Structural characterization of a PCP-R di-domain from an archaeal non-ribosomal peptide synthetase reveals novel inter-domain interactions.

Non-ribosomal peptide synthetases (NRPSs) are multi-modular enzymes that produce a wide range of bio-active peptides such as siderophores, toxins, and antibacterial and insecticidal agents. NRPSs are dynamic proteins characterized by extensive inter-domain communications as a consequence of their assembly-line mode of synthesis. Hence, crystal structures of multi-domain fragments of NRPSs have aided in elucidating crucial inter-domain interactions that occur during different steps of the NRPS catalytic cycle. One crucial yet unexplored interaction is that between the reductase (R) domain and the peptide carrier protein (PCP) domain. Reductase domains are members of the short chain dehydrogenase/reductase (SDR) family and function as termination domains that catalyze the reductive release of the final peptide product from the terminal PCP domain of the NRPS. Here we report the crystal structure of an archaeal NRPS PCP-R di-domain construct. This is the first NRPS reductase domain structure to be determined together with the upstream PCP domain and is also the first structure of an archaeal NRPS to be reported. The structure reveals that a novel helix-turn-helix motif, found in NRPS reductase domains but not in other SDR family members, plays a major role in the interface between the PCP and reductase domains. The information derived from the described PCP-R interface will aid in gaining further mechanistic insights into the peptide termination reaction catalyzed by the reductase domain and may have implications in engineering NRPSs to synthesize novel peptide products.