State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, China.
School of Food Science and Technology, Jiangnan University, Wuxi 214122, China.
Int J Mol Sci. 2019 Mar 30;20(7):1603. doi: 10.3390/ijms20071603.
Although various ω-3 fatty acid desaturases (ω3Des) have been identified and well-studied regarding substrate preference and regiospecificity, the molecular mechanism of their substrate specificities remains to be investigated. Here we compared two ω3Des, FADS15 from and oRiFADS17 from , which possessed a substrate preference for linoleic acid and arachidonic acid, respectively. Their sequences were divided into six sections and a domain-swapping strategy was used to test the role of each section in catalytic activity. Heterologous expression and fatty acid experiments of hybrid enzymes in indicated that the sequences between his-boxes I and II played critical roles in influencing substrate preference. Based on site-directed mutagenesis and molecular docking, the amino acid substitutions W129T and T144W, located in the upper part of the hydrocarbon chain, were found to be involved in substrate specificity, while V137T and V152T were confirmed to interfere with substrate recognition. This study provides significant insight into the structure-function relationship of ω3Des.
虽然已经鉴定出了各种ω-3 脂肪酸去饱和酶（ω3Des），并且针对其底物偏好和区域特异性进行了很好的研究，但它们的底物特异性的分子机制仍有待研究。在这里，我们比较了两种ω3Des，一种来自 的 FADS15 和另一种来自 的 oRiFADS17，它们分别对亚油酸和花生四烯酸具有底物偏好。它们的序列被分为六个部分，并使用结构域交换策略来测试每个部分在催化活性中的作用。在 中异源表达和脂肪酸实验表明，His 框 I 和 II 之间的序列在影响底物偏好方面起着关键作用。基于定点突变和分子对接，发现位于烃链上部的氨基酸替换 W129T 和 T144W 参与了底物特异性，而 V137T 和 V152T 被证实干扰了底物识别。这项研究为 ω3Des 的结构-功能关系提供了重要的见解。
