The Structure of a Streptomyces avermitilis α-L-Rhamnosidase Reveals a Novel Carbohydrate-binding Module CBM67 within the Six-domain Arrangement
The Structure of a Streptomyces avermitilis α-L-Rhamnosidase Reveals a Novel Carbohydrate-binding Module CBM67 within the Six-domain Arrangement
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DOI：
10.1074/jbc.m113.460097
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发表时间：
2013-04-26
影响因子：
4.8
通讯作者：
Kaneko, Satoshi
中科院分区：
文献类型：
--
作者：
Fujimoto, Zui;Jackson, Adam;Kaneko, Satoshi
α-L-鼠李糖苷酶从寡糖或多糖水解α-连接的L-鼠李糖苷。我们测定了阿维链霉菌糖苷水解酶家族78 α-L-鼠李糖苷酶（SaRha 78 A）的游离和L-鼠李糖复合物形式的晶体结构，其显示存在六个结构域N、D、E、F、A和C。在配体配合物中，L-鼠李糖结合在催化模块的活性位点上，揭示了SaRha 78 A可能的催化机理。预测Glu（636）将质子提供给糖苷氧，并且Glu（895）是可能的催化通用碱，激活亲核水，表明该酶通过反转机制操作。Glu（636）和Glu（895）的替换导致α-鼠李糖苷酶活性的显著损失。结构域D也以钙依赖性方式结合L-鼠李糖，K-D为135 μ M。因此，结构域D是非催化性碳水化合物结合模块（命名为SaCBM 67）。突变和结构数据鉴定了SaCBM 67中的氨基酸，其靶向L-鼠李糖的特征，将其与植物细胞壁中存在的其他主要糖区分开来。SaCBM 67的失活导致SaRha 78 A对多糖复合物阿拉伯树胶的活性显著降低，但对芳基鼠李糖苷没有，表明SaCBM 67有助于酶对不溶性底物的功能。
alpha-L-Rhamnosidases hydrolyze alpha-linked L-rhamnosides from oligosaccharides or polysaccharides. We determined the crystal structure of the glycoside hydrolase family 78 Streptomyces avermitilis alpha-L-rhamnosidase (SaRha78A) in its free and L-rhamnose complexed forms, which revealed the presence of six domains N, D, E, F, A, and C. In the ligand complex, L-rhamnose was bound in the proposed active site of the catalytic module, revealing the likely catalytic mechanism of SaRha78A. Glu(636) is predicted to donate protons to the glycosidic oxygen, and Glu(895) is the likely catalytic general base, activating the nucleophilic water, indicating that the enzyme operates through an inverting mechanism. Replacement of Glu(636) and Glu(895) resulted in significant loss of alpha-rhamnosidase activity. Domain D also bound L-rhamnose in a calcium-dependent manner, with a K-D of 135 mu M. Domain D is thus a non-catalytic carbohydrate binding module (designated SaCBM67). Mutagenesis and structural data identified the amino acids in SaCBM67 that target the features of L-rhamnose that distinguishes it from the other major sugars present in plant cell walls. Inactivation of SaCBM67 caused a substantial reduction in the activity of SaRha78A against the polysaccharide composite gum arabic, but not against aryl rhamnosides, indicating that SaCBM67 contributes to enzyme function against insoluble substrates.