Daisuke Fujinami*, Ayumi Mizui, Azusa Miyata and Sohei Ito*,
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引用次数: 0
摘要
糖基转移酶在糖蛋白和糖治疗药物的生物合成过程中发挥着重要作用。在这项研究中,我们研究了属于 GT2 家族的蛋白糖基转移酶 FlgGT1。GT2 家族包括参与抗菌肽生物合成的半胱氨酸 S-糖基转移酶,它们共享保守的催化结构域,同时表现出不同的 C 端结构域。我们的体外研究发现,FlgGT1 在糖基化鞭毛蛋白 Hag 时识别的是结构基序而不是特定的氨基酸序列。值得注意的是,FlgGT1 选择丝氨酸或苏氨酸 O 型糖基化,而不是半胱氨酸 S 型糖基化。分子动力学模拟揭示了 FlgGT1 能够容纳各种糖核苷酸作为供体底物的结构基础。对 FlgGT1 的突变实验表明,截断相对较大的 C 端结构域会导致鞭毛蛋白糖基化活性的丧失。我们根据序列相似性网络分析和 AlphaFold2 结构预测进行的分类表明,C 端结构域的获得是 GT2 家族中糖基转移酶赋予不同底物特异性的关键进化适应。
In Vitro Characterization of an O-Specific Glycosyltransferase Involved in Flagellin Glycosylation
Glycosyltransferases play a fundamental role in the biosynthesis of glycoproteins and glycotherapeutics. In this study, we investigated protein glycosyltransferase FlgGT1, belonging to the GT2 family. The GT2 family includes cysteine S-glycosyltransferases involved in antimicrobial peptide biosyntheses, sharing conserved catalytic domains while exhibiting diverse C-terminal domains. Our in vitro studies revealed that FlgGT1 recognizes structural motifs rather than specific amino acid sequences when glycosylating the flagellin protein Hag. Notably, FlgGT1 is selective for serine or threonine O-glycosylation over cysteine S-glycosylation. Molecular dynamics simulations provided insights into the structural basis of FlgGT1’s ability to accommodate various sugar nucleotides as donor substrates. Mutagenesis experiments on FlgGT1 demonstrated that truncating the relatively large C-terminal domain resulted in a loss of flagellin glycosylation activity. Our classification based on sequence similarity network analysis and AlphaFold2 structural predictions suggests that the acquisition of the C-terminal domain is a key evolutionary adaptation conferring distinct substrate specificities on glycosyltransferases within the GT2 family.
期刊介绍:
ACS Chemical Biology provides an international forum for the rapid communication of research that broadly embraces the interface between chemistry and biology.
The journal also serves as a forum to facilitate the communication between biologists and chemists that will translate into new research opportunities and discoveries. Results will be published in which molecular reasoning has been used to probe questions through in vitro investigations, cell biological methods, or organismic studies.
We welcome mechanistic studies on proteins, nucleic acids, sugars, lipids, and nonbiological polymers. The journal serves a large scientific community, exploring cellular function from both chemical and biological perspectives. It is understood that submitted work is based upon original results and has not been published previously.