College of Pharmacy, State Key Laboratory of Medicinal Chemical Biology and Tianjin Key Laboratory of Molecular Drug Research, Nankai University, Tianjin, China.
PLoS One. 2013 May 21;8(5):e63452. doi: 10.1371/journal.pone.0063452. Print 2013.
O-Linked β-N-acetylglucosaminyl transferase (OGT) plays an important role in the glycosylation of proteins, which is involved in various cellular events. In human, three isoforms of OGT (short OGT [sOGT]; mitochondrial OGT [mOGT]; and nucleocytoplasmic OGT [ncOGT]) share the same catalytic domain, implying that they might adopt a similar catalytic mechanism, including sugar donor recognition. In this work, the sugar-nucleotide tolerance of sOGT was investigated. Among a series of uridine 5'-diphosphate-N-acetylglucosamine (UDP-GlcNAc) analogs tested using the casein kinase II (CKII) peptide as the sugar acceptor, four compounds could be used by sOGT, including UDP-6-deoxy-GlcNAc, UDP-GlcNPr, UDP-6-deoxy-GalNAc and UDP-4-deoxy-GlcNAc. Determined values of Km showed that the substitution of the N-acyl group, deoxy modification of C6/C4-OH or epimerization of C4-OH of the GlcNAc in UDP-GlcNAc decreased its affinity to sOGT. A molecular docking study combined with site-directed mutagenesis indicated that the backbone carbonyl oxygen of Leu653 and the hydroxyl group of Thr560 in sOGT contributed to the recognition of the sugar moiety via hydrogen bonds. The close vicinity between Met501 and the N-acyl group of GlcNPr, as well as the hydrophobic environment near Met501, were responsible for the selective binding of UDP-GlcNPr. These findings illustrate the interaction of OGT and sugar nucleotide donor, providing insights into the OGT catalytic mechanism.
O-连接β-N-乙酰氨基葡萄糖基转移酶（OGT）在蛋白质的糖基化中起着重要作用，而糖基化参与了各种细胞事件。在人类中，OGT 有三种同工型（短型 OGT [sOGT]；线粒体 OGT [mOGT]；核质型 OGT [ncOGT]），它们具有相同的催化结构域，这意味着它们可能采用相似的催化机制，包括糖供体识别。在这项工作中，研究了 sOGT 对糖核苷酸的耐受性。在所测试的一系列尿嘧啶 5'-二磷酸-N-乙酰氨基葡萄糖（UDP-GlcNAc）类似物中，有 4 种化合物可以被 sOGT 利用，包括 UDP-6-脱氧-GlcNAc、UDP-GlcNPr、UDP-6-脱氧-GalNAc 和 UDP-4-脱氧-GlcNAc。通过测定 Km 值发现，GlcNAc 中 N-酰基、C6/C4-OH 脱氧或 C4-OH 差向异构化的取代降低了其与 sOGT 的亲和力。分子对接研究结合定点突变表明，sOGT 中 Leu653 的骨架羰基氧和 Thr560 的羟基通过氢键参与了糖部分的识别。Met501 与 GlcNPr 的 N-酰基以及 Met501 附近的疏水环境之间的紧密接近，负责 UDP-GlcNPr 的选择性结合。这些发现阐明了 OGT 与糖核苷酸供体的相互作用，为 OGT 催化机制提供了深入的见解。
