The deoxy sugar l-fucose is frequently found as a glycan constituent on and outside living cells, and in mammals it is involved in a wide range of biological processes including leukocyte trafficking, histo-blood group antigenicity and antibody effector functions. The manipulation of fucose levels in those biomedically important systems may provide novel insights and therapeutic leads. However, despite the large established sequence diversity of natural fucosidases, so far, very few enzymes have been characterized. We explored the diversity of the α-l-fucosidase-containing CAZY family GH29 by bio-informatic analysis, and by the recombinant production and exploration for fucosidase activity of a subset of 82 protein sequences that represent the family’s large sequence diversity. After establishing that most of the corresponding proteins can be readily expressed in E. coli, more than half of the obtained recombinant proteins (57% of the entire subset) showed activity towards the simple chromogenic fucosylated substrate 4-nitrophenyl α-l-fucopyranoside. Thirty-seven of these active GH29 enzymes (and the GH29 subtaxa that they represent) had not been characterized before. With such a sequence diversity-based collection available, it can easily be used to screen for fucosidase activity towards biomedically relevant fucosylated glycoproteins. As an example, the subset was used to screen GH29 members for activity towards the naturally occurring sialyl-Lewis x-type epitope on glycoproteins, and several such enzymes were identified. Together, the results provide a significant increase in the diversity of characterized GH29 enzymes, and the recombinant enzymes constitute a resource for the further functional exploration of this enzyme family.

中文翻译：

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GH29 岩藻糖苷酶家族的功能探索。

脱氧糖l-岩藻糖经常被发现是活细胞内外的聚糖成分，在哺乳动物中，它参与了广泛的生物过程，包括白细胞运输、组织血型抗原性和抗体效应功能。在那些生物医学上重要的系统中操纵岩藻糖水平可能会提供新的见解和治疗线索。然而，尽管天然岩藻糖苷酶具有大量已建立的序列多样性，但迄今为止，很少有酶被表征。我们探索了α- l的多样性-岩藻糖苷酶的 CAZY 家族 GH29 通过生物信息学分析，以及对代表该家族大序列多样性的 82 个蛋白质序列子集的岩藻糖苷酶活性的重组生产和探索。在确定大多数相应的蛋白质可以在大肠杆菌中轻松表达后，获得的重组蛋白质中有一半以上（整个子集的 57%）对简单的显色岩藻糖基化底物 4-硝基苯基 α- l显示出活性-吡喃岩藻糖苷。其中 37 种活性 GH29 酶（以及它们所代表的 GH29 亚类群）以前没有被表征过。有了这样一个基于序列多样性的集合，它可以很容易地用于筛选岩藻糖苷酶对生物医学相关岩藻糖基化糖蛋白的活性。例如，该子集用于筛选 GH29 成员对糖蛋白上天然存在的唾液酸-刘易斯 x 型表位的活性，并鉴定了几种此类酶。总之，这些结果显着增加了特征性 GH29 酶的多样性，重组酶构成了对该酶家族进一步功能探索的资源。