A computational and experimental study of O-glycosylation. Catalysis by human UDP-GalNAc polypeptide:GalNAc transferase-T2.
A computational and experimental study of O-glycosylation. Catalysis by human UDP-GalNAc polypeptide:GalNAc transferase-T2.
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DOI：
10.1039/c3ob42569j
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发表时间：
2014-05-07
影响因子：
3.2
通讯作者：
Masgrau L
中科院分区：
文献类型：
--
作者：
Gómez H;Rojas R;Patel D;Tabak LA;Lluch JM;Masgrau L
据估计，50%的蛋白质是用糖标签糖基化的，这种标签可以通过所谓的糖码来调节蛋白质的活动。在这里，我们提出了首次对人GalNAc-T2的QM/MM计算，GalNAc-T2是一种保留糖基转移酶，它启动粘蛋白O-糖聚糖的生物合成。重要的是，我们已经表征了糖基受体β-磷酸和糖受体(EA2肽)Thr7的主链酰胺基团之间的氢键，它促进了催化作用，我们认为这可能是一种通过保留糖基转移酶而用于多肽O-糖基化的一般催化策略。还发现了其他重要的底物-底物相互作用，例如，二磷酸二核苷酸的β-磷酸与来自受体底物的攻击羟基以及与转移糖C2‘位的取代基之间的相互作用。我们的结果支持该酶的正面攻击机制，在QM(M05-2X/TZVP//BP86/SVP)/CHARMM22水平上的势垒高度约为20kcal摩尔−1，与实验动力学数据吻合较好。实验和电子突变表明，转移酶活性对Glu334、Asn335和Arg362残基的变化非常敏感。此外，我们对不同供体底物的计算表明，如果使用2‘-脱氧-Gal或2’-氧甲基-Gal，则人GalNAc-T2将失去活性，而UDP-Gal被确认为有效的糖供体。最后，本文的分析强调了底物-底物和酶-底物的相互作用主要集中在稳定UDP离开基团在接近过渡态时产生的负电荷，确定这是保留糖基转移酶催化的关键方面。
It is estimated that >50% of proteins are glycosylated with sugar tags that can modulate protein activity through what has been called the sugar code. Here we present the first QM/MM calculations of human GalNAc-T2, a retaining glycosyltransferase, which initiates the biosynthesis of mucin-type O-glycans. Importantly, we have characterized a hydrogen bond between the β-phosphate of UDP and the backbone amide group from the Thr7 of the sugar acceptor (EA2 peptide) that promotes catalysis and that we propose could be a general catalytic strategy used in peptide O-glycosylation by retaining glycosyltransferases. Additional important substrate–substrate interactions have been identified, for example, between the β-phosphate of UDP with the attacking hydroxyl group from the acceptor substrate and with the substituent at the C2′ position of the transferred sugar. Our results support a front-side attack mechanism for this enzyme, with a barrier height of ~20 kcal mol−1 at the QM(M05-2X/TZVP//BP86/SVP)/CHARMM22 level, in reasonable agreement with the experimental kinetic data. Experimental and in silico mutations show that transferase activity is very sensitive to changes in residues Glu334, Asn335 and Arg362. Additionally, our calculations for different donor substrates suggest that human GalNAc-T2 would be inactive if 2′-deoxy-Gal or 2′-oxymethyl-Gal were used, while UDP-Gal is confirmed as a valid sugar donor. Finally, the analysis herein presented highlights that both the substrate–substrate and the enzyme–substrate interactions are mainly concentrated on stabilizing the negative charge developing at the UDP leaving group as the transition state is approached, identifying this as a key aspect of retaining glycosyltransferases catalysis.