Unraveling the difference between invertases and fructan exohydrolases: A single amino acid (Asp-239) substitution transforms Arabidopsis cell wall invertase1 into a fructan 1-exohydrolase
Unraveling the difference between invertases and fructan exohydrolases: A single amino acid (Asp-239) substitution transforms Arabidopsis cell wall invertase1 into a fructan 1-exohydrolase
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DOI：
10.1104/pp.107.105049
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发表时间：
2007-11-01
期刊：
影响因子：
7.4
通讯作者：
Van den Ende, Wim
中科院分区：
文献类型：
--
作者：
Le Roy, Katrien;Lammens, Willem;Van den Ende, Wim
植物细胞壁转化酶和果聚糖外水解酶(FEHS)在分子水平和结构水平上是非常相似的酶(糖苷水解酶32家族)，但它们的功能不同，在植物中发挥着不同的作用。转化酶优先水解蔗糖(Suc)中的葡萄糖(GLC)-果糖(Fu)键，而植物FeHs没有转移酶活性，只在果糖中分裂末端的Fru-Fu键。最近，拟南芥细胞壁倒置1(AtcwINV1)和菊苣(菊苣)1-FEH IIa的三维结构被解析。到目前为止，在糖苷键的水解反应中，哪些氨基酸残基决定了蔗糖或果聚糖作为给体底物仍是未知的。在本文中，我们提供了基于AtcwINV1的定点突变数据，表明天冬氨酸(Asp)-239残基在Suc的结合和水解中发挥着重要作用。此外，研究还发现，活性中心边缘的疏水区的存在对于Suc的最佳和稳定结合是重要的。令人惊讶的是，D239A突变体扮演着1-FEH的角色，优先降解1-酮糖，这表明缺乏转化酶活性的植物FeH可能是通过几个突变变化从细胞壁转化酶类型的祖先进化而来的。一般来说，含有Asp-239功能同系物的32和68家族酶以Suc为优先底物，而缺乏该同系物的酶以果聚糖为供体底物。这种Asp-239同源基因的存在与否被认为是区分真正的蔗糖酶和缺陷的蔗糖酶/Fehs的一个可靠的决定因素。
Plant cell wall invertases and fructan exohydrolases (FEHs) are very closely related enzymes at the molecular and structural level (family 32 of glycoside hydrolases), but they are functionally different and are believed to fulfill distinct roles in plants. Invertases preferentially hydrolyze the glucose (Glc)-fructose (Fru) linkage in sucrose (Suc), whereas plant FEHs have no invertase activity and only split terminal Fru-Fru linkages in fructans. Recently, the three-dimensional structures of Arabidopsis (Arabidopsis thaliana) cell wall Invertase1 (AtcwINV1) and chicory (Cichorium intybus) 1-FEH IIa were resolved. Until now, it remained unknown which amino acid residues determine whether Suc or fructan is used as a donor substrate in the hydrolysis reaction of the glycosidic bond. In this article, we present site-directed mutagenesis-based data on AtcwINV1 showing that the aspartate (Asp)-239 residue fulfills an important role in both binding and hydrolysis of Suc. Moreover, it was found that the presence of a hydrophobic zone at the rim of the active site is important for optimal and stable binding of Suc. Surprisingly, a D239A mutant acted as a 1-FEH, preferentially degrading 1-kestose, indicating that plant FEHs lacking invertase activity could have evolved from a cell wall invertase-type ancestor by a few mutational changes. In general, family 32 and 68 enzymes containing an Asp-239 functional homolog have Suc as a preferential substrate, whereas enzymes lacking this homolog use fructans as a donor substrate. The presence or absence of such an Asp-239 homolog is proposed as a reliable determinant to discriminate between real invertases and defective invertases/ FEHs.