Yep Alejandra, Ballicora Miguel A, Preiss Jack
Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824, USA.
Arch Biochem Biophys. 2006 Sep 15;453(2):188-96. doi: 10.1016/j.abb.2006.07.003. Epub 2006 Jul 31.
Bacterial glycogen/starch synthases are retaining GT-B glycosyltransferases that transfer glucosyl units from ADP-Glc to the non-reducing end of glycogen or starch. We modeled the Escherichia coli glycogen synthase based on the coordinates of the inactive form of the Agrobacterium tumefaciens glycogen synthase and the active form of the maltodextrin phosphorylase, a retaining GT-B glycosyltransferase belonging to a different family. In this model, we identified a set of conserved residues surrounding the sugar nucleotide substrate, and we replaced them with different amino acids by means of site-directed mutagenesis. Kinetic analysis of the mutants revealed the involvement of these residues in ADP-Glc binding. Replacement of Asp21, Asn246 or Tyr355 for Ala decreased the apparent affinity for ADP-Glc 18-, 45-, and 31-fold, respectively. Comparison with other crystallized retaining GT-B glycosyltransferases confirmed the striking similarities among this group of enzymes even though they use different substrates.
细菌糖原/淀粉合酶属于保留型GT-B糖基转移酶，可将葡糖基单位从ADP-葡萄糖转移至糖原或淀粉的非还原端。我们基于根癌土壤杆菌糖原合酶的无活性形式以及麦芽糖糊精磷酸化酶（一种属于不同家族的保留型GT-B糖基转移酶）的活性形式的坐标，对大肠杆菌糖原合酶进行了建模。在该模型中，我们确定了围绕糖核苷酸底物的一组保守残基，并通过定点诱变将它们替换为不同的氨基酸。对突变体的动力学分析揭示了这些残基在ADP-葡萄糖结合中的作用。将Asp21、Asn246或Tyr355替换为丙氨酸分别使对ADP-葡萄糖的表观亲和力降低了18倍、45倍和31倍。与其他结晶的保留型GT-B糖基转移酶进行比较，证实了这组酶之间存在显著相似性，尽管它们使用不同的底物。
