Consensus mutagenesis and computational simulation provide insight into the desaturation catalytic mechanism for delta 6 fatty acid desaturase

Fatty acid desaturase (FADS) generates double bond at a certain position of the corresponding polyunsaturated fatty acids (PUFAs) with high selectivity, the enzyme activity and PUFAs products of which are essential to biological systems and are associated with a variety of physiological diseases. Little is known about the structure of FADSs and their amino acid residues related to catalytic activities. Identifying key residues of Micromonas pusilla delta 6 desaturase (MpFADS6) provides a point of departure for a better understanding of desaturation. In this study, conserved amino acids were anchored through gene consensus analysis, thereby generating corresponding variants by site-directed mutagenesis. To achieve stable and high-efficiency expression of MpFADS6 and its variants in Saccharomyces cerevisiae, the key points of induced expression were optimized. The contribution of conserved residues to the function of enzyme was determined by analyzing enzyme activity of the variants. Molecular modeling indicated that these residues are essential to catalytic activities, or substrate binding. Mutants MpFADS6([Q409R]) and MpFADS6([M242P]) abolished desaturation, while MpFADS6([F419V]) and MpFADS6([A374Q]) significantly reduced catalytic activities. Given that certain residues have been identified to have a significant impact on MpFADS6 activities, it is put forward that histidine-conserved region III of FADS6 is related to electronic transfer during desaturation, while histidine-conserved regions I and II are related to desaturation. These findings provide new insights and methods to determine the structure, mechanism and directed transformation of membrane-bound desaturases. (C) 2021 Elsevier Inc. All rights reserved.

Automated summary

Fatty acid desaturase (FADS) is crucial in generating double bonds in polyunsaturated fatty acids (PUFAs) and its activity is associated with various physiological diseases. In this study, the key residues of MpFADS6 were identified and their contributions to enzyme function were investigated. The results provide new insights into the structure, mechanism, and directed transformation of membrane-bound desaturases.