BPS2026 – Structural and biochemical characterization of a standalone homodimeric non-ribosomal peptide synthetase from gut bacteria

Non-ribosomal peptide synthetases (NRPSs) synthesize natural products including antibiotics and siderophores. Recently, an NRPS-like pathway was discovered in human gut bacteria that synthesizes fatty acid amides (FAAs), which affect GPCRs. This FAA system features a unique minimalistic architecture where each domain exists independently and lacks an acceptor peptidyl carrier protein (PCP). We performed solution scattering and crystallography to reveal that the FAA condensation domain (OaaC) from Coprococcus eutactus forms an atypical homodimer that occludes the donor acyl carrier protein (OaaACP) binding site predicted from conventional multidomain NRPS structures. To understand OaaC homodimer-OaaACP interaction, we employed mass photometry with apo, holo, and loaded OaaACP states. Strong dimer dissociation occurred only with loaded OaaACP, suggesting a regulatory mechanism whereby FAA NRPS systems ensure only functional ACPs bind C domains. To understand how this non-canonical system accommodates these structural differences, we examined catalytic motifs. While canonical NRPS condensation domains contain the HHxxxDG motif, OaaC contains DHxxxDS. Liganded OaaC crystal structures revealed fourth position serine interactions with the carboxyl group of acceptor substrate 5-aminovaleric acid. We investigated whether condensation domains using small-molecule acceptors not bound to acceptor PCPs correlate with glycine substitution at the fourth catalytic position. Analysis of C domain sequences from various NRPS systems revealed a clear correlation: FAA C domains and others interacting directly with small-molecule amines lack fourth-position glycine, while canonical NRPS C domains using acceptor PCPs predominantly retain glycine. These findings provide insights into structural adaptations driving substrate specificity in non-canonical NRPS systems and may inform engineering efforts to produce novel FAA bioactive products with therapeutic potential.