Crystallographic structure of ChitA, a glycoside hydrolase family 19, plant class IV chitinase from Zea mays
Crystallographic structure of ChitA, a glycoside hydrolase family 19, plant class IV chitinase from Zea mays
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DOI：
10.1002/pro.2437
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发表时间：
2014-05-01
期刊：
影响因子：
8
通讯作者：
Rose, David R.
中科院分区：
文献类型：
--
作者：
Chaudet, Marcia M.;Naumann, Todd A.;Rose, David R.
玉米 ChitA 几丁质酶由连接到羧基末端几丁质酶结构域的小的、hevein 样结构域组成。在真菌性耳腐病期间，类橡胶素结构域被分泌的真菌蛋白酶切割，产生截短形式的 ChitA。在这里，我们报告了截短的 ChitA (ChitA N) 的结构和生化特征，它缺乏 hevein 样结构域。 ChitA N 及其突变体 (ChitA N-EQ) 得到表达和纯化；酶测定表明 ChitA N 活性与全长酶相当。 Glu62 突变为 Gln (ChitA N-EQ) 在不破坏底物结合的情况下消除了几丁质酶活性，表明 Glu62 直接参与催化。 ChitA N-EQ 的晶体结构为 Glu62、Arg177 和 Glu165 在水解中的关键作用以及 Ser103 和 Tyr106 在底物结合中的关键作用提供了强有力的支持。这些发现表明，hevein 样结构域对于酶活性来说不是必需的。此外，这种植物 IV 类几丁质酶的晶体结构与较大的 I 类和 II 类酶的结构的比较表明 IV 类几丁质酶已经进化以适应较短的底物。PDB 代码：
Maize ChitA chitinase is composed of a small, hevein-like domain attached to a carboxy-terminal chitinase domain. During fungal ear rot, the hevein-like domain is cleaved by secreted fungal proteases to produce truncated forms of ChitA. Here, we report a structural and biochemical characterization of truncated ChitA (ChitA N), which lacks the hevein-like domain. ChitA N and a mutant form (ChitA N-EQ) were expressed and purified; enzyme assays showed that ChitA N activity was comparable to the full-length enzyme. Mutation of Glu62 to Gln (ChitA N-EQ) abolished chitinase activity without disrupting substrate binding, demonstrating that Glu62 is directly involved in catalysis. A crystal structure of ChitA N-EQ provided strong support for key roles for Glu62, Arg177, and Glu165 in hydrolysis, and for Ser103 and Tyr106 in substrate binding. These findings demonstrate that the hevein-like domain is not needed for enzyme activity. Moreover, comparison of the crystal structure of this plant class IV chitinase with structures from larger class I and II enzymes suggest that class IV chitinases have evolved to accommodate shorter substrates.PDB Code(s):