Reaction mechanism and substrate specificity for nucleotide sugar of mammalian α1,6-fucosyltransferase -: a large-scale preparation and characterization of recombinant human FUT8
Reaction mechanism and substrate specificity for nucleotide sugar of mammalian α1,6-fucosyltransferase -: a large-scale preparation and characterization of recombinant human FUT8
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DOI：
10.1093/glycob/cwj068
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发表时间：
2006-04-01
期刊：
影响因子：
4.3
通讯作者：
Taniguchi, N
中科院分区：
文献类型：
--
作者：
Ihara, H;Ikeda, Y;Taniguchi, N
FUT8是哺乳动物α-1，6-岩藻糖基转移酶，通过α-1，6-键催化5‘-二磷酸鸟苷-β-L-岩藻糖将岩藻糖残基转移到天冬酰胺连接的低聚糖核心结构的还原型末端。FUT8是一种典型的II型膜蛋白，定位于高尔基体。我们以前已经证明，在α1，2-，α1，6-和蛋白O-岩藻糖基转移酶之间保守的两个相邻的精氨酸残基在供体底物结合中起着重要的作用。然而，除了受体的底物专一性外，对FUT8的催化和反应机理以及三元结构的细节还不清楚。为了更好地了解FUT8，我们建立了大规模的重组人FUT8生产系统，其中该酶是由杆状病毒感染的昆虫细胞以可溶性形式生产的。动力学分析和GDPβ-L-岩藻糖衍生物的抑制研究表明，FUT8对该反应的催化作用依赖于快速平衡随机机理，并强烈识别GDPβ-L-岩藻糖的碱基和二磷酰基。这些结果也适用于其他岩藻糖基转移酶和糖基转移酶。
FUT8, mammalian alpha 1,6-fucosyltransferase, catalyzes the transfer of a fucose residue from the donor substrate, guanosine 5'-diphosphate (GDP)-beta-l-fucose, to the reducing terminal GlcNAc of the core structure of asparagine-linked oligosaccharide via an alpha 1,6-linkage. FUT8 is a typical type II membrane protein, which is localized in the Golgi apparatus. We have previously shown that two neighboring arginine residues that are conserved among alpha 1,2-, alpha 1,6-, and protein O-fucosyltransferases play an important role in donor substrate binding. However, details of the catalytic and reaction mechanisms and the ternary structure of FUT8 are not understood except for the substrate specificity of the acceptor. To develop a better understanding of FUT8, we established a large-scale production system for recombinant human FUT8, in which the enzyme is produced in soluble form by baculovirus-infected insect cells. Kinetic analyses and inhibition studies using derivatives of GDP-beta-l-fucose revealed that FUT8 catalyzes the reaction which depends on a rapid equilibrium random mechanism and strongly recognizes the base portion and diphosphoryl group of GDP-beta-l-fucose. These results may also be applicable to other fucosyltransferases and glycosyltransferases.