Abstract
Enzyme catalyzed desaturation of intracellular fatty acids plays an important role in various physiological and pathological processes related to lipids. Limited to the multiple transmembrane domains, it is difficult to obtain their three-dimensional structure of fatty acid desaturases. So how they interact with their substrates is unclear. Here, we predicted the complex of Micromonas pusilla delta 6 desaturase (MpFADS6) with the substrate linoleinyl-CoA (ALA-CoA) by trRosetta software and docking poses by Dock 6 software. The potential enzyme-substrate binding sites were anchored by analysis of the complex. Then, site-directed mutagenesis and activity verification clarified that W290, W224, and F352 were critical residues of the substrate tunnel and directly bonded to ALA-CoA. H94 and H69 were indispensable for transporting electrons with heme. H452, N445, and H358 significantly influenced the recognition and attraction of MpFADS6 to the substrate. These findings provide new insights and methods to determine the structure, mechanisms and directed transformation of membrane-bound desaturases.