The side chain of a glycosylated asparagine residue is important for the stability of isopullulanase.
The side chain of a glycosylated asparagine residue is important for the stability of isopullulanase.
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DOI：
10.1093/jb/mvu065
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发表时间：
2015-04
影响因子：
2.7
通讯作者：
T. Miyazaki;H. Yashiro;A. Nishikawa;T. Tonozuka
中科院分区：
文献类型：
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作者：
T. Miyazaki;H. Yashiro;A. Nishikawa;T. Tonozuka
已证明N-糖基化对于某些糖蛋白的稳定性很重要。异普鲁兰酶（伊普）是一种多糖水解酶，是一种高度N-糖基化的蛋白质，伊普去糖基化导致热稳定性降低。为了研究N-聚糖在伊普中的功能，我们将重点放在位于活性位点Asn 448附近的N-糖基化残基上。三种伊普变体Y 440 A、N448 A和S450 A的热稳定性比野生型酶低0.5-8.4°C。测定了内切糖苷酶H（Endo H）处理的N448 A变体的晶体结构。在不对称单元中有四个伊普分子，Mol-A、B、C和D。在Mol-C中由氨基酸残基435-455组成的环的构象与野生型伊普相同，而在Mol-A、Mol-B和Mol-D中该环的构象彼此不同。这些结果表明Asn 448侧链对伊普的稳定性是主要重要的。我们的研究结果表明，只有N-糖基化的天冬酰胺残基的突变可能会导致不正确的结论，N-聚糖的功能评价。通常，N-糖基化位点的结构在伊普中形成延伸构型;然而，Asn 448位点具有缺乏这种构型的非典型结构。
N-glycosylation has been shown to be important for the stability of some glycoproteins. Isopullulanase (IPU), a polysaccharide-hydrolyzing enzyme, is a highly N-glycosylated protein, and IPU deglycosylation results in a decrease in thermostability. To investigate the function of N-glycan in IPU, we focused on an N-glycosylated residue located in the vicinity of the active site, Asn448. The thermostabilities of three IPU variants, Y440A, N448A and S450A, were 0.5-8.4°C lower than the wild-type enzyme. The crystal structure of endoglycosidase H (Endo H)-treated N448A variant was determined. There are four IPU molecules, Mol-A, B, C and D, in the asymmetric unit. The conformation of a loop composed of amino acid residues 435-455 in Mol-C was identical to wild-type IPU, whereas the conformations of this loop in Mol-A, Mol-B and Mol-D were different from each other. These results suggest that the Asn448 side chain is primarily important for the stability of IPU. Our results indicate that mutation of only N-glycosylated Asn residue may lead to incorrect conclusion for the evaluation of the function of N-glycan. Usually, the structures of N-glycosylation sites form an extended configuration in IPU; however, the Asn448 site had an atypical structure that lacked this configuration.