Department of Food and Nutritional Sciences, Graduate School of Nutritional and Environmental Sciences, University of Shizuoka, 52-1 Yada, Suruga-Ku, Shizuoka 422-8526, Japan.
J Mol Biol. 2011 Apr 29;408(2):177-86. doi: 10.1016/j.jmb.2011.02.028. Epub 2011 Feb 25.
Glucansucrase (GSase) from Streptococcus mutans is an essential agent in dental caries pathogenesis. Here, we report the crystal structure of S. mutans glycosyltransferase (GTF-SI), which synthesizes soluble and insoluble glucans and is a glycoside hydrolase (GH) family 70 GSase in the free enzyme form and in complex with acarbose and maltose. Resolution of the GTF-SI structure confirmed that the domain order of GTF-SI is circularly permuted as compared to that of GH family 13 α-amylases. As a result, domains A, B and IV of GTF-SI are each composed of two separate polypeptide chains. Structural comparison of GTF-SI and amylosucrase, which is closely related to GH family 13 amylases, indicated that the two enzymes share a similar transglycosylation mechanism via a glycosyl-enzyme intermediate in subsite -1. On the other hand, novel structural features were revealed in subsites +1 and +2 of GTF-SI. Trp517 provided the platform for glycosyl acceptor binding, while Tyr430, Asn481 and Ser589, which are conserved in family 70 enzymes but not in family 13 enzymes, comprised subsite +1. Based on the structure of GTF-SI and amino acid comparison of GTF-SI, GTF-I and GTF-S, Asp593 in GTF-SI appeared to be the most critical point for acceptor sugar orientation, influencing the transglycosylation specificity of GSases, that is, whether they produced insoluble glucan with α(1-3) glycosidic linkages or soluble glucan with α(1-6) linkages. The structural information derived from the current study should be extremely useful in the design of novel inhibitors that prevent the biofilm formation by GTF-SI.
变形链球菌的葡聚糖蔗糖酶（GSase）是龋齿发病机制中的重要因素。在这里，我们报告了变形链球菌糖基转移酶（GTF-SI）的晶体结构，它合成可溶性和不溶性葡聚糖，并且在游离酶形式和与阿卡波糖和麦芽糖复合物中是糖苷水解酶（GH）家族 70 的 GSase。GTF-SI 结构的解析证实，与 GH 家族 13 α-淀粉酶相比，GTF-SI 的结构域顺序是环状排列的。结果，GTF-SI 的结构域 A、B 和 IV 分别由两条单独的多肽链组成。GTF-SI 与 GH 家族 13 淀粉酶密切相关的淀粉蔗糖酶的结构比较表明，这两种酶通过糖苷-酶中间体在亚位点-1 上共享类似的转糖基化机制。另一方面，在 GTF-SI 的 +1 和 +2 亚位点揭示了新的结构特征。色氨酸 517 为糖基受体结合提供了平台，而酪氨酸 430、天冬酰胺 481 和丝氨酸 589 则在家族 70 酶中保守，但在家族 13 酶中不存在，它们构成了 +1 亚位点。基于 GTF-SI 的结构和 GTF-SI、GTF-I 和 GTF-S 的氨基酸比较，GTF-SI 中的天冬氨酸 593 似乎是影响 GSase 转糖基特异性的最关键因素，即它们是否产生具有 α（1-3）糖苷键的不溶性葡聚糖或具有 α（1-6）键的可溶性葡聚糖。本研究获得的结构信息对于设计防止 GTF-SI 生物膜形成的新型抑制剂将非常有用。
