| 刘春妮,肖菲,李文利.粉蝶霉素糖基转移酶GT1507受体结合口袋关键残基探究[J].中国海洋药物,2025,44(1):49-56. |
| 粉蝶霉素糖基转移酶GT1507受体结合口袋关键残基探究 |
| Probing the active residues in acceptor binding pocket of Piericidin-glycosyltransferase GT1507 |
| 投稿时间：2023-05-12 修订日期：2023-06-16 |
| DOI：10.13400/j.cnki.cjmd.2025.01.009 |
| 中文关键词: 糖基化修饰 糖基转移酶 粉蝶霉素 |
| English Keywords:glycosylation modification glycosyltransferases Piericidin A1 |
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| 中文摘要: |
| 目的：粉蝶霉素类化合物具有良好的抗菌、抗肿瘤等活性,糖基化修饰显著影响其生物活性。本研究旨在探究海洋链霉菌OUC6819中粉蝶霉素类化合物糖基转移酶GT1507受体结合口袋中关键活性残基,为将GT1507开发为工具酶提供理论基础。方法：采用多序列比对方式分析GT1507及其同源蛋白的受体结合位点；通过定点突变构建GT1507的突变体酶；随后采用高效液相色谱检测不同突变体酶催化粉蝶霉素(Piericidin A1, PA)加载葡萄糖基和N-乙酰葡萄糖胺基团的效率。结果：Nβ3-Nα3区域内的L80F和Nβ5-Nα5区域内V182F催化PA加载葡萄糖基活性显著降低,Y139F和A159E与野生型酶催化活性相当；当以UDP-N-乙酰葡萄糖胺为糖基供体时,突变体酶L80F的催化效率提高,V182F的活性与野生型相当,而Y139F和A159E催化活性显著降低。结论：Leu80、Tyr139、Ala159和Val182是GT1507与受体结合相关的氨基酸,不同糖基供体与蛋白的结合会影响受体结合口袋的关键氨基酸,从而影响突变体酶的催化效率,以上研究为GT1507进一步酶工程改造提供了理论基础。 |
| English Summary: |
| Objective: Piericidin has good antibacterial, antifungal and antitumor activities, and glycosylation modification significantly affects its biological activities. This study aimed to explore the active residues in the acceptor binding pocket of the piericidin-glycosyltransferase GT1507 in Streptomyces youssoufiensis OUC6819 and to provide a theoretical basis for the development of GT1507 as a tool enzyme. Methods: The residues related to acceptor binding were selected based on the multiple sequence alignment of GT1507 and its homolog. The mutants of GT1507 were constructed by site-directed mutagenesis. The efficiency of different mutants catalyzing the loading of glucosyl and N-acetylglucosamine on Piericidin A1 (PA) was detected by high-performance liquid chromatography. Results: Leu80 in Nβ3-Nα3 region and V182F in Nβ5-Nα5 region significantly reduced the catalytic activity of PA loading glucose group, and Tyr139 and Ala159 were equivalent to wild-type enzyme catalytic activity. When UDP-N-acetylglucosamine was used as a glycosyl donor, the catalytic efficiency of the mutant L80F was improved, the activity of V182F was comparable to that of the wild type, while the catalytic activity of Y139F and A159E was significantly reduced. Conclusion: Leu80, Tyr139, Ala159 and Val182 are the key amino acids for GT1507 receptor binding. Different glycosyl donors change the key amino acids of receptor binding by affecting the offset angle of acceptor pocket. The above research provides a theoretical basis for further enzyme engineering of GT1507. |
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