Structure of a Stand-Alone Homodimeric Nonribosomal Peptide Synthetase Condensation Domain Reveals Occlusion of the Canonical Carrier-Protein Interface

Abstract Fatty acid amides (FAAs) produced by gut-resident bacteria act as potent modulators of host G-protein coupled receptor signaling, yet the enzymatic mechanisms underlying their biosynthesis remain poorly understood. In many bacteria from the gut microbiome, including Coprococcus eutactus, FAA production is mediated by a nonribosomal peptide synthetase (NRPS)–like pathway that includes OaaC, a free-standing condensation domain that catalyzes amide bond formation between acyl carrier protein (ACP) tethered fatty acids and small-molecule amine acceptors. Here, we combine structural, biophysical, biochemical, and evolutionary analyses to interrogate the molecular basis of OaaC function. Solution scattering and X-ray crystallography reveal that OaaC adopts an atypical homodimeric architecture that occludes the canonical ACP-binding surface and donor access pathways. Mass photometry demonstrates that this homodimer is stable in the absence of substrates and is insensitive to free fatty acids, free amines, and apo-ACP. In contrast, holo or acyl-loaded OaaACP selectively destabilizes the homodimer forming the OaaC-OaaACP complex population. LC–MS reconstitution assays confirm that OaaC catalyzes fatty acid amide formation in vitro and can utilize acyl donors spanning multiple chain lengths and saturation states. Phylogenetic and sequence analyses place FAA-associated condensation domains in a distinct clade most closely related to starter condensation domains and reveal a conserved noncanonical active site motif that differentiates them from PCP-dependent NRPS condensation domains. Together, these findings support a model in which OaaC activity is regulated through substrate-dependent modulation of oligomeric state, providing a model framework for understanding FAA biosynthesis in gut microbes and expanding the known functional diversity of NRPS condensation domains.