State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi, 214122, People's Republic of China; School of Food Science and Technology, Jiangnan University, Wuxi, 214122, PR China.
State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi, 214122, People's Republic of China; School of Food Science and Technology, Jiangnan University, Wuxi, 214122, PR China; National Engineering Research Center for Functional Food, Jiangnan University, Wuxi, 214122, PR China; Wuxi Translational Medicine Research Center and Jiangsu Translational Medicine Research Institute Wuxi Branch, Wuxi, 214122, PR China.
Biochem Biophys Res Commun. 2022 Jan 1;586:74-80. doi: 10.1016/j.bbrc.2021.11.050. Epub 2021 Nov 20.
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 and MpFADS6 abolished desaturation, while MpFADS6 and MpFADS6 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.
脂肪酸去饱和酶（FADS）具有高度的选择性，可在相应多不饱和脂肪酸（PUFAs）的特定位置生成双键，其酶活性和 PUFAs 产物对生物系统至关重要，并与多种生理疾病相关。目前人们对 FADS 的结构及其与催化活性相关的氨基酸残基知之甚少。确定 Micromonas pusilla delta 6 去饱和酶（MpFADS6）的关键残基为深入了解去饱和作用提供了一个起点。在这项研究中，通过基因共识分析锚定保守氨基酸，从而通过定点诱变生成相应的变体。为了在酿酒酵母中稳定高效地表达 MpFADS6 及其变体，优化了诱导表达的关键点。通过分析变体的酶活性来确定保守残基对酶功能的贡献。分子建模表明，这些残基对催化活性或底物结合至关重要。突变体 MpFADS6 和 MpFADS6 丧失了去饱和作用，而 MpFADS6 和 MpFADS6 显著降低了催化活性。鉴于某些残基已被确定对 MpFADS6 活性有重大影响，因此提出 FADS6 的组氨酸保守区 III 与去饱和过程中的电子转移有关，而组氨酸保守区 I 和 II 与去饱和有关。这些发现为确定膜结合去饱和酶的结构、机制和定向转化提供了新的见解和方法。
