Yang Jiangyu, Li Kun, Rong Yongheng, Liu Zhaoxi, Liu Xiaoyu, Yu Yue, Shi Wenjing, Kong Yun, Chen Min
State Key Laboratory of Microbial Technology, Shandong University, Qingdao 266237, China.
National Glycoengineering Research Center, Shandong University, Qingdao 266237, China.
Eng Microbiol. 2023 Nov 30;4(1):100134. doi: 10.1016/j.engmic.2023.100134. eCollection 2024 Mar.
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 -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.
碳水化合物活性酶的模块化使得具有不同功能的酶具有相似的片段。然而，由于酶活性位点的结构复杂以及各种突变对酶活性的上位效应，使用传统方法设计具有多个突变位点的酶很困难。在本研究中，我们通过在α-糖基转移酶（ApNGT）的供体-受体结合口袋中进行片段替换来设计多点突变体，以获得新特性。候选片段从定制的糖基转移酶数据库中选择。与野生型ApNGT相比，计算了三个片段替换突变体的稳定性和底物结合能，并选择稳定性排名靠前且底物结合能排名中等的突变体进行优先实验验证。我们发现一个名为F13的突变体，它将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。一方面，这项工作表明在酶的供体-受体结合口袋中替换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突变体有望在靶向药物递送中发挥重要作用。
