Homologous glycosyltransferases GTA and GTB perform the final step in human ABO(H) blood group A and B antigen synthesis by transferring the sugar moiety from donor UDP-GalNAc/UDP-Gal to the terminal H antigen disaccharide acceptor. Like other GT-A fold family 6 glycosyltransferases, GTA and GTB undergo major conformational changes in two mobile regions, the C-terminal tail and internal loop, to achieve the closed, catalytic state. These changes are known to establish a salt bridge network among conserved active site residues Arg188, Asp211 and Asp302, which move to accommodate a series of discrete donor conformations while promoting loop ordering and formation of the closed enzyme state. However, the individual significance of these residues in linking these processes remains unclear. Here, we report the kinetics and high-resolution structures of GTA/GTB mutants of residues 188 and 302. The structural data support a conserved salt bridge network critical to mobile polypeptide loop organization and stabilization of the catalytically competent donor conformation. Consistent with the X-ray crystal structures, the kinetic data suggest that disruption of this salt bridge network has a destabilizing effect on the transition state, emphasizing the importance of Arg188 and Asp302 in the glycosyltransfer reaction mechanism. The salt bridge network observed in GTA/GTB structures during substrate binding appears to be conserved not only among other Carbohydrate Active EnZyme family 6 glycosyltransferases but also within both retaining and inverting GT-A fold glycosyltransferases. Our findings augment recently published crystal structures, which have identified a correlation between donor substrate conformational changes and mobile loop ordering.

中文翻译：

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保守的残基Arg188和Asp302对于ABO（H）血型A和B糖基转移酶的活性位点组织和催化至关重要^†

同源糖基转移酶GTA和GTB通过将糖部分从供体UDP-GalNAc / UDP-Gal转移到末端H抗原二糖受体上，完成了人类ABO（H）血型A和B抗原合成的最后一步。像其他GT-A折叠家族6糖基转移酶一样，GTA和GTB在两个移动区域（C端尾部和内部环）中经历主要构象变化，以实现封闭的催化状态。已知这些变化可在保守的活性位点残基Arg188，Asp211和Asp302之间建立盐桥网络，这些残基移动以适应一系列离散的供体构象，同时促进环的有序排列和闭合酶态的形成。然而，这些残基在连接这些过程中的个体重要性仍不清楚。这里，我们报道了残基188和302的GTA / GTB突变体的动力学和高分辨率结构。结构数据支持保守的盐桥网络，对移动多肽环的组织和催化活性供体构象的稳定至关重要。与X射线晶体结构一致，动力学数据表明该盐桥网络的破坏对过渡态具有不稳定作用，强调了Arg188和Asp302在糖基转移反应机理中的重要性。在底物结合过程中，在GTA / GTB结构中观察到的盐桥网络不仅在其他碳水化合物活性酶家族6糖基转移酶中而且在保留和反转GT-A折叠糖基转移酶中都是保守的。我们的发现增强了最近发表的晶体结构，