Gusenda_2025_Protein.Sci_34_e70229

Branched-chain fatty acids (BCFAs) exhibit enhanced oxidative stability, lower melting points, and reduced viscosity compared to straight-chain fatty acids (StCFAs), making them valuable for biological systems and industrial applications. Previous studies have shown that metazoan fatty acid synthase (mFAS) can produce BCFAs through the incorporation of branched starter units and branched extender substrates such as methylmalonyl-CoA (metmal-CoA). However, the mechanistic and kinetic underpinnings of BCFA biosynthesis in metazoans remain poorly understood. Here, we address this by characterizing the kinetic parameters of mFAS-catalyzed BCFA synthesis using NADPH consumption assays and analyzing the synthesized products via GC-MS. These experiments revealed a lower turnover number of mFAS with metmal-CoA and a shift toward medium-chain fatty acids compared to the native StCFA biosynthesis with malonyl-CoA. The ketoacyl synthase (KS) kinetic measurements revealed low elongation rates, indicating that the KS domain dictates the substrate specificity and speed of BCFA production. To gain a better understanding of the reaction mechanism, we performed molecular dynamic simulations, which revealed key KS: substrate interactions by conserved threonine residues. These insights support a refined decarboxylative condensation mechanism and illuminate how substrate specificity arises within mFAS. These findings offer a foundation for future protein engineering and inhibitor design strategies.