α-l-Fucosidases are widely occurring enzymes that remove fucose residues from N- and O-fucosylated glycoproteins. Comparison of amino acid sequences of fucosidases reveals that although the nucleophile is conserved among all α-l-fucosidases, the position of the acid/base residue is quite variable. Although several site-directed mutation studies have previously been performed on bacterial fucosidases, the only eukaryotic fucosidase so studied was the human fucosidase. Recent alignments indicate that human and Arthropoda α-l-fucosidases share at least 50% identity and the acid/base residue seems to be conserved among them suggesting a common acid/base residue in Metazoa. Here we describe the cloning and expression in Pichia pastoris of a very active α-l-fucosidase from the spider Nephilingis cruentata (NcFuc) with a K_m value for pNPFuc of 0.4 mM. NcFuc hydrolyzed fucoidan, 2´fucosyllactose and also lacto-N-difucohexaose II. Mutants modified at the conserved residues D214N, E209A, E59A were expressed and characterized. The 500-fold lower k_cat of D214N than the wild type was consistent with a role in catalysis, as was the 8000-fold lower k_cat value of E59A. This was supported by the 57-fold increase in the k_cat of E59A upon addition of azide. A complex pH/rate profile was seen for the wild-type and mutant forms of NcFuc, similar to those measured previously for the Sulfolobus fucosidase. The non-conservative catalytic structure and distinct active site organization reinforce the necessity of structural studies of new fucosidases.

中文翻译：

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蜘蛛Nephilingis cruentata GH29岩藻糖苷酶中非保守酸/碱催化残基的鉴定和作用

α- 1-岩藻糖苷酶是广泛存在的酶，其从N-和O-岩藻糖基化的糖蛋白中去除岩藻糖残基。岩藻糖苷酶的氨基酸序列的比较表明，尽管亲核试剂在所有α- 1-岩藻糖苷酶中是保守的，但是酸/碱残基的位置是相当可变的。尽管以前已经对细菌岩藻糖苷酶进行了一些定点突变研究，但如此研究的唯一的真核岩藻糖苷酶是人岩藻糖苷酶。最近的比对表明，人和节肢动物α- 1-岩藻糖苷酶具有至少50％的同一性，并且其中的酸/碱残基似乎是保守的，表明后生动物中存在常见的酸/碱残基。在这里我们描述了克隆和表达来自蜘蛛Nephilingis cruentata（NcFuc）的非常活跃的α- 1-岩藻糖苷酶的巴斯德毕赤酵母，pNPFuc的K _m值为0.4 mM。NcFuc水解了岩藻依聚糖，2′岩藻糖基乳糖以及乳糖N-二岩藻糖糖II。表达并表征了在保守残基D214N，E209A，E59A处修饰的突变体。与野生型相比，D214N的k _cat值低500倍，与催化作用一致，E59A的k _cat值也低8000倍。k _cat的增加57倍支持了这一点加入叠氮化物后得到的E59A。NcFuc的野生型和突变体形式具有复杂的pH /速率分布，类似于先前对Sulfolobus岩藻糖苷酶的测量。非保守的催化结构和独特的活性位点组织加强了对新型岩藻糖苷酶进行结构研究的必要性。