X-ray structure determination and deuteration of nattokinase.
X-ray structure determination and deuteration of nattokinase.
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X射线结构的确定和nattokinase的侵蚀。
DOI：
10.1107/s0909049513020700
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发表时间：
2013-11
影响因子：
2.5
通讯作者：
Morimoto Y
中科院分区：
文献类型：
--
作者：
Yanagisawa Y;Chatake T;Naito S;Ohsugi T;Yatagai C;Sumi H;Kawaguchi A;Chiba-Kamosida K;Ogawa M;Adachi T;Morimoto Y
对纳豆激酶进行了X射线结构测定和氢化反应，以便于进行中子结晶学分析。纳豆激酶(NK)是一种由纳豆枯草芽孢杆菌大量产生的强纤溶酶。虽然NK是枯草杆菌毒素家族的一员，但与其他枯草杆菌毒素相比，它表现出不同的底物特异性。分子模拟结果预测，NK的底物专一性可能与活性中心Ser221附近的氢排列有关。因此，中子结晶学分析应该为揭示NK的酶机制提供有价值的信息。在本报告中，我们确定了非氢形式的未氢NK的X-射线结构，并成功地制备出了非氢形式的NK。非氢NK结构是在1.74 ä分辨率下确定的。枯草芽孢杆菌DB104的NK和枯草杆菌毒素 E的三维结构基本相同。将纳豆枯草芽孢杆菌(Bacillussubtilis Natto)培养在氢化培养基中进行NK的氢化反应。通过连续几轮培养，获得了耐D2O的纳豆芽孢杆菌菌株，其培养基中的D2O浓度逐渐升高。从培养上清液中分离纯化NK细胞，用纤维蛋白平板法测定其活性。这些结果为中子蛋白质结晶学分析奠定了基础。
X-ray structure determination and deuteration of nattokinase were performed to facilitate neutron crystallographic analysis. Nattokinase (NK) is a strong fibrinolytic enzyme, which is produced in abundance by Bacillus subtilis natto. Although NK is a member of the subtilisin family, it displays different substrate specificity when compared with other subtilisins. The results of molecular simulations predict that hydrogen arrangements around Ser221 at the active site probably account for the substrate specificity of NK. Therefore, neutron crystallographic analysis should provide valuable information that reveals the enzymatic mechanism of NK. In this report, the X-ray structure of the non-hydrogen form of undeuterated NK was determined, and the preparation of deuterated NK was successfully achieved. The non-hydrogen NK structure was determined at 1.74 Å resolution. The three-dimensional structures of NK and subtilisin E from Bacillus subtilis DB104 are near identical. Deuteration of NK was carried out by cultivating Bacillus subtilis natto in deuterated medium. The D2O resistant strain of Bacillus subtilis natto was obtained by successive cultivation rounds, in which the concentration of D2O in the medium was gradually increased. NK was purified from the culture medium and its activity was confirmed by the fibrin plate method. The results lay the framework for neutron protein crystallography analysis.