Thraustochytrid PUFA synthase ER domains form a stable heterodimer

Omega-3 polyunsaturated fatty acids (PUFAs) are essential nutrients for humans and are synthesized de novo by specialized enzymes known as PUFA synthases (Pfas). The domains of these enzymes are structurally related to those of mammalian or bacterial fatty acid synthases (FAS), as well as microbial polyketide synthases (PKS). Pfas are typically composed of three polypeptides in thraustochytrids and myxobacteria, or four in marine gammaproteobacteria. The enoyl-ACP reductase (ER) domain plays a key role in PUFA synthesis by catalyzing the reduction of carbon‑carbon double bonds during modification reactions. In gammaproteobacteria, a single ER domain is present as a standalone protein (PfaD) within the megasynthase. However, in thraustochytrids ER domains are found in both PfaB (ERb) and PfaC (ERc), although their specific functional roles remain unclear. Previous studies have shown that ER domains act as homodimers in Pfas, FAS, and PKS systems. Here, we investigate the PUFA synthase from the thraustochytrid Schizochytrium sp. and demonstrate that ERb and ERc interact to form a heterodimer, as confirmed by size-exclusion chromatography with multi-angle light scattering (SEC-MALS) and by a crystal structure solved at 2.2 Å resolution with bound flavin mononucleotide (FMN). These findings indicate that ER domains may facilitate dimerization between PfaB and PfaC. Furthermore, molecular docking and structural alignments support a ping-pong mechanism involving FMN and NADH for ERb and ERc activity. To our knowledge, this is the first reported crystal structure of a PUFA synthase ER-domain complex from thraustochytrids, providing new insights into the mechanism of action of these enzymes.