Structure and dynamics of an α-fucosidase reveal a mechanism for highly efficient IgG transfucosylation.
Structure and dynamics of an α-fucosidase reveal a mechanism for highly efficient IgG transfucosylation.
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DOI：
10.1038/s41467-020-20044-z
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发表时间：
2020-12-04
影响因子：
16.6
通讯作者：
Sundberg EJ
中科院分区：
文献类型：
--
作者：
Klontz EH;Li C;Kihn K;Fields JK;Beckett D;Snyder GA;Wintrode PL;Deredge D;Wang LX;Sundberg EJ
岩藻糖基化对于具有生物技术和医学应用的许多蛋白质的功能是重要的。α-岩藻糖苷酶包括一个大的酶家族，其识别这些蛋白质上具有不同α-键的岩藻糖基化底物。干酪乳杆菌产生一种α-岩藻糖苷酶，称为AlfC，对α（1，6）-岩藻糖具有特异性，这是在人N-聚糖核心岩藻糖基化中发现的唯一键。AlfC及其某些点突变体已用于从单克隆抗体N-聚糖中添加和去除岩藻糖，对其效应子功能具有显著影响。尽管AlfC具有潜在的用途，但对其机制知之甚少。在这里，我们提出的晶体结构的AlfC，结合突变和动力学分析，氢氘交换质谱，分子动力学模拟，和转岩藻糖基化实验，以确定的分子机制的活动AlfC及其转岩藻糖苷酶突变体。我们的研究结果表明，AlfC在活性位点附近产生了一个芳香亚位点，该亚位点特异性地容纳α（1，6）-键中的GlcNAc，表明酶活性受活性位点环的不同开放和闭合构象控制，某些突变使平衡向开放构象移动，以促进岩藻糖基转移而不是水解，并为合理产生AlfC岩藻糖苷转移酶突变体提供了潜在的可推广的框架。AlfC岩藻糖苷转移酶用于调节修饰单克隆抗体的聚糖的岩藻糖基化。在此，结构和生物物理特性揭示了AlfC的酶促机制，并为设计具有新特异性和功能的AlfC突变体提供了蓝图。
Fucosylation is important for the function of many proteins with biotechnical and medical applications. Alpha-fucosidases comprise a large enzyme family that recognizes fucosylated substrates with diverse α-linkages on these proteins. Lactobacillus casei produces an α-fucosidase, called AlfC, with specificity towards α(1,6)-fucose, the only linkage found in human N-glycan core fucosylation. AlfC and certain point mutants thereof have been used to add and remove fucose from monoclonal antibody N-glycans, with significant impacts on their effector functions. Despite the potential uses for AlfC, little is known about its mechanism. Here, we present crystal structures of AlfC, combined with mutational and kinetic analyses, hydrogen–deuterium exchange mass spectrometry, molecular dynamic simulations, and transfucosylation experiments to define the molecular mechanisms of the activities of AlfC and its transfucosidase mutants. Our results indicate that AlfC creates an aromatic subsite adjacent to the active site that specifically accommodates GlcNAc in α(1,6)-linkages, suggest that enzymatic activity is controlled by distinct open and closed conformations of an active-site loop, with certain mutations shifting the equilibrium towards open conformations to promote transfucosylation over hydrolysis, and provide a potentially generalizable framework for the rational creation of AlfC transfucosidase mutants. AlfC transfucosidase is used to modulate fucosylation of glycans decorating monoclonal antibodies. Herein, structural and biophysical characterization reveals the enzymatic mechanism of AlfC and a blueprint for the design of AlfC mutants with novel specificities and functions.
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影响因子：
28.5
作者：
Mellor JD;Brown MP;Irving HR;Zalcberg JR;Dobrovic A
通讯作者：
Dobrovic A
脑膜脓毒症伊丽莎白金菌核心岩藻糖苷酶的鉴定和表征
影响因子：
4.8
作者：
Li，Tiansheng;Li，Mengjie;Chen，Li
通讯作者：
Chen，Li
影响因子：
4.3
作者：
Koval'ova, Terezia;Koval, Tomas;Dohnalek, Jan
通讯作者：
Dohnalek, Jan
影响因子：
3.4
作者：
McGibbon, Robert T.;Beauchamp, Kyle A.;Pande, Vijay S.
通讯作者：
Pande, Vijay S.
DOI：
10.1107/s0907444909052925
发表时间：
2010-02
期刊：
Acta crystallographica. Section D, Biological crystallography
影响因子：
--
作者：
Adams PD;Afonine PV;Bunkóczi G;Chen VB;Davis IW;Echols N;Headd JJ;Hung LW;Kapral GJ;Grosse-Kunstleve RW;McCoy AJ;Moriarty NW;Oeffner R;Read RJ;Richardson DC;Richardson JS;Terwilliger TC;Zwart PH
通讯作者：
Zwart PH