Small angle x-ray studies reveal that Aspergillus niger glucoamylase has a defined extended conformation and can form dimers in solution
Small angle x-ray studies reveal that Aspergillus niger glucoamylase has a defined extended conformation and can form dimers in solution
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DOI：
10.1074/jbc.m801709200
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发表时间：
2008-05-23
影响因子：
4.8
通讯作者：
Vestergaard, Bente
中科院分区：
文献类型：
--
作者：
Jorgensen, Anders Dysted;Nohr, Jane;Vestergaard, Bente
工业上重要的葡糖淀粉酶 1 是一种外切作用糖苷酶，其底物优先选择淀粉非还原端的 α-1,4 和 α-1,6 连接。它由淀粉结合结构域和散布高度糖基化多肽接头的催化结构域组成。连接子功能知之甚少，结构上也未描述，有关域组织和分子内功能协同性的数据是相互冲突的或不全面的。在此，我们报告了对黑曲霉葡糖淀粉酶 1、缺乏淀粉结合结构域的葡糖淀粉酶 2 以及具有短接头的葡糖淀粉酶 1 的工程低糖基化变体的组合小角 X 射线散射和量热研究。低分辨率溶液结构表明，连接子采用紧凑的结构，为葡糖淀粉酶提供了明确的扩展整体构象。我们证明，同时针对催化和淀粉结合结构域的短异二齿抑制剂的结合导致葡糖淀粉酶的二聚化，而不是如之前所暗示的由接头柔性介导的分子内构象重排。我们的结果表明，葡糖淀粉酶在不溶性底物水解过程中通过瞬时二聚体形成发挥作用，并解决了淀粉结合和水解的协同效应问题。
The industrially important glucoamylase 1 is an exo-acting glycosidase with substrate preference for alpha-1,4 and alpha-1,6 linkages at non-reducing ends of starch. It consists of a starch binding and a catalytic domain interspersed by a highly glycosylated polypeptide linker. The linker function is poorly understood and structurally undescribed, and data regarding domain organization and intramolecular functional cooperativity are conflicting or non-comprehensive. Here, we report a combined small angle x-ray scattering and calorimetry study of Aspergillus niger glucoamylase 1, glucoamylase 2, which lacks a starch binding domain, and an engineered low-glycosylated variant of glucoamylase 1 with a short linker. Low resolution solution structures show that the linker adopts a compact structure rendering a well defined extended overall conformation to glucoamylase. We demonstrate that binding of a short heterobidentate inhibitor simultaneously directed toward the catalytic and starch binding domains causes dimerization of glucoamylase and not, as suggested previously, an intramolecular conformational rearrangement mediated by linker flexibility. Our results suggest that glucoamylase functions via transient dimer formation during hydrolysis of insoluble substrates and address the question of the cooperative effect of starch binding and hydrolysis.