DataSheet1_Biochemical and Structural Characterization of Thermostable GH159 Glycoside Hydrolases Exhibiting α-L-Arabinofuranosidase Activity.PDF

Functional, biochemical, and preliminary structural properties are reported for three glycoside hydrolases of the recently described glycoside hydrolase (GH) family 159. The genes were cloned from the genomic sequences of different Caldicellulosiruptor strains. This study extends the spectrum of functions of GH159 enzymes. The only activity previously reported for GH159 was hydrolytic activity on β-galactofuranosides. Activity screening using a set of para-nitrophenyl (pNP) glycosides suggested additional arabinosidase activity on substrates with arabinosyl residues, which has not been previously reported for members of GH159. Even though the thermophilic enzymes investigated—Cs_Gaf159A, Ch_Gaf159A, and Ck_Gaf159A—cleaved pNP-α-l-arabinofuranoside, they were only weakly active on arabinogalactan, and they did not cleave arabinose from arabinan, arabinoxylan, or gum arabic. However, the enzymes were able to hydrolyze the α-1,3-linkage in different arabinoxylan-derived oligosaccharides (AXOS) with arabinosylated xylose at the non-reducing end (A3X, A2,3XX), suggesting their role in the intracellular hydrolysis of oligosaccharides. Crystallization and structural analysis of the apo form of one of the Caldicellulosiruptor enzymes, Ch_Gaf159A, enabled the elucidation of the first 3D structure of a GH159 member. This work revealed a five-bladed β-propeller structure for GH159 enzymes. The 3D structure and its substrate-binding pocket also provides an explanation at the molecular level for the observed exo-activity of the enzyme. Furthermore, the structural data enabled the prediction of the catalytic amino acids. This was supported by the complete inactivation by mutation of residues D19, D142, and E190 of Ch_Gaf159A.

本报告详述了近期描述的糖苷水解酶家族159中的三种糖苷水解酶的功能性、生化性质及初步的构象特性。相关基因已被克隆自不同嗜热菌属Caldicellulosiruptor的基因组序列中。本研究扩展了GH159酶类功能谱系。此前对于GH159酶类唯一报道的活性为对β-半乳糖基呋喃糖的水解活性。通过一套对硝基苯基（pNP）糖苷的活性筛选，提示GH159成员可能具有对含阿拉伯糖基残基底物的阿拉伯糖苷酶活性，该活性此前未曾见于GH159家族成员。尽管所研究的嗜热酶类——Cs_Gaf159A、Ch_Gaf159A和Ck_Gaf159A——能够切割pNP-α-l-阿拉伯呋喃糖，但它们在阿拉伯半乳聚糖上的活性较弱，且不能从阿拉伯聚糖、阿拉伯木聚糖或阿拉伯胶中切割阿拉伯糖。然而，这些酶能够水解含有非还原端阿拉伯糖基化的木糖的不同阿拉伯木聚糖衍生的寡糖（AXOS）中的α-1,3-糖苷键，这表明它们在寡糖的细胞内水解中发挥作用。嗜热菌属酶Ch_Gaf159A的无活性形式结晶及结构分析，首次阐明了GH159成员的三维结构。这项工作揭示了GH159酶类的五叶β-螺旋结构。三维结构和其底物结合口袋也为酶观察到的外切活性提供了分子层面的解释。此外，结构数据还允许预测催化氨基酸。这一预测得到了Ch_Gaf159A中的D19、D142和E190残基突变后完全失活的支持。