Department of Life Science, College of Natural Sciences, Hanyang University, Seoul 04763, Korea.
Eugene Biotech Co. Ltd., Daejeon 34051, Korea.
Mar Drugs. 2018 Nov 1;16(11):418. doi: 10.3390/md16110418.
The mutant of marine microalga accumulates zeaxanthin under normal growth conditions, and its phenotype has been speculated to be related to zeaxanthin epoxidase (ZEP). In this study, we isolated the ZEP gene from both wild-type and the mutant. We found that the mutant has a point mutation of the 1337th nucleotide of the ZEP sequence (a change from guanine to adenine), resulting in a change of glycine to aspartate in a highly conserved region in the catalytic domain. Similar expression levels of ZEP mRNA and protein in both wild-type and were confirmed by using qRT-PCR and western blot analysis, respectively. Additionally, the enzyme activity analysis of ZEPs in the presence of cofactors showed that the inactivation of ZEP in was not caused by deficiency in the levels of cofactors. From the predicted three-dimensional ZEP structure of , we observed a conformational change on the substrate-binding site in the ZEP. A comparative analysis of the ZEP structures suggested that the conformational change induced by a single amino acid mutation might impact the interaction between the substrate and substrate-binding site, resulting in loss of zeaxanthin epoxidase function.
海洋微藻突变体在正常生长条件下积累玉米黄质，其表型被推测与玉米黄质环氧化酶（ZEP）有关。在本研究中，我们从野生型和突变体中分离出 ZEP 基因。我们发现突变体在 ZEP 序列的第 1337 个核苷酸处发生点突变（由鸟嘌呤变为腺嘌呤），导致催化结构域中高度保守区域的甘氨酸变为天冬氨酸。通过 qRT-PCR 和 Western blot 分析分别证实野生型和突变体中 ZEP mRNA 和蛋白的表达水平相似。此外，在存在辅因子的情况下对 ZEPs 的酶活性分析表明，突变体中 ZEP 的失活不是由于辅因子水平的缺乏所致。从突变体 ZEP 的预测三维结构中，我们观察到在底物结合位点上的构象变化。对 ZEP 结构的比较分析表明，单个氨基酸突变引起的构象变化可能影响底物与底物结合位点之间的相互作用，导致玉米黄质环氧化酶功能丧失。
