Crystal structures and inhibitor binding studies of plant class V chitinases: the cycad enzyme exhibits unique strutural and functional features.
Crystal structures and inhibitor binding studies of plant class V chitinases: the cycad enzyme exhibits unique strutural and functional features.
复制标题
植物 V 类几丁质酶的晶体结构和抑制剂结合研究：苏铁酶表现出独特的结构和功能特征。
DOI：
10.1111/tpj.12785
复制
发表时间：
2015
期刊：
影响因子：
--
通讯作者：
and Tamo Fukamizo.
中科院分区：
文献类型：
--
作者：
Naoyuki Umemoto;Yuka Kanda;Takayuki Ohnuma;Takuo Osawa;Tomoyuki Numata;Shohei Sakuda;Toki Taira;and Tamo Fukamizo.
来自苏铁Cycas revoluta的V类（糖苷水解酶家族18）几丁质酶（CrChiA）是已报道具有有效转糖基化（TG）活性的植物几丁质酶。我们解析了CrChiA的晶体结构，并将其与不能有效催化TG反应的烟草（Nicotiana tabacum）（NtChiV）和拟南芥（Arabidopsis thaliana）（AtChiC）的Ⅴ类几丁质酶进行了比较。3种几丁质酶均具有相似的（α/β）8桶折叠结构，并具有一个（α + β）插入结构域。在CrChiA的受体结合位点（+1，+2和+3）中，发现Trp 168侧链与+3糖面对面堆叠。然而，在NtChiV和AtChiC的相同区域中没有发现这种相互作用。在DxDxE基序中，这是催化所必需的，中间Asp（Asp 117）的羧基总是朝向CrChiA中的催化酸Glu 119，而NtChiV和AtChiC中相应的Asp朝向第一个Asp。CrChiA的这些结构特征似乎是有效的TG活性的原因。当使用等温滴定量热法评估抑制剂别氨基菌素的结合时，发现三种几丁质酶的结合自由能的变化彼此相似，即在−9.5和−9.8 kcal mol−1之间。然而，溶剂化和构象熵变CrChiA显着不同的NtChiV和AtChiC，但类似的几丁质酶A从粘质沙雷氏菌（SmChiA），这也表现出显着的TG活性。这些结果提供了深入了解TG反应的分子机制和从细菌几丁质酶到植物V类几丁质酶的分子进化。
A class V (glycoside hydrolase family 18) chitinase from the cycadCycas revoluta(CrChiA) is a plant chitinase that has been reported to possess efficient transglycosylation (TG) activity. We solved the crystal structure of CrChiA, and compared it with those of class V chitinases fromNicotiana tabacum(NtChiV) andArabidopsis thaliana(AtChiC), which do not efficiently catalyze the TG reaction. All three chitinases had a similar (α/β)8barrel fold with an (α + β) insertion domain. In the acceptor binding site (+1, +2 and +3) of CrChiA, the Trp168 side chain was found to stack face‐to‐face with the +3 sugar. However, this interaction was not found in the identical regions of NtChiV and AtChiC. In the DxDxE motif, which is essential for catalysis, the carboxyl group of the middle Asp (Asp117) was always oriented toward the catalytic acid Glu119 in CrChiA, whereas the corresponding Asp in NtChiV and AtChiC was oriented toward the first Asp. These structural features of CrChiA appear to be responsible for the efficient TG activity. When binding of the inhibitor allosamidin was evaluated using isothermal titration calorimetry, the changes in binding free energy of the three chitinases were found to be similar to each other, i.e. between −9.5 and −9.8 kcal mol−1. However, solvation and conformational entropy changes in CrChiA were markedly different from those in NtChiV and AtChiC, but similar to those of chitinase A fromSerratia marcescens(SmChiA), which also exhibits significant TG activity. These results provide insight into the molecular mechanism underlying the TG reaction and the molecular evolution from bacterial chitinases to plant class V chitinases.