背景:α-l-岩藻糖赋予含岩藻糖的生物分子例如人乳寡糖的独特功能。α-1-岩藻糖苷酶可作为岩藻糖基化糖应用中的理想工具。发现新型α-1-岩藻糖苷酶并阐明其酶特性始终是值得的任务。
结果:从海洋细菌温英庄岩藻的基因组中克隆了一个名为Afc95A_Wf的GH95家族α-l-岩藻糖苷酶,并在大肠杆菌中表达。它在40°C和pH7.5下表现出最大活性。Afc95A_Wf在报道的α-1-岩藻糖苷酶中定义了不同的底物特异性,能够水解CNP-岩藻糖中的α-岩藻苷,Fucα1-2gβ1-4Glc和Galβ1-4(Fucα1-3)Glc,并显示出对α1,2-岩藻糖苷连接的偏好。它采用了氨基酸序列中第391位的Asp残基,这与以前公认的Asn残基不同。预测的三级结构和定点诱变表明Asp391参与了Afc95A_Wf的催化。底物特异性和催化位点的差异揭示了Afc95A_Wf在催化中采用了一种新机制。
结论:克隆并表达了GH95家族α-1-岩藻糖苷酶(Afc95A_Wf)。它显示了α1,2-岩藻苷连接对α1,3-岩藻苷连接的切割偏好。与已知的GH95家族蛋白相比,Afc95A_Wf表现出不同的底物特异性和重要催化位点的残基,揭示了GH95家族催化机理的多样性。©2024化学工业学会。
BACKGROUND: α-l-Fucose confers unique functions for fucose-containing biomolecules such as human milk oligosaccharides. α-l-Fucosidases can serve as desirable tools in the application of fucosylated saccharides. Discovering novel α-l-fucosidases and elucidating their enzyme properties are always worthy tasks.
RESULTS: A GH95 family α-l-fucosidase named Afc95A_Wf was cloned from the genome of the marine bacterium Wenyingzhuangia fucanilytica and expressed in Escherichia coli. It exhibited maximum activity at 40 °C and pH 7.5. Afc95A_Wf defined a different substrate specificity among reported α-l-fucosidases, which was capable of hydrolyzing α-fucoside in CNP-fucose, Fucα1-2Galβ1-4Glc and Galβ1-4(Fucα1-3)Glc, and showed a preference for α1,2-fucosidic linkage. It adopted Asp residue in the amino acid sequence at position 391, which was distinct from the previously acknowledged residue of Asn. The predicted tertiary structure and site-directed mutagenesis revealed that Asp391 participates in the catalysis of Afc95A_Wf. The differences in the substrate specificity and catalytic site shed light on that Afc95A_Wf adopted a novel mechanism in catalysis.
CONCLUSIONS: A GH95 family α-l-fucosidase (Afc95A_Wf) was cloned and expressed. It showed a cleavage preference for α1,2-fucosidic linkage to α1,3-fucosidic linkage. Afc95A_Wf demonstrated a different substrate specificity and a residue at an important catalytic site compared with known GH95 family proteins, which revealed the occurrence of diversity on catalytic mechanisms in the GH95 family. © 2024 Society of Chemical Industry.