The State Key Laboratory of Microbial Technology, National Glycoengineering Research Centers, Shandong University, Qingdao, Shandong 266237, China.
College of Life Sciences, Shandong Normal University, Jinan, Shandong 250013, China.
Enzyme Microb Technol. 2022 Mar;154:109949. doi: 10.1016/j.enzmictec.2021.109949. Epub 2021 Nov 25.
N-glycosylation is one of the most important post-translational modifications of proteins. Cytoplasmic soluble N-glycosyltransferase (NGT) exists in bacteria, which is able to transfer different monosaccharide from sugar nucleotide to the NXS/T(X ≠ Pro) consensus sequence in a polypeptide. At present, the NGT enzymes reported could transfer a variety of different sugars to protein, which will lead to the heterogeneity of the sugar chain and the complexity and instability of the structure and function of glycopeptides. According to the FuncLib algorithm, we obtained mutant ApNGT-P1 from ApNGT (the NGT from Actinobacillus pleuropneumoniae) with increased substrate specificity. Compared with the wild-type ApNGT, mutant ApNGT-P1 could only utilize UDP-Glc as sugar donors. The optimum temperature of ApNGT-P1 was about 40 °C and the optimum pH was 7.5-8.0 in PBS buffer. ApNGT-P1 exhibited better tolerance for K, Mn, Ca, and Mg, but was strongly inhibited by Na, Cu and Zn. The mutant can be applied to the efficient production of glycosylated peptides or proteins with uniform glucose at their glycosylation sites. Besides, this work provided a feasible pathway for further studies on the improving donor substrates selectivity of NGTs.
N-糖基化是蛋白质最重要的翻译后修饰之一。细胞质可溶性 N-糖基转移酶（NGT）存在于细菌中，它能够将不同的单糖从糖核苷酸转移到多肽中的 NXS/T（X≠脯氨酸）共有序列。目前，已报道的 NGT 酶可以将各种不同的糖转移到蛋白质上，这将导致糖链的异质性以及糖肽结构和功能的复杂性和不稳定性。根据 FuncLib 算法，我们从胸膜肺炎放线杆菌（Actinobacillus pleuropneumoniae）的 NGT（即 NGT）中获得了具有增加的底物特异性的突变体 ApNGT-P1。与野生型 ApNGT 相比，突变体 ApNGT-P1 只能利用 UDP-Glc 作为糖供体。ApNGT-P1 的最适温度约为 40°C，在 PBS 缓冲液中的最适 pH 值为 7.5-8.0。ApNGT-P1 对 K、Mn、Ca 和 Mg 表现出更好的耐受性，但强烈抑制 Na、Cu 和 Zn。该突变体可用于在糖基化位点高效生产具有均匀葡萄糖的糖基化肽或蛋白质。此外，这项工作为进一步研究 NGT 对供体底物选择性的改善提供了可行的途径。
