the key role in substrate recognition and activation, the condensation domain (C) which is responsible for substrate linkage and stereochemical filtering recently became the subject of debate - with its attributed role as a "gatekeeper" being called into question. Since we have thoroughly investigated different combinations of C-A didomains in a series of in vitro, in vivo, and in situ experiments suggesting an important role to the C-A interface for the activity and specificity of the downstream A domain and not the C domain as such, we would like to contribute to this discussion. The role of the C-A interface, termed extended gatekeeping, due to structural features of the C domains, can also be transferred to other NRPSs by engineering, was finally investigated and characterised in an in silico approach on 30 wild-type and recombinant C-A interfaces. With these data, we not only would like to offer a new perspective on the specificity of C domains, but also to revise our previously established NRPS engineering and construction rules.