Determination of FAT-1 desaturase activity and substrate preference

n-3 polyunsaturated fatty acids (n-3 PUFAs) possess numerous health benefits. The FAT-1 desaturase in the model organism Caenorhabditis elegans is a Δ15-desaturase that converts n-6 PUFAs into n-3 PUFAs. Transgenic expression of FAT-1 has been used in organisms such as pigs, mice, and fish to improve n-3 PUFA levels. However, the determination of FAT-1 activity and substrate preference per se remains unclear. AlphaFold structure prediction revealed that FAT-1 is an integral membrane protein located in the endoplasmic reticulum, and it consists of four transmembrane helices (TM1-4) with a functional CYTB5 domain in the N-terminus and a desaturase domain containing three histidine-rich sequences (His boxes) in the C-terminus. A small region in the desaturase domain containing amino acids 210-217, especially G212, G216, and S217, are essential for its activity. FAT-1 can convert all four n-6 PUFAs to corresponding or downstream n-3 PUFAs in both C. elegans and mammalian cells, and may prioritize the conversion of C20:4n6 (arachidonic acid, ARA) to C20:5n3 (eicosapentaenoic acid, EPA). These results uncover the significant mechanism of the activity and substrate preference of the FAT-1 desaturase, providing insights into the transgenic application of FAT-1.