Singh, J.; Grant, T. D.; Gulick, A. M.

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-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.