Structure and enzymatic properties of a two-domain family GH19 chitinase from Japanese cedar (Cryptomeria japonica) pollen
Structure and enzymatic properties of a two-domain family GH19 chitinase from Japanese cedar (Cryptomeria japonica) pollen
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日本柳杉花粉双域家族 GH19 几丁质酶的结构和酶学特性
DOI：
10.1021/acs.jafc.8b01140
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发表时间：
2018
影响因子：
6.1
通讯作者：
T.
中科院分区：
文献类型：
--
作者：
Takashima;T.;Numata;T.;Taira;T.;Fukamizo;T. and Ohnuma;T.
CJP-4是一种在日本杉木（cryptomeria japonica）花粉中发现的过敏原。该蛋白是一个双结构域家族GH19 （IV类）几丁质酶，由n端CBM18结构域和GH19催化结构域组成。在这里，我们制备了重组cbp -4和cbp - 18截断的cbp -4 （cbp -4- cat）蛋白。除了通过x射线晶体学解析cbp -4- cat的晶体结构外，我们还分析了这两种蛋白水解几丁质寡糖（GlcNAc）n、多糖底物、乙二醇几丁质和β-几丁质纳米纤维的能力，并检测了它们对真菌生长的抑制活性。截断CBM18结构域对（GlcNAc）水解模式无显著影响。然而，当我们使用多糖底物时，观察到显著的效果。CJP-4对可溶性底物乙二醇甲壳素的活性低于CJP-4- cat。相比之下，CJP-4对不溶性底物β-几丁质纳米纤维的活性高于CJP-4- cat。CJP-4对真菌生长有明显抑制作用，而CJP-4- cat对真菌生长无明显抑制作用。这些结果表明，CBM18结构域通过与几丁质结合来促进不溶性底物的水解和CJP-4-Cat的抗真菌作用。据报道，CJP-4-Cat只有两个环（环I和环III），这是一种来自玉米的致敏性IV类几丁质酶ChiA。
CJP-4 is an allergen found in pollen of the Japanese cedarCryptomeria japonica. The protein is a two-domain family GH19 (class IV) Chitinase consisting of an N-terminal CBM18 domain and a GH19 catalytic domain. Here, we produced recombinant CJP-4 and CBM18-truncated CJP-4 (CJP-4-Cat) proteins. In addition to solving the crystal structure of CJP-4-Cat by X-ray crystallography, we analyzed the ability of both proteins to hydrolyze chitin oligosaccharides, (GlcNAc)n, polysaccharide substrates, glycol chitin, and β-chitin nanofiber and examined their inhibitory activity toward fungal growth. Truncation of the CBM18 domain did not significantly affect the mode of (GlcNAc)nhydrolysis. However, significant effects were observed when we used the polysaccharide substrates. The activity of CJP-4 toward the soluble substrate, glycol chitin, was lower than that of CJP-4-Cat. In contrast, CJP-4 exhibited higher activity toward β-chitin nanofiber, an insoluble substrate, than did CJP-4-Cat. Fungal growth was strongly inhibited by CJP-4 but not by CJP-4-Cat. These results indicate that the CBM18 domain assists the hydrolysis of insoluble substrate and the antifungal action of CJP-4-Cat by binding to chitin. CJP-4-Cat was found to have only two loops (loops I and III), as reported for ChiA, an allergenic class IV Chitinase from maize.