Active site complementation and hexameric arrangement in the GH family 29; a structure-function study of α-l-fucosidase isoenzyme 1 from Paenibacillus thiaminolyticus
Active site complementation and hexameric arrangement in the GH family 29; a structure-function study of α-l-fucosidase isoenzyme 1 from Paenibacillus thiaminolyticus
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DOI：
10.1093/glycob/cwy078
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发表时间：
2019-01-01
期刊：
影响因子：
4.3
通讯作者：
Dohnalek, Jan
中科院分区：
文献类型：
--
作者：
Koval'ova, Terezia;Koval, Tomas;Dohnalek, Jan
来自解硫胺类芽孢杆菌细菌的 α-L-岩藻糖苷酶同工酶 1 是糖苷水解酶家族 GH29 的成员，能够从寡糖和复合糖的非还原末端裂解 L-岩藻糖。在这里，我们展示了该蛋白质的第一个晶体结构，揭示了该家族内的新型四元态。该蛋白质处于独特的六聚体组装中，揭示了第一个观察到的活性位点与该家族中相邻单体的残基互补的情况。互补色氨酸残基的突变引起催化特性的变化，包括最适pH值的变化、对人工显色底物的亲和力的变化以及对天然底物的反应速率的降低。野生型酶对大多数测试的天然存在的寡糖具有活性，并且能够在多种受体分子上进行转糖基化，包括糖类、醇类或显色底物。互补残基的突变既不改变底物特异性，也不改变对转糖基化受体分子类型的偏好；然而，两种情况下反应的产率均较低。与晶体结构中的酶结合的麦芽糖分子确定了表面碳水化合物结合位点，可能参与较大寡糖的结合。
alpha-l-Fucosidase isoenzyme 1 from bacterium Paenibacillus thiaminolyticus is a member of the glycoside hydrolase family GH29 capable of cleaving l-fucose from nonreducing termini of oligosaccharides and glycoconjugates. Here we present the first crystal structure of this protein revealing a novel quaternary state within this family. The protein is in a unique hexameric assembly revealing the first observed case of active site complementation by a residue from an adjacent monomer in this family. Mutation of the complementing tryptophan residue caused changes in the catalytic properties including a shift of the pH optimum, a change of affinity to an artificial chromogenic substrate and a decreased reaction rate for a natural substrate. The wild-type enzyme was active on most of the tested naturally occurring oligosaccharides and capable of transglycosylation on a variety of acceptor molecules, including saccharides, alcohols or chromogenic substrates. Mutation of the complementing residue changed neither substrate specificity nor the preference for the type of transglycosylation acceptor molecule; however, the yields of the reactions were lower in both cases. Maltose molecules bound to the enzyme in the crystal structure identified surface carbohydrate-binding sites, possibly participating in binding of larger oligosaccharides.