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

Publisher: Elsevier Country of Publication: United States NLM ID: 0372516 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1090-2104 (Electronic) Linking ISSN: 0006291X NLM ISO Abbreviation: Biochem Biophys Res Commun Subsets: MEDLINE

Publication: <2002- >: San Diego, CA : Elsevier
Original Publication: New York, Academic Press.

Molecular Docking Simulation*; Algal Proteins/*chemistry ; Chlorophyta/*enzymology ; Fatty Acid Desaturases/*chemistry ; Linoleic Acid/*chemistry ; Saccharomyces cerevisiae/*genetics; Algal Proteins/genetics ; Algal Proteins/metabolism ; Amino Acid Sequence ; Biocatalysis ; Catalytic Domain ; Chlorophyta/chemistry ; Cloning, Molecular ; Fatty Acid Desaturases/genetics ; Fatty Acid Desaturases/metabolism ; Gene Expression ; Genetic Vectors/chemistry ; Genetic Vectors/metabolism ; Linoleic Acid/metabolism ; Mutagenesis, Site-Directed ; Protein Binding ; Protein Conformation ; Protein Interaction Domains and Motifs ; Recombinant Proteins/chemistry ; Recombinant Proteins/genetics ; Recombinant Proteins/metabolism ; Saccharomyces cerevisiae/enzymology ; Sequence Alignment ; Sequence Homology, Amino Acid ; Substrate Specificity

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.
Competing Interests: Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
(Copyright © 2021 Elsevier Inc. All rights reserved.)

Keywords: Desaturation; Enzymatic activity; Ligand binding site; Molecular docking; Site-directed mutagenesis

0 (Algal Proteins)
0 (Recombinant Proteins)
9KJL21T0QJ (Linoleic Acid)
EC 1.14.19.- (Fatty Acid Desaturases)

Date Created: 20211127 Date Completed: 20220112 Latest Revision: 20220112

20221213

10.1016/j.bbrc.2021.11.050

34837835