Gómez Hansel, Rojas Raúl, Patel Divya, Tabak Lawrence A, Lluch José M, Masgrau Laura
Institut de Biotecnologia i de Biomedicina, Universitat Autònoma de Barcelona, 08193 Bellaterra (Cerdanyola del Vallès), Barcelona, Spain.
Org Biomol Chem. 2014 May 7;12(17):2645-55. doi: 10.1039/c3ob42569j.
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.
据估计，超过50%的蛋白质被糖标签糖基化，这些糖标签可通过所谓的糖密码调节蛋白质活性。在此，我们展示了对人GalNAc-T2（一种保留型糖基转移酶）的首次量子力学/分子力学（QM/MM）计算，该酶启动粘蛋白型O-聚糖的生物合成。重要的是，我们表征了UDP的β-磷酸与糖受体（EA2肽）的苏氨酸7的主链酰胺基团之间的氢键，该氢键促进催化作用，并且我们提出这可能是保留型糖基转移酶在肽O-糖基化中使用的一种通用催化策略。还确定了其他重要的底物-底物相互作用，例如，UDP的β-磷酸与受体底物的进攻羟基以及转移糖的C2'位置的取代基之间的相互作用。我们的结果支持该酶的正面进攻机制，在QM（M05-2X/TZVP//BP86/SVP）/CHARMM22水平下势垒高度约为20 kcal mol⁻¹，与实验动力学数据合理吻合。实验和计算机模拟突变表明，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，人GalNAc-T2将无活性，而UDP-Gal被确认为有效的糖供体。最后，本文的分析突出表明，底物-底物和酶-底物相互作用主要集中在接近过渡态时稳定UDP离去基团上发展的负电荷，将此确定为保留型糖基转移酶催化的一个关键方面。
