Yep Alejandra, Ballicora Miguel A, Preiss Jack
Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824, USA.
Biochem Biophys Res Commun. 2004 Apr 9;316(3):960-6. doi: 10.1016/j.bbrc.2004.02.136.
Bacterial glycogen synthases transfer a glucosyl unit, retaining the anomeric configuration, from ADP-glucose to the non-reducing end of glycogen. We modeled the Escherichia coli glycogen synthase based on three glycosyltransferases with a GT-B fold. Comparison between the model and the structure of the active site of crystallized retaining GT-B glycosyltransferases identified conserved residues with the same topology. To confirm the importance of these residues predicted by the model, we studied them in E. coli glycogen synthase by site-directed mutagenesis. Mutations D137A, R300A, K305A, and H161A decreased the specific activity 8100-, 2600-, 1200-, and 710-fold, respectively. None of these mutations increased the Km for glycogen and only H161A and R300A had a higher Km for ADP-Glc of 11- and 8-fold, respectively. These residues were essential, validating the model that shows a strong similarity between the active site of E. coli glycogen synthase and the other retaining GT-B glycosyltransferases known to date.
细菌糖原合酶将一个保留异头构型的葡糖基单元从 ADP-葡萄糖转移至糖原的非还原端。我们基于三种具有 GT-B 折叠的糖基转移酶对大肠杆菌糖原合酶进行了建模。该模型与已结晶的保留型 GT-B 糖基转移酶活性位点结构之间的比较确定了具有相同拓扑结构的保守残基。为了证实该模型预测的这些残基的重要性，我们通过定点诱变在大肠杆菌糖原合酶中对它们进行了研究。D137A、R300A、K305A 和 H161A 突变分别使比活性降低了 8100 倍、2600 倍、1200 倍和 710 倍。这些突变均未增加糖原的 Km 值，只有 H161A 和 R300A 对 ADP-Glc 的 Km 值分别升高了 11 倍和 8 倍。这些残基至关重要，验证了该模型，该模型表明大肠杆菌糖原合酶的活性位点与迄今为止已知的其他保留型 GT-B 糖基转移酶之间具有很强的相似性。
