Abstract

O-GlcNAcylation is an important post-translational modification of proteins. O-GlcNAcylated proteins have crucial roles in several cellular contexts both in eukaryotes and bacteria. O-GlcNActransferase (OGT) is the enzyme instrumental in O-GlcNAcylation of proteins. OGT is conserved across eukaryotes. The first bacterial OGT discovered is GmaR in Listeria monocytogenes. GmaR is a GT-2 family bifunctional protein that catalyzes glycosylation of the flagellin protein FlaA and controls transcription of flagellar motility genes in a temperature-dependent manner. Here, we provide methods for heterologous expression and purification of recombinant GmaR and FlaA, in vivo/in vitro glycosylation assays, analysis of the molecular form of recombinant GmaR and detailed enzyme kinetics. We study the structure and functional dynamics of GmaR. Using solution small-angle X-ray scattering and molecular modeling, we show that GmaR adopts an extended shape with two distinctly spaced structural units in the presence of cofactor Mg²⁺ and with donor UDP-GlcNAc and cofactor combined. Comparisons of restored structures revealed that in-solution binding of Mg²⁺ ions brings about shape rearrangements and induces structural-rigidity in hyper-variable regions at the N-terminus of GmaR protein. Taking function and shape data together, we describe that Mg²⁺ binding enables GmaR to adopt a shape that can bind the substrate. The manuscript provides the first 3D solution structure of a bacterial OGT of GT-2 family and detailed biochemical characterization of GmaR to facilitate its future applications.

中文翻译：

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单核细胞增生李斯特菌EGD-e双功能O-GlcNAc转移酶的形状-功能洞察

摘要

O -GlcNAcylation 是一种重要的蛋白质翻译后修饰。O -GlcNAcylated 蛋白在真核生物和细菌中的几种细胞环境中发挥着至关重要的作用。O -GlcNAc转移酶（OGT）是蛋白质O -GlcNAc化的重要酶。OGT 在真核生物中是保守的。第一个发现的细菌 OGT 是李斯特菌中的 GmaR. GmaR 是一种 GT-2 家族双功能蛋白，可催化鞭毛蛋白 FlaA 的糖基化，并以温度依赖性方式控制鞭毛运动基因的转录。在这里，我们提供了重组 GmaR 和 FlaA 的异源表达和纯化方法、体内/体外糖基化测定、重组 GmaR 分子形式的分析和详细的酶动力学。我们研究 GmaR 的结构和功能动力学。使用溶液小角 X 射线散射和分子建模，我们表明 GmaR 在辅因子 Mg ²⁺和供体 UDP-GlcNAc 和辅因子结合的情况下采用具有两个明显间隔的结构单元的扩展形状。恢复结构的比较表明，Mg ^{2+的溶液内结合}离子导致形状重排并在 GmaR 蛋白 N 端的高变区诱导结构刚性。结合功能和形状数据，我们描述了 Mg ²⁺结合使 GmaR 能够采用可以结合底物的形状。该手稿提供了 GT-2 家族细菌 OGT 的第一个 3D 解决方案结构和 GmaR 的详细生化表征，以促进其未来的应用。