利用片段置换法合理设计高活性 N-糖基转移酶突变体

Jiangyu Yang , Kun Li , Yongheng Rong , Zhaoxi Liu , Xiaoyu Liu , Yue Yu , Wenjing Shi , Yun Kong , Min Chen
{"title":"利用片段置换法合理设计高活性 N-糖基转移酶突变体","authors":"Jiangyu Yang ,&nbsp;Kun Li ,&nbsp;Yongheng Rong ,&nbsp;Zhaoxi Liu ,&nbsp;Xiaoyu Liu ,&nbsp;Yue Yu ,&nbsp;Wenjing Shi ,&nbsp;Yun Kong ,&nbsp;Min Chen","doi":"10.1016/j.engmic.2023.100134","DOIUrl":null,"url":null,"abstract":"<div><p>The modularity of carbohydrate-active enzymes facilitates that enzymes with different functions have similar fragments. However, because of the complex structure of the enzyme active sites and the epistatic effects of various mutations on enzyme activity, it is difficult to design enzymes with multiple mutation sites using conventional methods. In this study, we designed multi-point mutants by fragment replacement in the donor-acceptor binding pocket of <em>Actinobacillus pleuropneumoniae N</em>-glycosyltransferase (ApNGT) to obtain novel properties. Candidate fragments were selected from a customized glycosyltransferase database. The stability and substrate-binding energy of the three fragment replacement mutants were calculated in comparison with wild-type ApNGT, and mutants with top-ranking stability and middle-ranking substrate-binding energy were chosen for priority experimental verification. We found that a mutant called F13, which increased the glycosylation efficiency of the natural substrate by 1.44 times, the relative conversion of UDP-galactose by 14.2 times, and the relative conversion of UDP-xylose from almost 0 to 78.6%. Most importantly, F13 mutant acquired an entirely new property, the ability to utilize UDP-glucuronic acid. On one hand, this work shows that replacing similar fragments in the donor-acceptor binding pocket of the enzyme might provide new ideas for designing mutants with new properties; on the other hand, F13 mutant is expected to play an important role in targeted drug delivery.</p></div>","PeriodicalId":100478,"journal":{"name":"Engineering Microbiology","volume":"4 1","pages":"Article 100134"},"PeriodicalIF":0.0000,"publicationDate":"2023-11-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2667370323000668/pdfft?md5=9d882243453dbe21a97fd045b480ba38&pid=1-s2.0-S2667370323000668-main.pdf","citationCount":"0","resultStr":"{\"title\":\"Rational design of a highly active N-glycosyltransferase mutant using fragment replacement approach\",\"authors\":\"Jiangyu Yang ,&nbsp;Kun Li ,&nbsp;Yongheng Rong ,&nbsp;Zhaoxi Liu ,&nbsp;Xiaoyu Liu ,&nbsp;Yue Yu ,&nbsp;Wenjing Shi ,&nbsp;Yun Kong ,&nbsp;Min Chen\",\"doi\":\"10.1016/j.engmic.2023.100134\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><p>The modularity of carbohydrate-active enzymes facilitates that enzymes with different functions have similar fragments. However, because of the complex structure of the enzyme active sites and the epistatic effects of various mutations on enzyme activity, it is difficult to design enzymes with multiple mutation sites using conventional methods. In this study, we designed multi-point mutants by fragment replacement in the donor-acceptor binding pocket of <em>Actinobacillus pleuropneumoniae N</em>-glycosyltransferase (ApNGT) to obtain novel properties. Candidate fragments were selected from a customized glycosyltransferase database. The stability and substrate-binding energy of the three fragment replacement mutants were calculated in comparison with wild-type ApNGT, and mutants with top-ranking stability and middle-ranking substrate-binding energy were chosen for priority experimental verification. We found that a mutant called F13, which increased the glycosylation efficiency of the natural substrate by 1.44 times, the relative conversion of UDP-galactose by 14.2 times, and the relative conversion of UDP-xylose from almost 0 to 78.6%. Most importantly, F13 mutant acquired an entirely new property, the ability to utilize UDP-glucuronic acid. On one hand, this work shows that replacing similar fragments in the donor-acceptor binding pocket of the enzyme might provide new ideas for designing mutants with new properties; on the other hand, F13 mutant is expected to play an important role in targeted drug delivery.</p></div>\",\"PeriodicalId\":100478,\"journal\":{\"name\":\"Engineering Microbiology\",\"volume\":\"4 1\",\"pages\":\"Article 100134\"},\"PeriodicalIF\":0.0000,\"publicationDate\":\"2023-11-30\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://www.sciencedirect.com/science/article/pii/S2667370323000668/pdfft?md5=9d882243453dbe21a97fd045b480ba38&pid=1-s2.0-S2667370323000668-main.pdf\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Engineering Microbiology\",\"FirstCategoryId\":\"1085\",\"ListUrlMain\":\"https://www.sciencedirect.com/science/article/pii/S2667370323000668\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"\",\"JCRName\":\"\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Engineering Microbiology","FirstCategoryId":"1085","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S2667370323000668","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"","JCRName":"","Score":null,"Total":0}
引用次数: 0

摘要

碳水化合物活性酶的模块性使具有不同功能的酶具有相似的片段。然而,由于酶活性位点结构复杂,各种突变对酶活性的影响具有表观效应,因此很难用传统方法设计出具有多个突变位点的酶。在这项研究中,我们通过在胸膜肺炎放线杆菌 N-糖基转移酶(ApNGT)的供体-受体结合袋中进行片段置换,设计出多点突变体,以获得新的特性。候选片段是从定制的糖基转移酶数据库中筛选出来的。与野生型 ApNGT 相比,我们计算了三个片段置换突变体的稳定性和底物结合能,并选择了稳定性排名第一、底物结合能排名居中的突变体优先进行实验验证。我们发现,一个名为F13的突变体,其天然底物的糖基化效率提高了1.44倍,UDP-半乳糖的相对转化率提高了14.2倍,UDP-木糖的相对转化率从几乎为0提高到78.6%。最重要的是,F13 突变体获得了一种全新的特性,即利用 UDP-葡萄糖醛酸的能力。这项工作一方面表明,替换酶的供体-受体结合口袋中的相似片段可能为设计具有新特性的突变体提供新思路;另一方面,F13突变体有望在靶向给药方面发挥重要作用。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

Rational design of a highly active N-glycosyltransferase mutant using fragment replacement approach

Rational design of a highly active N-glycosyltransferase mutant using fragment replacement approach

The modularity of carbohydrate-active enzymes facilitates that enzymes with different functions have similar fragments. However, because of the complex structure of the enzyme active sites and the epistatic effects of various mutations on enzyme activity, it is difficult to design enzymes with multiple mutation sites using conventional methods. In this study, we designed multi-point mutants by fragment replacement in the donor-acceptor binding pocket of Actinobacillus pleuropneumoniae N-glycosyltransferase (ApNGT) to obtain novel properties. Candidate fragments were selected from a customized glycosyltransferase database. The stability and substrate-binding energy of the three fragment replacement mutants were calculated in comparison with wild-type ApNGT, and mutants with top-ranking stability and middle-ranking substrate-binding energy were chosen for priority experimental verification. We found that a mutant called F13, which increased the glycosylation efficiency of the natural substrate by 1.44 times, the relative conversion of UDP-galactose by 14.2 times, and the relative conversion of UDP-xylose from almost 0 to 78.6%. Most importantly, F13 mutant acquired an entirely new property, the ability to utilize UDP-glucuronic acid. On one hand, this work shows that replacing similar fragments in the donor-acceptor binding pocket of the enzyme might provide new ideas for designing mutants with new properties; on the other hand, F13 mutant is expected to play an important role in targeted drug delivery.

求助全文
通过发布文献求助,成功后即可免费获取论文全文。 去求助
来源期刊
CiteScore
3.90
自引率
0.00%
发文量
0
×
引用
GB/T 7714-2015
复制
MLA
复制
APA
复制
导出至
BibTeX EndNote RefMan NoteFirst NoteExpress
×
提示
您的信息不完整,为了账户安全,请先补充。
现在去补充
×
提示
您因"违规操作"
具体请查看互助需知
我知道了
×
提示
确定
请完成安全验证×
copy
已复制链接
快去分享给好友吧!
我知道了
右上角分享
点击右上角分享
0
联系我们:info@booksci.cn Book学术提供免费学术资源搜索服务,方便国内外学者检索中英文文献。致力于提供最便捷和优质的服务体验。 Copyright © 2023 布克学术 All rights reserved.
京ICP备2023020795号-1
ghs 京公网安备 11010802042870号
Book学术文献互助
Book学术文献互助群
群 号:481959085
Book学术官方微信