Skp1, a subunit of E3 Skp1/Cullin-1/F-box protein ubiquitin ligases, is modified by a prolyl hydroxylase that mediates O2 regulation of the social amoeba Dictyostelium and the parasite Toxoplasma gondii. The full effect of hydroxylation requires modification of the hydroxyproline by a pentasaccharide that, in Dictyostelium, influences Skp1 structure to favor assembly of Skp1/F-box protein subcomplexes. In Toxoplasma, the presence of a contrasting penultimate sugar assembled by a different glycosyltransferase enables testing of the conformational control model. To define the final sugar and its linkage, here we identified the glycosyltransferase that completes the glycan and found that it is closely related to glycogenin, an enzyme that may prime glycogen synthesis in yeast and animals. However, the Toxoplasma enzyme catalyzes formation of a Galα1,3Glcα linkage rather than the Glcα1,4Glcα linkage formed by glycogenin. Kinetic and crystallographic experiments showed that the glycosyltransferase Gat1 is specific for Skp1 in Toxoplasma and also in another protist, the crop pathogen Pythium ultimum. The fifth sugar is important for glycan function as indicated by the slow-growth phenotype of gat1Δ parasites. Computational analyses indicated that, despite the sequence difference, the Toxoplasma glycan still assumes an ordered conformation that controls Skp1 structure and revealed the importance of nonpolar packing interactions of the fifth sugar. The substitution of glycosyltransferases in Toxoplasma and Pythium by an unrelated bifunctional enzyme that assembles a distinct but structurally compatible glycan in Dictyostelium is a remarkable case of convergent evolution, which emphasizes the importance of the terminal α-galactose and establishes the phylogenetic breadth of Skp1 glycoregulation.

中文翻译：

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末端 α3-半乳糖修饰可调节弓形虫中的 E3 泛素连接酶亚基。


Skp1 是 E3 Skp1/Cullin-1/F-box 蛋白泛素连接酶的一个亚基，由脯氨酰羟化酶修饰，介导社会性阿米巴原虫盘基网柄菌和寄生虫弓形虫的 O2 调节。羟基化的全部效果需要用五糖修饰羟脯氨酸，在盘基网柄菌中，五糖会影响 Skp1 结构以有利于 Skp1/F-box 蛋白亚复合物的组装。在弓形虫中，由不同糖基转移酶组装的对比倒数第二个糖的存在使得能够测试构象控制模型。为了定义最终的糖及其连接，我们鉴定了完成聚糖的糖基转移酶，并发现它与糖原蛋白密切相关，糖原蛋白是一种可能引发酵母和动物糖原合成的酶。然而，弓形虫酶催化Galα1,3Glcα键的形成，而不是由糖原形成的Glcα1,4Glcα键的形成。动力学和晶体学实验表明，糖基转移酶 Gat1 对弓形虫以及另一种原生生物（作物病原体终极腐霉）中的 Skp1 具有特异性。正如 gat1Δ 寄生虫的缓慢生长表型所示，第五种糖对于聚糖功能很重要。计算分析表明，尽管存在序列差异，弓形虫聚糖仍然呈现控制 Skp1 结构的有序构象，并揭示了第五种糖非极性堆积相互作用的重要性。 弓形虫和腐霉中的糖基转移酶被一种不相关的双功能酶取代，该酶在盘基网柄菌中组装出独特但结构相容的聚糖，这是趋同进化的一个显着案例，它强调了末端 α-半乳糖的重要性，并建立了 Skp1 糖调节的系统发育广度。