Synthetic UDP-galactofuranose analogs reveal critical enzyme-substrate interactions in GlfT2-catalyzed mycobacterial galactan assembly
Synthetic UDP-galactofuranose analogs reveal critical enzyme-substrate interactions in GlfT2-catalyzed mycobacterial galactan assembly
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DOI：
10.1039/c2ob25159k
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发表时间：
2012-01-01
影响因子：
3.2
通讯作者：
Lowary, Todd L.
中科院分区：
文献类型：
--
作者：
Poulin, Myles B.;Zhou, Ruokun;Lowary, Todd L.
分枝杆菌细胞壁半乳聚糖由β -(1- > - 5)和β -(1- > - 6)半乳糖呋喃基残基交替组成，通过两种双功能半乳糖呋喃基转移酶GlfT1和GlfT2的作用组装而成，这两种酶以udp -半乳糖呋喃糖（UDP-Galf）为供体底物。合成的UDP-Galf类似物的动力学分析确定了GlfT2（一种程序性聚合糖基转移酶）识别供体底物的关键相互作用。甲基化UDP-Galf类似物的测试表明，供体底物结合袋是立体拥挤的。对脱氧UDP-Galf类似物的评价表明，C-6羟基对底物活性不是必需的，与UDP-Galf C-3羟基的相互作用使底物定向转换，但在底物识别中似乎没有作用，使3-脱氧类似物成为酶的中等竞争性抑制剂。此外，在生长的半乳糖链上加入C-5或C-6脱氧的半乳糖残基，或l -阿拉伯糖铀糖残基，会产生“死胡同”反应产物，不再作为酶的受体。这一发现表明GlfT2活性需要受体底物的末端C-5和C-6羟基的双重识别，这与最近基于酶的晶体结构开发的模型一致。这些观察结果为GlfT2活性位点的特定蛋白质-碳水化合物相互作用提供了见解，并可能促进未来抑制剂的设计。
Mycobacterial cell wall galactan, composed of alternating beta-(1-->5) and beta-(1-->6) galactofuranosyl residues, is assembled by the action of two bifunctional galactofuranosyltransferases, GlfT1 and GlfT2, which use UDP-galactofuranose (UDP-Galf) as the donor substrate. Kinetic analysis of synthetic UDP-Galf analogs identified critical interactions involved in donor substrate recognition by GlfT2, a processive polymerizing glycosyltransferase. Testing of methylated UDP-Galf analogs showed the donor substrate-binding pocket is sterically crowded. Evaluation of deoxy UDP-Galf analogs revealed that the C-6 hydroxyl group is not essential for substrate activity, and that interactions with the UDP-Galf C-3 hydroxyl group orient the substrate for turnover but appears to play no role in substrate recognition, making the 3-deoxy-analog a moderate competitive inhibitor of the enzyme. Moreover, the addition of a Galf residue deoxygenated at C-5 or C-6, or an L-arabinofuranose residue, to the growing galactan chain resulted in "dead end" reaction products, which no longer act as an acceptor for the enzyme. This finding shows dual recognition of both the terminal C-5 and C-6 hydroxyl groups of the acceptor substrate are required for GlfT2 activity, which is consistent with a recent model developed based upon a crystal structure of the enzyme. These observations provide insight into specific protein-carbohydrate interactions in the GlfT2 active site and may facilitate the design of future inhibitors.